CN102630490A - Artificial cultivation method for cordyceps militaris mycelium for increasing cordycepin content - Google Patents
Artificial cultivation method for cordyceps militaris mycelium for increasing cordycepin content Download PDFInfo
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Abstract
The invention discloses an artificial cultivation method for cordyceps militaris mycelium for increasing cordycepin content. The artificial cultivation method comprises the steps of test tube slant strain cultivation, test tube liquid strain cultivation, triangular flask liquid seed cultivation, seed jar cultivation and fermentation tank cultivation. The artificial cultivation method is characterized in that: during the step of fermentation tank cultivation, when the yield of mycelium is 0.4-0.8% from the seed jar cultivation, the fermentation tank cultivation is started; 1%-9% of manganese sulfate by weight is contained in a culture medium; the temperature is at 23-26 DEG C; aerating is at a ratio of 1:0.5; the stirring speed is 120rpm-150rpm; and when the yield of the mycelium is 1.2-1.8% and the content of raw sugar in the culture medium is at most 0.2%, the jar is released and the cordyceps militaris mycelium is harvested. According to the artificial cultivation method disclosed by the invention, under the stressing action of manganese ions, the cordycepin content in the cordyceps militaris mycelium is increased by above 0.8 times than that in a reference culture medium without manganese sulfate, an effective method for increasing the yield is supplied to the production of the cordycepin products, and the market prospect is wide.
Description
Technical field:
The present invention relates to a kind ofly coerce the mycelial artificial culture method of Cordyceps militaris that improves cordycepin output, belong to technical field of bioengineering through manganese ion.
Background technology:
Cordycepin (cordycepin, i.e. 3 '-desoxyadenossine) is the characteristic chemical constituent of some Cordyceps sinensis fungus, also is one of most important active component in the Cordyceps sinensis fungus.
Chinese caterpillar fungus have functions such as immunological regulation, antitumor, antifatigue, adjusting cardiopulmonary.Cordycepin is as a kind of novel broad spectrum antibiotic, and with its distinctive antimicrobial antiviral activity, the RNA that it can suppress virus synthesizes; Hay bacillus and mycobacterium tuberculosis avium all there is inhibitory action; HIV-I type virus also there is lethal effect; Especially multiple solid malignant there is very strong inhibitory action.Cordycepin demonstrates fabulous pharmaceutical applications prospect, and the research of cordycepin is at present just becoming extremely active field in the pharmaceutical chemistry at present.
The content of cordycepin in Cordyceps sinensis is atomic, and some other Cordyceps sinensis fungus such as Cordyceps militaris, Cordyceps kyushuensis etc., its cordycepin is much higher.So current cordycepin product generally extracts from Cordyceps militaris and obtains.
The method that improves the cordycepin content of Cordyceps sinensis fungus has two types; One type is the modes such as mutation breeding through bacterial strain; Improve the cordycepin yield level of original bacterial classification; Like " seed selection of a kind of method of producing cordycepin and high-yield cordyceps bacterial strain BYB-08 with use CN200810101715 ", seed selection one plant height produce bacterial strain BYB-08; In " a kind of industrial method for culturing north winter CN200610117998 of worm summer herb with high-content of cordycepin ", seed selection bacterial strain Cordyceps militaris COB201, CGMCC No.1823, all improve than original starting strain output.Another kind of method is to improve the output of cordycepin through the mode of adding inducer in condition of culture optimization and the incubation.Like " a kind of fermentation process CN200910027839 that is used for high cordyceps militaris biomass and high cordycepin content "; Through adding biotype elicitor Xylaria gingko endogenous fungus extract in the incubation; With abiotic type elicitor sodium acetate, ammonium sulfate, promote the generation of cordycepin in the thalline.
No matter these modes are the modes of mutation breeding or the mode of endogenetic fungus elicitor improves content of cordycepin, and it is all cumbersome to operate, and cordycepin content is not very high.
The applicant is in the research work of being engaged in Cordyceps sinensis fungus for a long time; Found that Cordyceps sinensis fungus has superpower patience and enriched character for zinc ion, manganese ion; In the further research aweto fungus Mechanism of Physiological and Biochemical for the superpower patience of zinc ion and manganese ion and enriched character, the manganese ion of discovery higher concentration has inducing action and facilitation for the generation of cordycepin.This discovery has fabulous application prospect in the production of cordycepin.
At present, also do not report about coerce the artificial culture method that improves cordycepin output through manganese ion both at home and abroad.
Summary of the invention:
The objective of the invention is to overcome the deficiency of above-mentioned prior art and a kind of simple to operate, cordycepin content is high a kind of mycelial artificial culture method of Cordyceps militaris that improves cordycepin content of coercing through manganese ion is provided.
The object of the invention can reach through following measure: a kind of mycelial artificial culture method of Cordyceps militaris that improves cordycepin content; It may further comprise the steps: test tube slant spawn culture, test tube strain cultivation, the cultivation of triangular flask liquid seeds, seed tank culture, fermentation tank culture; It is characterized in that in the described fermentation tank culture step when seed tank culture to mycelium recovery rate 0.4-0.8%, changing fermentation tank culture over to, contain 1%-9% manganese sulphate in its medium by weight percentage; 23 ℃ ~ 26 ℃ of temperature; Ventilated 1: 0.5, speed of agitator 120 rpm ~ 150 rpm are cultured to mycelium recovery rate 1.2-1.8%; Medium content of reducing sugar 0.2% o'clock is at the most put jar results.
In order further to carry out the object of the invention, described fermentation tank culture medium is made up of glucose, peptone, yeast extract, manganese sulphate, water.
In order further to carry out the object of the invention, described fermentation tank culture medium is made up of sucrose, corn steep liquor, manganese sulphate, water.
In order further to carry out the object of the invention, described fermentation tank culture medium is made up of corn flour, soybean cake powder, manganese sulphate, water.
In order further to carry out the object of the invention, described fermentation tank culture medium is made up of sucrose, fish meal, manganese sulphate, water.
The present invention compares with prior art can produce following good effect: the applicant is based on to the research of cordyceps filament to the manganese ion enrichment mechanism; Recognize that the cordyceps filament has extremely strong accumulation ability to manganese ion; And find to coerce down at the high concentration manganese ion; Can improve the output of mycelium cordycepin, and best manganese ion concentration scope is arranged.Adopt cultural method of the present invention, not only simple, and cordycepin output is high, cordycepin content exceeds more than 0.8 times in the mycelium that the high concentration manganese ion coerces than not adding.
Embodiment: following specific embodiments of the invention elaborates:
Embodiment 1:
Bacterial classification: Cordyceps militaris spawn Cordyceps militaris 5.270 derives from microorganism fungus kind preservation center, Guangdong Province.
Test tube slant spawn culture (activation) (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, agar powder 15-20g, water 890-971g, its pH value is 5.0-7.0, is sub-packed in the test tube; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, 15 ~ 30 ℃ of dark cultivations 5-10 days;
Test tube strain cultivation (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, is sub-packed in the test tube sterilization; Insert activation back bevel bacterial classification, 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
Carry out the triangular flask liquid seeds and cultivate (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g; Magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986 g; Its pH value is 5.0-7.0, the prepared culture medium branch is packed in the 500ml conical flask into every bottled 200ml; Seal the back and under 120 ~ 130 ℃ of conditions, sterilized 18 ~ 30 minutes, take out medium, 5.0-7.0 is cooled to 20 ~ 30 ℃; Every bottle graft is gone into the cultured test tube liquid spawn of 10ml-20ml under aseptic condition, and 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
Seed tank culture (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g; Potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, in batches sterilization; Insert cultured triangular flask liquid spawn, the volume 60%-80% that feeds intake, 15 ~ 30 ℃ of temperature; Ventilated 1: 0.5, and in the 100L seeding tank, cultivated 2-5 days for 15 ~ 30 ℃, and be extended in 10 tons of jars stir culture step by step 2-5 days;
Fermentation tank culture:
When seed tank culture to mycelium recovery rate 0.4-0.8%, change 50 tons of fermentation tank culture over to, feed intake 40 tons, feed intake (medium) is: glucose 800-2000 kg; Peptone 160-400 kg, yeast extract 160-400 kg, manganese sulphate 400 kg-3600kg, water surplus; Its pH value is 5.0-7.0, and 23 ℃ ~ 26 ℃ of temperature were ventilated 1: 0.5, speed of agitator 120 rpm ~ 150 rpm; Be cultured to mycelium recovery rate 1.2-1.8%, medium content of reducing sugar 0.2% o'clock is at the most put jar results.Cordycepin content reaches 2.31mg/g in the mycelium, and cordycepin content reaches 0.69mg/g in the mycelium and do not add under the condition of manganese sulphate, increases by 2.3 times after adding manganese sulphate.
Embodiment 2:
Bacterial classification: Cordyceps militaris spawn Cordyceps militaris 5.701 derives from Chinese common micro-organisms culture presevation administrative center.
Test tube slant spawn culture (activation) (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, agar powder 15-20g, water 890-971g, its pH value is 5.0-7.0, is sub-packed in the test tube; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, 15 ~ 30 ℃ of dark cultivations 5-10 days;
Test tube strain cultivation (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, is sub-packed in the test tube sterilization; Insert activation back bevel bacterial classification, 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
The triangular flask liquid seeds is cultivated (also can cultivate according to other conventional methods):
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g; Magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986 g; Its pH value is 5.0-7.0, the prepared culture medium branch is packed in the 500ml conical flask into every bottled 200ml; Seal the back and under 120 ~ 130 ℃ of conditions, sterilized 18 ~ 30 minutes, take out medium, 5.0-7.0 is cooled to 20 ~ 30 ℃; Every bottle graft is gone into the cultured test tube liquid spawn of 10ml-20ml under aseptic condition, and 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
Seed tank culture (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g; Potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, in batches sterilization; Insert cultured triangular flask liquid spawn, the volume 60%-80% that feeds intake, 15 ~ 30 ℃ of temperature; Ventilated 1: 0.5, and in the 100L seeding tank, cultivated 2-5 days for 15 ~ 30 ℃, and be extended in 10 tons of jars stir culture step by step 2-5 days;
Fermentation tank culture:
When seed tank culture to mycelium recovery rate 0.4-0.8%, change 50 tons of fermentation tank culture over to.Feed intake 40 tons, feed intake (medium) is: sucrose 800-2000 kg, fish meal 200-400 kg; Manganese sulphate 400 kg-3600kg, water surplus, its pH value is 5.0-7.0; 23 ℃ ~ 26 ℃ of temperature were ventilated 1: 0.5, speed of agitator 120 rpm ~ 150 rpm; Be cultured to mycelium recovery rate 1.2-1.8%, medium content of reducing sugar 0.2% o'clock is at the most put jar results.Cordycepin content reaches 1.17mg/g in the mycelium, and cordycepin content reaches 0.65mg/g in the mycelium and do not add under the condition of manganese sulphate, has increased by 0.8 times after adding manganese sulphate.
Embodiment 3:
Bacterial classification: Cordyceps militaris spawn Cordyceps militaris 5.270 derives from microorganism fungus kind preservation center, Guangdong Province.
Test tube slant spawn culture (activation) (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 20g, peptone 6g, yeast extract 5g, magnesium sulfate 1g, potassium dihydrogen phosphate 1g, agar powder 15g, water 952g, its pH value is 5.0-7.0, is sub-packed in the test tube; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, 15 ~ 30 ℃ of dark cultivations 5-10 days;
Test tube strain cultivation (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, is sub-packed in the test tube sterilization; Insert activation back bevel bacterial classification, 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
The triangular flask liquid seeds is cultivated (also can cultivate according to other conventional methods):
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g; Magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986g; Its pH value is 5.0-7.0, the prepared culture medium branch is packed in the 500ml conical flask into every bottled 200ml; Seal the back and under 120 ~ 130 ℃ of conditions, sterilized 18 ~ 30 minutes, take out medium, 5.0-7.0 is cooled to 20 ~ 30 ℃; Every bottle graft is gone into the cultured test tube liquid spawn of 10ml-20ml under aseptic condition, and 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
Seed tank culture (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g; Potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, in batches sterilization; Insert cultured triangular flask liquid spawn, the volume 60%-80% that feeds intake, 15 ~ 30 ℃ of temperature; Ventilated 1: 0.5, and in the 100L seeding tank, cultivated 2-5 days for 15 ~ 30 ℃, and be extended in 10 tons of jars stir culture step by step 2-5 days;
Fermentation tank culture:
When seed tank culture to mycelium recovery rate 0.4-0.8%, change 50 tons of fermentation tank culture over to.Feed intake 40 tons, feed intake (medium) is: sucrose 800-2000 kg, corn steep liquor 200-1000 kg, manganese sulphate 400 kg-3600kg; Water surplus, its pH value is 5.0-7.0,23 ℃ ~ 26 ℃ of temperature; Ventilated speed of agitator 120 rpm ~ 150 rpm 1: 0.5; Be cultured to mycelium recovery rate 1.2-1.8%, medium content of reducing sugar 0.2% o'clock is at the most put jar results.Cordycepin content reaches 1.92mg/g in the mycelium, and cordycepin content reaches 0.55mg/g in the mycelium and do not add under the condition of manganese sulphate, has increased by 2.50 times after adding manganese sulphate.
Embodiment 4:
Bacterial classification: Cordyceps militaris spawn Cordyceps militaris 5.270 derives from microorganism fungus kind preservation center, Guangdong Province.
Test tube slant spawn culture (activation) (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, agar powder 15-20g, water 890-971g, its pH value is 5.0-7.0, is sub-packed in the test tube; Cordyceps militaris spawn is inserted the test tube slant under aseptic condition, 15 ~ 30 ℃ of dark cultivations 5-10 days;
Test tube strain cultivation (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, is sub-packed in the test tube sterilization; Insert activation back bevel bacterial classification, 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
The triangular flask liquid seeds is cultivated (also can cultivate according to other conventional methods):
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g; Magnesium sulfate 1-5g, potassium dihydrogen phosphate 1-5g, water 910-986 g; Its pH value is 5.0-7.0, the prepared culture medium branch is packed in the 500ml conical flask into every bottled 200ml; Seal the back and under 120 ~ 130 ℃ of conditions, sterilized 18 ~ 30 minutes, take out medium, 5.0-7.0 is cooled to 20 ~ 30 ℃; Every bottle graft is gone into the cultured test tube liquid spawn of 10ml-20ml under aseptic condition, and 15 ~ 30 ℃, speed of agitator 120rpm cultivated 5 days ~ 10 days;
Seed tank culture (also can cultivate) according to other conventional methods:
Preparing culture medium: glucose 10-60g, peptone 1-10g, yeast extract 1-10g, magnesium sulfate 1-5g; Potassium dihydrogen phosphate 1-5g, water 910-986g, its pH value is 5.0-7.0, in batches sterilization; Insert cultured triangular flask liquid spawn, the volume 60%-80% that feeds intake, 15 ~ 30 ℃ of temperature; Ventilated 1: 0.5, and in the 100L seeding tank, cultivated 2-5 days for 15 ~ 30 ℃, and be extended in 10 tons of jars stir culture step by step 2-5 days;
Fermentation tank culture:
When seed tank culture to mycelium recovery rate 0.4-0.8%, change 50 tons of fermentation tank culture over to, feed intake 40 tons, feed intake (medium) is: corn flour 800-2000 kg; Soybean cake powder 200-400 kg, manganese sulphate 400 kg-3600kg, water surplus, its pH value is 5.0-7.0; 23 ℃ ~ 26 ℃ of temperature were ventilated 1: 0.5, speed of agitator 120 rpm ~ 150 rpm; Be cultured to mycelium recovery rate 1.2-1.8%, medium content of reducing sugar 0.2% o'clock is at the most put jar results.Cordycepin content reaches 2.32mg/g in the mycelium, and cordycepin content reaches 0.67mg/g in the mycelium and do not add under the condition of manganese sulphate, has increased by 2.46 times after adding manganese sulphate.
Claims (5)
1. mycelial artificial culture method of Cordyceps militaris that improves cordycepin content, it may further comprise the steps: test tube slant spawn culture, test tube strain cultivation, the cultivation of triangular flask liquid seeds, seed tank culture, fermentation tank culture is characterized in that in the described fermentation tank culture step when seed tank culture to mycelium recovery rate 0.4-0.8%; Change fermentation tank culture over to; Contain 1%-9% manganese sulphate in its medium by weight percentage, 23 ℃ ~ 26 ℃ of temperature were ventilated 1: 0.5; Speed of agitator 120 rpm ~ 150 rpm; Be cultured to mycelium recovery rate 1.2-1.8%, medium content of reducing sugar 0.2% o'clock is at the most put jar results.
2. a kind of mycelial artificial culture method of Cordyceps militaris that improves cordycepin content according to claim 1 is characterized in that described fermentation tank culture medium is made up of glucose, peptone, yeast extract, manganese sulphate, water.
3. a kind of mycelial artificial culture method of Cordyceps militaris that improves cordycepin content according to claim 1 is characterized in that described fermentation tank culture medium is made up of sucrose, corn steep liquor, manganese sulphate, water.
4. a kind of mycelial artificial culture method of Cordyceps militaris that improves cordycepin content according to claim 1 is characterized in that described fermentation tank culture medium is made up of corn flour, soybean cake powder, manganese sulphate, water.
5. a kind of mycelial artificial culture method of Cordyceps militaris that improves cordycepin content according to claim 1 is characterized in that described fermentation tank culture medium is made up of sucrose, fish meal, manganese sulphate, water.
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| CN103081723A (en) * | 2013-02-17 | 2013-05-08 | 哈尔滨伟平科技开发有限公司 | Preparation method for Gloeostereum incarnatum powder |
| CN103609329A (en) * | 2013-11-05 | 2014-03-05 | 昆山市康乐虫草专业合作社 | Cordyceps militaris culturing method capable of improving cordycepin content |
| CN104969771A (en) * | 2014-04-04 | 2015-10-14 | 张治平 | High-cordycepin-content cordyceps militaris strain breeding technology |
| CN103897990A (en) * | 2014-04-14 | 2014-07-02 | 曾化伟 | Process for producing high anticancer component-cordyceps militaris L.Fr Link fungus powder in large scale through submerged fermentation |
| CN104221717A (en) * | 2014-09-26 | 2014-12-24 | 桂林丰茂源农业技术开发有限公司 | Method for increasing content of cordycepin in cordyceps militaris sporocarps |
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| CN104663252A (en) * | 2015-03-19 | 2015-06-03 | 张东海 | Breeding method and production process for cordyceps militaris strain with high cordycepin content |
| CN110024618A (en) * | 2019-05-22 | 2019-07-19 | 江苏康淼源生物科技有限公司 | A kind of segment wood cultivated method of polyporus rhinoceros cooke |
| CN110024618B (en) * | 2019-05-22 | 2021-02-12 | 江苏康淼源生物科技有限公司 | Cut-log cultivation method of polyporusrhinocerus cooke |
| CN112005814A (en) * | 2019-05-30 | 2020-12-01 | 大叶大学 | Cordyceps sobolifera, solid and liquid culture method thereof and application of extract thereof |
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