CN103563651B - Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance - Google Patents
Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance Download PDFInfo
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- CN103563651B CN103563651B CN201310509054.6A CN201310509054A CN103563651B CN 103563651 B CN103563651 B CN 103563651B CN 201310509054 A CN201310509054 A CN 201310509054A CN 103563651 B CN103563651 B CN 103563651B
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Abstract
The present invention is applicable to Cordyceps militaris (L.) Link. production field, and providing a kind of Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance.The Cordyceps militaris (L.) Link. that seed selection obtains by the method the excellent mother of fecundity can plant provenance as initial provenance, cultivate liquid spawn according to a conventional method, after liquid provenance is cultivated, aseptically packing is carried out to liquid spawn, be then preserved in-72 DEG C to-86 DEG C super cryogenic refrigerators.Institute's preservation provenance needs to randomly draw 3 pipes, thaw step by step, a numerous liquid production provenance is expanded again as liquid provenance after thawing, cultivate Cordyceps militaris (L.) Link. according to a conventional method, if Cordyceps militaris (L.) Link. growth is normal after cultivation, liquid provenance so with batch preservation is exactly effectively can use bacterial classification, and the bacterial classification of freezing can long term storage (the longest preservation term is 3 years at present).It is fast that the present invention fundamentally solves excellent Cordyceps militaris (L.) Link. provenance catagen speed, can fecundity provenance production and supply phase short technical problem, achieves same batch of provenance one-time detection, the lasting target used.
Description
Technical field
The invention belongs to Cordyceps militaris (L.) Link. production field, particularly relating to a kind of Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance.
Background technology
Cordyceps militaris formal name used at school Cordyceps militaris (L.) Link..It is one of two type species in Cordyceps, now as unique substitute of Cordyceps sinensis.Cordyceps militaris main component has cordycepin, cordycepic acid, Cordyceps polysaccharide, SOD enzyme etc., has cancer-resisting, and effect of kidney tonifying benefit lung and raising human immunological competence is a kind of food medicine dual-purpose health-care good product having Development volue.
Cordyceps militaris found so far from 1985, experienced by imitative wild cocoon chrysalis cultivation and two stages of the complete artificial culture of rice medium, become better and approaching perfection day by day with extensive cordyceps militaris cultivation technology in recent years, also occurred a bottleneck technical problem that can not be ignored, that is exactly Cordyceps militaris spawn degenerate problem.
For solving spawn degeneration problem, can fecundity provenance come mainly through seed selection and the excellent Cordyceps militaris (L.) Link. of preservation at present.Seed selection refer to by separate tissue technology or thecaspore hybridization technique obtain out careless high, output shape good can fecundity Cordyceps militaris (L.) Link. provenance; Preservation is then by the how science preservation of the excellent species of seed selection.Test for many years according to us, find that Cordyceps militaris (L.) Link. mother plants provenance method for preserving routinely, as: low temperature inclined-plane solid preserving process, although there is certain delayed involution effect, preservation effect is very unstable, cannot realize effective preservation.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance, is intended to solve excellent Cordyceps militaris (L.) Link. provenance catagen speed fast, can fecundity provenance production and supply phase short technical problem.
The present invention is achieved in that a kind of Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance, comprise following step:
1) Cordyceps militaris (L.) Link. seed selection obtained the excellent mother of fecundity can plant provenance as initial experiment provenance, and provenance form is that inclined-plane solid mother plants;
2) liquid spawn culture medium is sub-packed in 250 milliliters of Erlenmeyer flasks or white dripping bottle, every bottled substratum 100 milliliters, loads in high-pressure sterilizing pot after sealing, at 1.05Kg/cm
2sterilizing 60 minutes under pressure, to be cooled to less than 25 DEG C start inoculation;
3) postvaccinal Cordyceps militaris bacterial classification shakes cultivation at 20 DEG C ± 1 DEG C temperature, concussion frequency 120 ~ 130 beats/min, cultured continuously 5 ~ 7 days; Select the liquid spawn that bacterium ball size is even, burr is sharp keen, quantity is many, medium liquid is as clear as crystal;
4) be aseptically dispensed in 25 milliliters of aseptic freeze pipes with cover by the liquid spawn that step 3) is cultivated, often pipe 10 milliliters, and be numbered, select 3 pipes at random, all the other are all deposited in-72 DEG C to-86 DEG C super cryogenic refrigerators simultaneously;
5) the 3 pipe liquid spawns selected at random are carried out according to a conventional method examination to plant, 3 bottles go out grass same batch of provenance all normally, and the same batch of provenance be deposited in super cryogenic refrigerator uses as productivity provenance.
Further, step 2) in liquid spawn culture medium formula be: glucose 20 grams, peptone 5 grams, yeast leaching 3 grams, powder, potassium primary phosphate 3 grams, 2 grams, magnesium sulfate, vitamins B
110 milligrams, vitamins B
25 milligrams, 1000 milliliters, water, boil and dissolve each component substances in formula.
Further, in super cryogenic refrigerator, liquid spawn is thawed before using step by step, concrete steps are: first liquid spawn freeze pipe is forwarded to temperature for depositing 24 hours in-24 DEG C to-30 DEG C refrigerator-freezers, then forwarding temperature to for depositing 24 hours in-10 DEG C to-16 DEG C refrigerator-freezers, finally putting into frozen water and mixing 0 DEG C of environment and thaw to liquid nutrient medium and all dissolve.
Compared with prior art, beneficial effect is in the present invention:
The Cordyceps militaris (L.) Link. preserved by the present invention can fecundity liquid provenance, and effective storage life can reach 3 years even more for a long time (the longest test preservation term is 3 years at present);
It is fast that the present invention fundamentally solves excellent Cordyceps militaris (L.) Link. provenance catagen speed, can fecundity provenance production and supply phase short technical problem, achieves same batch of provenance one-time detection, the lasting target used.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
A kind of Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance, carries out according to following step:
1) Cordyceps militaris (L.) Link. seed selection obtained the excellent mother of fecundity can plant provenance as initial experiment provenance, and provenance form is that inclined-plane solid mother plants;
2) liquid spawn culture medium is sub-packed in 250 milliliters of Erlenmeyer flasks or white dripping bottle, every bottled substratum 100 milliliters, loads in high-pressure sterilizing pot after sealing, at 1.05Kg/cm
2sterilizing 60 minutes under pressure, to be cooledly starts to inoculate according to a conventional method to less than 25 DEG C; Concrete inoculation method: in Bechtop gnotobasis, by good for female to liquid nutrient medium after sterilizing and inclined-plane solid all source preparation, open near spirit lamp flame and femalely plant tube tampon, get the bacterial classification 1 piece of soybean grain size with inoculating tool, access liquid nutrient medium, asepticly seals.
3) postvaccinal Cordyceps militaris bacterial classification shakes cultivation at 20 DEG C ± 1 DEG C temperature, concussion frequency 120 ~ 130 beats/min, cultured continuously 5 ~ 7 days; Select the liquid spawn that bacterium ball size is even, burr is sharp keen, quantity is many, medium liquid is as clear as crystal;
4) be aseptically dispensed in 25 milliliters of aseptic freeze pipes with cover by the liquid spawn that step 3) is cultivated, often pipe 10 milliliters, and be numbered, select 3 pipes at random, all the other are all deposited in-72 DEG C to-86 DEG C super cryogenic refrigerators simultaneously;
5) the 3 pipe liquid spawns selected at random are carried out according to a conventional method examination to plant, if 3 pipes go out grass all normally, the same batch of provenance be deposited in super cryogenic refrigerator can be used as productivity provenance and uses.
Wherein, step 2) in liquid spawn culture medium formula be: glucose 20 grams, peptone 5 grams, yeast leaching 3 grams, powder, potassium primary phosphate 3 grams, 2 grams, magnesium sulfate, vitamins B
110 milligrams, vitamins B
25 milligrams, 1000 milliliters, water, boil and dissolve each component substances in formula.
6) sample of above-mentioned preservation thaws before use step by step, concrete steps are: first liquid spawn freeze pipe is forwarded to temperature for depositing 24 hours in-24 DEG C to-30 DEG C refrigerator-freezers, then forwarding temperature to for depositing 24 hours in-10 DEG C to-16 DEG C refrigerator-freezers, finally putting into frozen water and mixing 0 DEG C of environment and thaw to liquid nutrient medium and all dissolve.
Take out liquid spawn freeze pipe after thawing and produce liquid spawn by 2,3 programsteps, cultured liquid spawn can be expanded once numerous by liquid spawn provenance as production again.
The effective storage life of sample of preservation of the present invention can reach 3 years even more for a long time (the longest test preservation term is 3 years at present).
Embodiment 2
Be with the difference of embodiment 1, in step 4), the liquid spawn that step 3) is cultivated aseptically is dispensed in 25 milliliters of aseptic freeze pipes with cover, often pipe 10 milliliters, and is numbered, select 3 pipes at random, all the other are all deposited in-86 DEG C of super cryogenic refrigerators simultaneously;
The sample of preservation thaws before use step by step, concrete steps are: first liquid spawn freeze pipe is forwarded to temperature for depositing 24 hours in-30 DEG C of refrigerator-freezers, then forwarding temperature to for depositing 24 hours in-16 DEG C of refrigerator-freezers, finally putting into frozen water and mixing 0 DEG C of environment and thaw to liquid nutrient medium and all dissolve.
Embodiment 3
Be with the difference of embodiment 1:
Aseptically be dispensed in 25 milliliters of aseptic freeze pipes with cover by the liquid spawn that step 3) is cultivated, often pipe 10 milliliters, and be numbered, select 3 pipes at random, all the other are all deposited in-72 DEG C of super cryogenic refrigerators simultaneously;
The sample of preservation thaws before use step by step, concrete steps are: concrete steps are: first liquid spawn freeze pipe is forwarded to temperature for depositing 24 hours in-24 DEG C of refrigerator-freezers, then forwarding temperature to for depositing 24 hours in-10 DEG C of refrigerator-freezers, finally putting into frozen water and mixing 0 DEG C of environment and thaw to liquid nutrient medium and all dissolve.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance, it is characterized in that, comprises following step:
1) Cordyceps militaris (L.) Link. seed selection obtained the excellent mother of fecundity can plant provenance as initial experiment provenance, and provenance form is that inclined-plane solid mother plants;
2) liquid spawn culture medium is sub-packed in 250 milliliters of Erlenmeyer flasks or white dripping bottle, every bottled substratum 100 milliliters, loads in high-pressure sterilizing pot after sealing, at 1.05Kg/cm
2sterilizing 60 minutes under pressure, to be cooled to less than 25 DEG C start inoculation;
3) postvaccinal Cordyceps militaris bacterial classification shakes cultivation at 20 DEG C ± 1 DEG C temperature, concussion frequency 120 ~ 130 beats/min, cultured continuously 5 ~ 7 days; Select the liquid spawn that bacterium ball size is even, burr is sharp keen, quantity is many, medium liquid is as clear as crystal;
4) by step 3) liquid spawn cultivated aseptically is dispensed in 25 milliliters of aseptic freeze pipes with cover, often pipe 10 milliliters, and be numbered, select 3 pipes at random, all the other are all deposited in-72 DEG C to-86 DEG C super cryogenic refrigerators simultaneously;
5) the 3 pipe liquid spawns selected at random are carried out according to a conventional method examination to plant, examination is planted 3 pipe bacterial classifications selected by result and is all normally gone out grass, then the same batch of provenance be deposited in super cryogenic refrigerator has can fecundity, can use as productivity provenance;
In super cryogenic refrigerator, liquid spawn is thawed before using step by step, concrete steps are: first liquid spawn freeze pipe is forwarded to temperature for depositing 24 hours in-24 DEG C to-30 DEG C refrigerator-freezers, then forwarding temperature to for depositing 24 hours in-10 DEG C to-16 DEG C refrigerator-freezers, finally putting into frozen water and mixing 0 DEG C of environment and thaw to liquid nutrient medium and all dissolve.
2. can the permanent method for preserving of fecundity production provenance according to Cordyceps militaris (L.) Link. according to claim 1, it is characterized in that, step 2) in liquid spawn culture medium formula be: glucose 20 grams, peptone 5 grams, yeast leaching 3 grams, powder, potassium primary phosphate 3 grams, 2 grams, magnesium sulfate, VB11 0 milligram, vitamins B
25 milligrams, 1000 milliliters, water, boil and dissolve each component substances in formula.
3. can the permanent method for preserving of fecundity production provenance according to Cordyceps militaris (L.) Link. according to claim 1, it is characterized in that, take out liquid spawn freeze pipe by step 2) and step 3) producing liquid spawn, cultured liquid spawn is expanded once numerous as production by liquid spawn provenance again.
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| CN104450521B (en) * | 2014-12-22 | 2017-07-07 | 东莞市生物技术研究所 | A kind of long-term method for preserving Cordyceps militaris spawn |
| CN104663251A (en) * | 2015-03-18 | 2015-06-03 | 景洪宏臻农业科技有限公司 | Boletus aereus mother strain long-time preservation method |
| CN107955794B (en) * | 2017-11-27 | 2021-04-13 | 沈阳农业大学 | High-quality preservation method of cordyceps militaris strains |
| CN108739053A (en) * | 2018-06-22 | 2018-11-06 | 辽东学院 | A kind of method of long-term preservation high cordycepin Cordyceps militaris spawn |
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| JPH0731B2 (en) * | 1989-09-08 | 1995-01-11 | ホクト産業株式会社 | Cryopreservation method for basidiomycetes |
| ES2342305T3 (en) * | 2004-08-12 | 2010-07-05 | Nippon Suisan Kaisha, Ltd. | METHOD OF PRODUCTION OF POLYINSATURATED FATTY ACIDS USING A NEW CELL CONSERVATION TECHNIQUE. |
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| CN1445360A (en) * | 2002-03-20 | 2003-10-01 | 中国科学院沈阳应用生态研究所 | Method of artificial planting northerly Chinese caterpillar fungus for increasing content of physiologically active substance |
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