CN103563651A - Long preservation method of fertile production seed source of cordyceps militaris - Google Patents
Long preservation method of fertile production seed source of cordyceps militaris Download PDFInfo
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- CN103563651A CN103563651A CN201310509054.6A CN201310509054A CN103563651A CN 103563651 A CN103563651 A CN 103563651A CN 201310509054 A CN201310509054 A CN 201310509054A CN 103563651 A CN103563651 A CN 103563651A
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Abstract
The invention is suitable for the field of cordyceps militaris production, and provides a long preservation method of a fertile production seed source of cordyceps militaris. The method comprises the steps that a fertile excellent parent seed source of the cordyceps militaris obtained by breeding serves as an initial seed source; a liquid strain is cultured by a conventional method; after a liquid seed source is cultured, the liquid strain is subpackaged under an aseptic condition, and then preserved in an ultra-low-temperature refrigerator (between -72 DEG C and -86 DEG C); three tubes of preserved seed source are required to be randomly extracted, and unfrozen step by step; the unfrozen seed source serves as the liquid seed source; the liquid production seed source is subjected to expanding propagation once; the cordyceps militaris is cultivated by the conventional method; if the cultivated cordyceps militaris grows normally, the liquid seed source preserved in the same batch is an effective and usable strain; and the strain subjected to freezing preservation can be preserved for a long time (the longest preservation period is three years at present). The method fundamentally solves the technical problems that a deterioration speed of the excellent cordyceps militaris seed source is high and a production supply period of the fertile seed source is short; and purposes of detecting the seed source in the same batch once and permanently using the seed source are achieved.
Description
Technical field
The invention belongs to Cordyceps militaris production field, relating in particular to a kind of Cordyceps militaris can the permanent method for preserving of fecundity production provenance.
Background technology
North Chinese caterpillar fungus formal name used at school Cordyceps militaris.It is one of two type sepecies in Cordyceps, now as unique substitute of Cordyceps sinensis.North Chinese caterpillar fungus main component has cordycepin, cordycepic acid, Cordyceps sinensis polysaccharide, SOD enzyme etc., has cancer-resisting, and kidney tonifying benefit lung and raising human immunological competence's effect is a kind of food medicine dual-purpose health-care good product that exploitation is worth that has.
Chinese caterpillar fungus was found so far from 1985 in north, imitative wild tussah chrysalis cultivation and two stages of the complete artificial cultivation of rice medium have been experienced, follow in recent years extensive cordyceps militaris cultivation technology to become better and approaching perfection day by day, also occurred a bottleneck technical problem can not be ignored, that is exactly Cordyceps militaris spawn degenerate problem.
For solving spawn degeneration problem, mainly by seed selection and the good Cordyceps militaris of preservation, can fecundity provenance complete at present.Seed selection refer to by organize isolation technics or ascospore hybridization technique obtain out careless high, output shape is good can fecundity Cordyceps militaris provenance; Preservation is by the how science preservation of the excellent species of seed selection.According to us, test for many years, find the female provenance method for preserving routinely of planting of Cordyceps militaris, 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 can the permanent method for preserving of fecundity production provenance, is intended to solve good Cordyceps militaris provenance catagen speed fast, can short technical problem of fecundity provenance production and supply phase.
The present invention is achieved in that a kind of Cordyceps militaris can the permanent method for preserving of fecundity production provenance,, comprise following step:
1) Cordyceps militaris seed selection being obtained can the good mother of fecundity be planted provenance as initial experiment provenance, and provenance form is the female kind of inclined-plane solid;
2) liquid spawn culture medium is sub-packed in 250 milliliters of Erlenmeyer flasks or white dripping bottle, 100 milliliters of every bottled medium, pack into after sealing in high-pressure sterilizing pot, at 1.05Kg/cm
2under pressure, sterilizing is 60 minutes, to be cooled to 25 ℃ start below inoculation;
3) postvaccinal Cordyceps militaris bacterial classification shakes cultivation at 20 ℃ ± 1 ℃ temperature, 120～130 beats/min of concussion frequencies, continuous culture 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) liquid spawn of step 3) being cultivated is divided and installs in 25 milliliters of aseptic cryovials with cover under aseptic condition, 10 milliliters of every pipes, and be numbered, select at random 3 pipes, all the other are all deposited in-72 ℃ to-86 ℃ of super low temperature refrigerators simultaneously;
5) the 3 pipe liquid spawns of selecting are at random tried to plant according to a conventional method, 3 bottles go out all normal same batch of provenances of grass, and the same batch of provenance being deposited in super low temperature refrigerator used as yielding ability provenance.
Further, step 2), liquid spawn culture medium formula is: 20 grams of glucose, 5 grams of peptones, yeast soak 3 grams, powder, 3 grams of potassium dihydrogen phosphates, 2 grams, magnesium sulfate, Cobastab
110 milligrams, Cobastab
21000 milliliters of 5 milligrams, water, boil and dissolve each component substances in formula.
Further, in super low temperature refrigerator, liquid spawn is thawed before using step by step, concrete steps are: first liquid spawn cryovial is forwarded to temperature for depositing 24 hours in-24 ℃ to-30 ℃ refrigerator-freezers, then forward temperature to for depositing 24 hours in-10 ℃ to-16 ℃ refrigerator-freezers, finally put into 0 ℃ of environment of frozen water mixing and thaw to liquid nutrient medium and all dissolve.
Compared with prior art, beneficial effect is in the present invention:
The Cordyceps militaris preserving by the present invention can fecundity liquid provenance, and effectively storage life can reach 3 years (the longest test preservation term is 3 years at present) even more for a long time;
It is fast that the present invention has fundamentally solved good Cordyceps militaris provenance catagen speed, can short technical problem of fecundity provenance production and supply phase, realized same batch of provenance one-time detection, the lasting target of using.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, 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, is not intended to limit the present invention.
Embodiment 1
Can the permanent method for preserving of fecundity production provenance, according to following step, carry out:
1) Cordyceps militaris seed selection being obtained can the good mother of fecundity be planted provenance as initial experiment provenance, and provenance form is the female kind of inclined-plane solid;
2) liquid spawn culture medium is sub-packed in 250 milliliters of Erlenmeyer flasks or white dripping bottle, 100 milliliters of every bottled medium, pack into after sealing in high-pressure sterilizing pot, at 1.05Kg/cm
2under pressure, sterilizing is 60 minutes, to be cooledly to 25 ℃, starts below to inoculate according to a conventional method; Concrete inoculation method: in superclean bench gnotobasis, the female all source preparation of liquid nutrient medium after sterilizing and inclined-plane solid is good, near alcolhol burner flame, open female tube tampon of planting, with inoculating tool, get 1 of the bacterial classification of soybean grain size, access liquid nutrient medium, aseptic sealing.
3) postvaccinal Cordyceps militaris bacterial classification shakes cultivation at 20 ℃ ± 1 ℃ temperature, 120～130 beats/min of concussion frequencies, continuous culture 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) liquid spawn of step 3) being cultivated is divided and installs in 25 milliliters of aseptic cryovials with cover under aseptic condition, 10 milliliters of every pipes, and be numbered, select at random 3 pipes, all the other are all deposited in-72 ℃ to-86 ℃ of super low temperature refrigerators simultaneously;
5) the 3 pipe liquid spawns of selecting are at random tried to plant according to a conventional method, all normal if 3 pipes go out grass, the same batch of provenance being deposited in super low temperature refrigerator can be used as the use of yielding ability provenance.
Wherein, step 2), liquid spawn culture medium formula is: 20 grams of glucose, 5 grams of peptones, yeast soak 3 grams, powder, 3 grams of potassium dihydrogen phosphates, 2 grams, magnesium sulfate, Cobastab
110 milligrams, Cobastab
21000 milliliters of 5 milligrams, 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 cryovial is forwarded to temperature for depositing 24 hours in-24 ℃ to-30 ℃ refrigerator-freezers, then forward temperature to for depositing 24 hours in-10 ℃ to-16 ℃ refrigerator-freezers, finally put into 0 ℃ of environment of frozen water mixing and thaw to liquid nutrient medium and all dissolve.
After thawing, take out liquid spawn cryovial and produce liquid spawn by 2,3 program steps, cultured liquid spawn can be used as to produce to be expanded once numerous by liquid spawn provenance again.
The effective storage life of sample of preservation of the present invention can reach 3 years (the longest test preservation term is 3 years at present) even more for a long time.
Embodiment 2
Be with the difference of embodiment 1, the liquid spawn of in step 4), step 3) being cultivated is divided and installs in 25 milliliters of aseptic cryovials with cover under aseptic condition, 10 milliliters of every pipes, and be numbered, select at random 3 pipes, all the other are all deposited in-86 ℃ of super low temperature refrigerators simultaneously;
The sample of preservation thaws before use step by step, concrete steps are: first liquid spawn cryovial is forwarded to temperature for depositing 24 hours in-30 ℃ of refrigerator-freezers, then forward temperature to for depositing 24 hours in-16 ℃ of refrigerator-freezers, finally put into frozen water and mix 0 ℃ of environment and thaw to liquid nutrient medium and all dissolve.
Embodiment 3
Be with the difference of embodiment 1:
The liquid spawn that step 3) is cultivated is divided and installs in 25 milliliters of aseptic cryovials with cover under aseptic condition, 10 milliliters of every pipes, and be numbered, select at random 3 pipes, all the other are all deposited in-72 ℃ of super low temperature refrigerators simultaneously;
The sample of preservation thaws before use step by step, concrete steps are: concrete steps are: first liquid spawn cryovial is forwarded to temperature for depositing 24 hours in-24 ℃ of refrigerator-freezers, then forward temperature to for depositing 24 hours in-10 ℃ of refrigerator-freezers, finally put into frozen water and mix 0 ℃ 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 modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
1. Cordyceps militaris can the permanent method for preserving of fecundity production provenance, it is characterized in that, comprises following step:
1) Cordyceps militaris seed selection being obtained can the good mother of fecundity be planted provenance as initial experiment provenance, and provenance form is the female kind of inclined-plane solid;
2) liquid spawn culture medium is sub-packed in 250 milliliters of Erlenmeyer flasks or white dripping bottle, 100 milliliters of every bottled medium, pack into after sealing in high-pressure sterilizing pot, at 1.05Kg/cm
2under pressure, sterilizing is 60 minutes, to be cooled to 25 ℃ start below inoculation;
3) postvaccinal Cordyceps militaris bacterial classification shakes cultivation at 20 ℃ ± 1 ℃ temperature, 120～130 beats/min of concussion frequencies, continuous culture 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) liquid spawn of step 3) being cultivated is divided and installs in 25 milliliters of aseptic cryovials with cover under aseptic condition, 10 milliliters of every pipes, and be numbered, select at random 3 pipes, all the other are all deposited in-72 ℃ to-86 ℃ of super low temperature refrigerators simultaneously;
5) the 3 pipe liquid spawns of selecting at random trys to plant according to a conventional method, examination is planted the selected 3 pipe bacterial classifications of result and is all normally gone out grass, and being deposited in that same batch of provenance in super low temperature refrigerator have can fecundity, can be used as the use of yielding ability provenance.
2. can the permanent method for preserving of fecundity production provenance according to Cordyceps militaris claimed in claim 1, it is characterized in that step 2) in liquid spawn culture medium formula be: 20 grams of glucose, 5 grams of peptones, yeast soak 3 grams, powder, 3 grams of potassium dihydrogen phosphates, 2 grams, magnesium sulfate, Cobastab
110 milligrams, Cobastab
21000 milliliters of 5 milligrams, water, boil and dissolve each component substances in formula.
3. can the permanent method for preserving of fecundity production provenance according to Cordyceps militaris claimed in claim 1, it is characterized in that, in super low temperature refrigerator, liquid spawn is thawed before using step by step, concrete steps are: first liquid spawn cryovial is forwarded to temperature for depositing 24 hours in-24 ℃ to-30 ℃ refrigerator-freezers, then forward temperature to for depositing 24 hours in-10 ℃ to-16 ℃ refrigerator-freezers, finally put into 0 ℃ of environment of frozen water mixing and thaw to liquid nutrient medium and all dissolve.
4. can the permanent method for preserving of fecundity production provenance according to Cordyceps militaris claimed in claim 3, it is characterized in that, take out liquid spawn cryovial by step 2) and step 3) production liquid spawn, cultured liquid spawn is expanded once numerous as producing by liquid spawn provenance again.
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| CN201310509054.6A CN103563651B (en) | 2013-10-23 | 2013-10-23 | Cordyceps militaris (L.) Link. can the permanent method for preserving of fecundity production provenance |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450521A (en) * | 2014-12-22 | 2015-03-25 | 东莞市生物技术研究所 | Method for preserving cordyceps militaris strain for long time |
| 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 |
| CN107955794A (en) * | 2017-11-27 | 2018-04-24 | 沈阳农业大学 | The high-quality method for preserving of Cordyceps militaris spawn |
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0398575A (en) * | 1989-09-08 | 1991-04-24 | Hokuto Sangyo Kk | Freeze preservation of basidiomycetes or the like |
| 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 |
| WO2006016702A1 (en) * | 2004-08-12 | 2006-02-16 | Suntory Limited | Method for polyunsaturated fatty acid production using novel cell preservation technique |
| CN102703323A (en) * | 2012-07-06 | 2012-10-03 | 浦城正大生化有限公司 | Strain preservation method |
-
2013
- 2013-10-23 CN CN201310509054.6A patent/CN103563651B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0398575A (en) * | 1989-09-08 | 1991-04-24 | Hokuto Sangyo Kk | Freeze preservation of basidiomycetes or the like |
| 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 |
| WO2006016702A1 (en) * | 2004-08-12 | 2006-02-16 | Suntory Limited | Method for polyunsaturated fatty acid production using novel cell preservation technique |
| CN102703323A (en) * | 2012-07-06 | 2012-10-03 | 浦城正大生化有限公司 | Strain preservation method |
Non-Patent Citations (3)
| Title |
|---|
| 吕和平等: "食用菌菌株的长期保藏方法", 《中国食用菌》 * |
| 方苏等: "冬虫夏草菌种保藏方法的比较", 《安徽农业科学》 * |
| 梁宁利: "微生物菌种保藏方法概述", 《农产品加工(学刊) 》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450521A (en) * | 2014-12-22 | 2015-03-25 | 东莞市生物技术研究所 | Method for preserving cordyceps militaris strain for long time |
| 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 |
| CN107955794A (en) * | 2017-11-27 | 2018-04-24 | 沈阳农业大学 | The high-quality method for preserving of Cordyceps militaris spawn |
| 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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