CN109526537A - A kind of Cordyceps militaris rejuvenation breeding technique - Google Patents
A kind of Cordyceps militaris rejuvenation breeding technique Download PDFInfo
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- 238000009395 breeding Methods 0.000 title claims abstract description 19
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- 230000001580 bacterial effect Effects 0.000 claims abstract description 25
- 230000004913 activation Effects 0.000 claims abstract description 16
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 15
- 241000209140 Triticum Species 0.000 claims abstract description 15
- 235000021307 Triticum Nutrition 0.000 claims abstract description 15
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- 230000007850 degeneration Effects 0.000 claims abstract description 12
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Abstract
The invention discloses a kind of Cordyceps militaris rejuvenation breeding techniques, the specific steps are as follows: the cordyceps militaris link bacterial strain of degeneration is carried out activation culture；Cordyceps militaris link bacterial strain after activation is inoculated in be inoculated on wheat or rice culture medium for cultivating after cultivating in Shake flask medium and is cultivated；Selection fructification interception tip, which is hung on culture medium for cultivating bottle cap, cultivates, until media surface spore germination is thorough, takes out fructification tip, continues to cultivate 30-40d；The mature Cordyceps militaris of culture is picked into drying, detects its principle active component cordycepin, is used as cultivation original seed until character is stablized.The present invention need not use spore collectors, simplify operation, saved breeding cost；Secondly as be monospore the purification and rejuvenation, therefore it can guarantee the purity of strain, directed screening goes out the stable excellent species of character, as cultivation original seed, suitable for being not suitable for and causing the bacterial strain rejuvenation of spawn degeneration due to spontaneous mutation and environmental factor.
Description
Technical field
The present invention relates to degeneration edible mushroom strains rejuvenation technique field, specifically a kind of Cordyceps militaris rejuvenation breeding techniques.
Background technique
Culturing edible fungus, needs to obtain high yield, and bacterial strain is crucial.Cordyceps militaris is Ascomycotina, ergot Zoopagales, ergot
Section, Cordyceps type sepecies.Cordyceps militaris also known as northern Chinese caterpillar Fungus, northern Cordyceps militaris are that cordyceps sinensis belongs to sibling species, effectively
Component is similar with cordyceps sinensis, and containing ucleosides, polysaccharide, sterols and SOD enzyme etc., and cordycepin content is even more to be higher than the winter
Worm summer grass has and improves the multiple pharmacological effects such as immunity of organisms, antitumor, anti-aging, protection cardiopulmonary tissue, tranquilizing soporific.
Therefore, Cordyceps militaris as cordyceps sinensis substitute on health medicine and food, the huge market demand.
In recent years, the key technology of China's Artificial Cultivation of Cordyceps militaris Link is broken through, so that extensive artificial culture pupa worm
Grass is achieved.However in production cultivation and practice process, decline or the degenerate problem of strain will necessarily be encountered, strain is made
Merit fails or loses to a certain extent.The decline of cordyceps militaris link bacterial strain is mainly manifested in, and mycelial growth rate slows down, bacterium
Silk is sparse, and fructification generation rate is low or fructification is individual short and small, distortion, and in fermentation process, the yield of required metabolite is substantially
Phenomena such as reduction.
The decline for leading to microbial strains is that many-sided reason causes, and be can be by the production of the quantitative change to qualitative change of spontaneous mutation
It is raw, it can be and be not suitable for causing to morph by environmental factor, or caused by planting by it the invasion of microorganism or preservation
Mode is improper, transposing simply, passage, and expands the minus variant in group constantly etc. caused by aspects reason.It degenerates and has
Body show themselves in that occur mycelia is sparse, the speed of growth is slack-off, oidium increases, fruit-body formation rate is low, lopsided amount is more, metabolism produce
Phenomena such as content of object cordycepin reduces.
After microbial strains decline, needs to take the method for rejuvenation to restore its original merit, generally use
Method is that the indexs such as its typical character, production performance are measured after carrying out Pure strain separation, filters out minority from the group to have failed
The individual not yet degenerated, or rejuvenation is carried out by other laboratory facilities, so that degenerative strain is restored original merit.
At this stage, the rejuvenation main method of edible mushroom has following several:
1. tissue separation: being that opposite fruiting time after degenerative strain fruiting, will be selected early, fructification appearance is healthy and strong, disease-resistant
The fructification for the characters such as ability is strong picks, and aseptically carries out tissue separation with PDA culture medium.Separate the bacterium obtained
Strain, measures through biology performance, selects the bacterial strain of merit, fruiting experiment is carried out, if the fructification after fruiting can be restored
Original character, the bacterial strain after being isolated can be used as subsequent cultivation original seed and use；If the fructification after fruiting is not replied also original
Character must then carry out tissue separation rejuvenation again.
2. mycelia purifying is separated with Tip Splitting: the mycelium tip portion of picking stalwartness carries out purifying culture, makes mycelia
Restore the purity to keep strain, so that strain is restored original vitality and merit, achieve the purpose that rejuvenation.
3. sexual spore separation screening: selection form is good, the medium well fructification of robust growth, using spore collectors
Sexual spore is collected, more spores or single spore separation culture are carried out.The uninucleate hyphae of acquisition also needs and another uninucleate hyphae that can be affine
Nucleated mycelium is formed after hybridization, then carries out mushroom producing culture.Spore separation needs repetition test, and fruiting performance is good, could conduct
Strain excellent puts into production in cultivation.
Strain decline how is solved during Cordyceps militaris factory culture, Mycelium growth rate slows down, and mycelia is dilute
It dredges, oidium amount increases, and fruit-body formation rate reduces or fructification individual is short and small, in deformity and fermentation process, required metabolism
The problems such as yield of product is greatly reduced, oneself is through becoming the critical issue of Cordyceps militaris factory culture.
Summary of the invention
The purpose of the present invention is to provide a kind of simple and easy, effective Cordyceps militaris rejuvenation breeding techniques, to solve
The problems mentioned above in the background art.
To achieve the above object, the invention provides the following technical scheme:
A kind of Cordyceps militaris rejuvenation breeding technique, the specific steps are as follows:
(1) cordyceps militaris link bacterial strain of degeneration is inoculated in PDA slant medium, 23 DEG C of progress activation cultures；
(2) cordyceps militaris link bacterial strain after activation is inoculated in Shake flask medium, 23 DEG C of culture 3-5d；
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 20-25 DEG C of culture 40-
50d or so；
(4) selection form is good, the medium well fructification of robust growth, and interception tip hangs on culture medium for cultivating bottle cap
On, 20-23 DEG C of culture 5-10d takes out fructification tip until media surface spore germination is thorough, continues to cultivate 30-40d；Together
When observe mycelial growth rate, produce surviving of son physical capabilities；
(5) continue to repeat (4) step operation 2-3 times, while the mature Cordyceps militaris of culture is picked into drying, detecting it mainly has
Ingredient cordycepin is imitated, is used as cultivation original seed until character is stablized.
As a further solution of the present invention: PDA slant medium in the step (1) are as follows: potato 200g/L, sucrose
20g/L, agar 15g/L, PH are natural.
As further scheme of the invention: seed culture medium in the step (2) are as follows: potato 200g/L, sucrose
20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1For 10mg/L.
As a further solution of the present invention: culture medium for cultivating in the step (3) are as follows: wheat or 30-50g/ bottles of rice,
40-70ml/ bottles of water.
As further scheme of the invention: the cordyceps militaris link bacterial strain is the great emigrant's 1# bacterial strain of Cordyceps militaris.
Compared with prior art, the beneficial effects of the present invention are:
The present invention need not use spore collectors, simplify operation, saved breeding cost；Secondly as being that monospore is pure
Change rejuvenation, therefore can guarantee the purity of strain, directed screening goes out the stable excellent species of character, as cultivation original seed, is suitable for
Since spontaneous mutation and environmental factor are not suitable for and cause the bacterial strain rejuvenation of spawn degeneration.The present invention breaches traditional bacterial strain
Rejuvenation method carries out rejuvenation to bacterial strain using spore ejection method, and the method operation is easy, simple and easy, and significant effect.By
Bacterial strain after such method rejuvenation, mycelia is sturdy, fine and close, and the speed of growth before rejuvenation compared to being enhanced, solid
In body cultivation, fruit-body formation rate is greatly improved, and Individual Size is more uniform, and abnormal rate reduces, compared to traditional rejuvenation method
Speech, the method is more efficient simple easily operated, practical.
Specific embodiment
The technical solution of the patent is explained in further detail With reference to embodiment.
Embodiment 1
A kind of Cordyceps militaris rejuvenation breeding technique, the specific steps are as follows:
(1) by the great emigrant's 1# strain inoculated of the Cordyceps militaris of degeneration in PDA slant medium, 23 DEG C of progress activation culture 8d；
The PDA slant medium are as follows: potato 200g/L, sucrose 20g/L, agar 15g/L, PH are natural.
(2) by the great emigrant's 1# strain inoculated of Cordyceps militaris after activation in Shake flask medium, 23 DEG C of culture 3d；The seed training
Support base are as follows: potato 200g/L, sucrose 20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1For 10mg/L.
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 20 DEG C of culture 40d or so；
Culture medium for cultivating are as follows: wheat or 30g/ bottles of rice, 40ml/ bottles of water.
(4) selection form is good, the medium well fructification of robust growth, and interception tip hangs on culture medium for cultivating bottle cap
On, 20 DEG C of culture 5d take out fructification tip until media surface spore germination is thorough, continue to cultivate 30d；Bacterium is observed simultaneously
The silk speed of growth, produce surviving of son physical capabilities；
(5) continue to repeat (4) step operation 2 times, while the mature Cordyceps militaris of culture is picked into drying, it is mainly effective to detect it
Ingredient cordycepin is used as cultivation original seed until character is stablized.
Embodiment 2
A kind of Cordyceps militaris rejuvenation breeding technique, the specific steps are as follows:
(1) by the great emigrant's 1# strain inoculated of the Cordyceps militaris of degeneration in PDA slant medium, 23 DEG C of progress activation culture 8d；
The PDA slant medium are as follows: potato 200g/L, sucrose 20g/L, agar 15g/L, PH are natural.
(2) by the great emigrant's 1# strain inoculated of Cordyceps militaris after activation in Shake flask medium, 23 DEG C of culture 3.5d；The seed
Culture medium are as follows: potato 200g/L, sucrose 20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1For 10mg/
L。
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 21 DEG C of culture 42d or so；
Culture medium for cultivating are as follows: wheat or 35g/ bottles of rice, 50ml/ bottles of water.
(4) selection form is good, the medium well fructification of robust growth, and interception tip hangs on culture medium for cultivating bottle cap
On, 21 DEG C of culture 6d take out fructification tip until media surface spore germination is thorough, continue to cultivate 32d；Bacterium is observed simultaneously
The silk speed of growth, produce surviving of son physical capabilities；
(5) continue to repeat (4) step operation 3 times, while the mature Cordyceps militaris of culture is picked into drying, it is mainly effective to detect it
Ingredient cordycepin is used as cultivation original seed until character is stablized.
Embodiment 3
A kind of Cordyceps militaris rejuvenation breeding technique, the specific steps are as follows:
(1) by the great emigrant's 1# strain inoculated of the Cordyceps militaris of degeneration in PDA slant medium, 23 DEG C of progress activation culture 8d；
The PDA slant medium are as follows: potato 200g/L, sucrose 20g/L, agar 15g/L, PH are natural.
(2) by the great emigrant's 1# strain inoculated of Cordyceps militaris after activation in Shake flask medium, 23 DEG C of culture 4d；The seed training
Support base are as follows: potato 200g/L, sucrose 20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1For 10mg/L.
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 22 DEG C of culture 45d or so；
Culture medium for cultivating are as follows: wheat or 40g/ bottles of rice, 55ml/ bottles of water.
(4) selection form is good, the medium well fructification of robust growth, and interception tip hangs on culture medium for cultivating bottle cap
On, 22 DEG C of culture 8d take out fructification tip until media surface spore germination is thorough, continue to cultivate 35d；Bacterium is observed simultaneously
The silk speed of growth, produce surviving of son physical capabilities；
(5) continue to repeat (4) step operation 2 times, while the mature Cordyceps militaris of culture is picked into drying, it is mainly effective to detect it
Ingredient cordycepin is used as cultivation original seed until character is stablized.
Embodiment 4
A kind of Cordyceps militaris rejuvenation breeding technique, the specific steps are as follows:
(1) by the great emigrant's 1# strain inoculated of the Cordyceps militaris of degeneration in PDA slant medium, 23 DEG C of progress activation culture 8d；
The PDA slant medium are as follows: potato 200g/L, sucrose 20g/L, agar 15g/L, PH are natural.
(2) by the great emigrant's 1# strain inoculated of Cordyceps militaris after activation in Shake flask medium, 23 DEG C of culture 4.5d；The seed
Culture medium are as follows: potato 200g/L, sucrose 20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1For 10mg/
L。
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 24 DEG C of culture 48d or so；
Culture medium for cultivating are as follows: wheat or 45g/ bottles of rice, 60ml/ bottles of water.
(4) selection form is good, the medium well fructification of robust growth, and interception tip hangs on culture medium for cultivating bottle cap
On, 22 DEG C of culture 9d take out fructification tip until media surface spore germination is thorough, continue to cultivate 38d；Bacterium is observed simultaneously
The silk speed of growth, produce surviving of son physical capabilities；
(5) continue to repeat (4) step operation 3 times, while the mature Cordyceps militaris of culture is picked into drying, it is mainly effective to detect it
Ingredient cordycepin is used as cultivation original seed until character is stablized.
Embodiment 5
A kind of Cordyceps militaris rejuvenation breeding technique, the specific steps are as follows:
(1) by the great emigrant's 1# strain inoculated of the Cordyceps militaris of degeneration in PDA slant medium, 23 DEG C of progress activation cultures；Institute
State PDA slant medium are as follows: potato 200g/L, sucrose 20g/L, agar 15g/L, PH are natural.
(2) by the great emigrant's 1# strain inoculated of Cordyceps militaris after activation in Shake flask medium, 23 DEG C of culture 5d；The seed training
Support base are as follows: potato 200g/L, sucrose 20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1For 10mg/L.
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 25 DEG C of culture 50d or so；
Culture medium for cultivating are as follows: wheat or 50g/ bottles of rice, 70ml/ bottles of water.
(4) selection form is good, the medium well fructification of robust growth, and interception tip hangs on culture medium for cultivating bottle cap
On, 23 DEG C of culture 10d take out fructification tip until media surface spore germination is thorough, continue to cultivate 40d；Bacterium is observed simultaneously
The silk speed of growth, produce surviving of son physical capabilities；
(5) continue to repeat (4) step operation 2 times, while the mature Cordyceps militaris of culture is picked into drying, detecting it mainly has
Ingredient cordycepin is imitated, is used as cultivation original seed until character is stablized.
The great emigrant's 1# bacterial strain of Cordyceps militaris used in embodiment, is researched and developed by Liaoning biotech inc Hong Qiao breeding
Center provides.
The present invention need not use spore collectors, simplify operation, saved breeding cost；Secondly as being that monospore is pure
Change rejuvenation, therefore can guarantee the purity of strain, directed screening goes out the stable excellent species of character, as cultivation original seed, is suitable for
Since spontaneous mutation and environmental factor are not suitable for and cause the bacterial strain rejuvenation of spawn degeneration.The present invention breaches traditional bacterial strain
Rejuvenation method carries out rejuvenation to bacterial strain using spore ejection method, and the method operation is easy, simple and easy, and significant effect.By
Bacterial strain after such method rejuvenation, mycelia is sturdy, fine and close, and the speed of growth before rejuvenation compared to being enhanced, solid
In body cultivation, fruit-body formation rate is greatly improved, and Individual Size is more uniform, and abnormal rate reduces, compared to traditional rejuvenation method
Speech, the method is more efficient simple easily operated, practical.
Experimental result shows that the bacterial strain after rejuvenation, for mycelial growth rate compared with greatly improving before rejuvenation, mycelium is sturdy,
It is fine and close；Strain inoculated after rejuvenation is on solid medium, and fruit-body formation rate greatly improves, and Individual Size is more uniform, deformity
Rate reduces；Cordycepin content is about 4.5-6.5 times before rejuvenation in fructification.
The preferred embodiment of the patent is described in detail above, but this patent is not limited to above-mentioned embodiment party
Formula within the knowledge of one of ordinary skill in the art can also be under the premise of not departing from this patent objective
Various changes can be made.
Claims (5)
1. a kind of Cordyceps militaris rejuvenation breeding technique, which is characterized in that specific step is as follows:
(1) cordyceps militaris link bacterial strain of degeneration is inoculated in PDA slant medium, 23 DEG C of progress activation cultures；
(2) cordyceps militaris link bacterial strain after activation is inoculated in Shake flask medium, 23 DEG C of culture 3-5d；
(3) it by cultured shake-flask seed liquid, is inoculated on wheat or rice culture medium for cultivating, 20-25 DEG C of culture 40-50d is left
It is right；
(4) selection form is good, the medium well fructification of robust growth, and interception tip is hung on culture medium for cultivating bottle cap, 20-
23 DEG C of culture 5-10d take out fructification tip until media surface spore germination is thorough, continue to cultivate 30-40d；It observes simultaneously
Mycelial growth rate, produce surviving of son physical capabilities；
(5) continue to repeat (4) step to operate 2-3 time, while the mature Cordyceps militaris picking of culture being dried, detect its mainly effectively at
Point cordycepin is used as cultivation original seed until character is stablized.
2. Cordyceps militaris rejuvenation breeding technique according to claim 1, which is characterized in that the inclined-plane PDA is trained in the step (1)
Support base are as follows: potato 200g/L, sucrose 20g/L, agar 15g/L, PH are natural.
3. Cordyceps militaris rejuvenation breeding technique according to claim 1, which is characterized in that seed culture in the step (2)
Base are as follows: potato 200g/L, sucrose 20g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1g/L, peptone 3g/L, VB1 10mg/L.
4. Cordyceps militaris rejuvenation breeding technique according to claim 1, which is characterized in that cultivation culture in the step (3)
Base are as follows: wheat or 30-50g/ bottles of rice, 40-70ml/ bottles of water.
5. Cordyceps militaris rejuvenation breeding technique according to claim 1, which is characterized in that the cordyceps militaris link bacterial strain is Cordyceps militaris
Great emigrant's 1# bacterial strain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113711842A (en) * | 2021-09-22 | 2021-11-30 | 辽宁省农业科学院 | Simple black fungus strain rejuvenation method |
| CN113875496A (en) * | 2021-11-10 | 2022-01-04 | 上海市农业科学院 | A kind of rejuvenation method of Cordyceps militaris strain |
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2017
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113711842A (en) * | 2021-09-22 | 2021-11-30 | 辽宁省农业科学院 | Simple black fungus strain rejuvenation method |
| CN113875496A (en) * | 2021-11-10 | 2022-01-04 | 上海市农业科学院 | A kind of rejuvenation method of Cordyceps militaris strain |
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Application publication date: 20190329 |