CN110024623A - L-PROLINE is improving the application in aweto blastopore quantity and hypha biomass - Google Patents
L-PROLINE is improving the application in aweto blastopore quantity and hypha biomass Download PDFInfo
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- CN110024623A CN110024623A CN201910362836.9A CN201910362836A CN110024623A CN 110024623 A CN110024623 A CN 110024623A CN 201910362836 A CN201910362836 A CN 201910362836A CN 110024623 A CN110024623 A CN 110024623A
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- aweto
- blastopore
- proline
- culture
- cordyceps sinensis
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- Mycology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The invention discloses L-PROLINEs to improve the application in aweto blastopore quantity and hypha biomass.Aiming at the problem that how the present invention improves hypha biomass and artificial bionic culture entomogenous fungi in conjunction with how to obtain a large amount of infection blastopores during cordyceps sinensis during aweto liquid fermentation, L-PROLINE is added in cordyceps sinensis bacterium culture medium, induction aweto efficiently generates blastopore, the culture medium of addition L-PROLINE blastopore quantity after accessing aweto Spore cultivation 30d is relatively not added with group 1.49~5.26 times of a raising, blastopore quantity after cultivating 45d improves 17~52.6 times, dry mycelial weight improves 15.67~66.44% after cultivating 60d.The more efficient acquisition blastopore of present invention energy can improve aweto liquid fermentation and obtain mycelial efficiency for infecting the bionical culture entomogenous fungi of bat moth in conjunction with cordyceps sinensis, shorten the time that cordyceps sinensis is manually cultivated, reduction toxigenic capacity.
Description
Technical field:
The invention belongs to fungal culture technical fields, and in particular to L-PROLINE is improving aweto blastopore number
Application in amount and hypha biomass.
Background technique:
Cordyceps sinensis Ophiocordyceps sinensis is the traditional rare traditional Chinese medicine in China, is praised with ginseng, pilose antler
It is big precious for Chinese medical three.Cordyceps sinensis is to infect bat moth by aweto (also known as Chinese coat spore, Hirsutella sinensis)
The bombys batryticatus and fungi stroma complex that section Hepialidae larva is formed.Cordyceps sinensis habitat is special (only in the high and cold grass of High aititude
The procreation of pasture naturally), the speed of growth is slow, and the period is longer, and natural resources is extremely limited.Although cordyceps sinensis has been put into
" national key protected wild plants register " second class protection species, but the excessive excavation of cordyceps sinensis is not held back effectively
System.The combined influence of a variety of factors such as degeneration, excessive development and utilization of global warming, especially cordyceps sinensis in habitat
Under, natural cordyceps resource reserves sharply decline (Hopping KA, Chignell SM, Lambin EF.The demise
ofcaterpillar fungus in the Himalayan region due to climate change and
overharvesting.Proc Natl Acad Sci USA.2018,115:11489-11494)。
China begins to the artificial culture technique of aweto from later period the 1970s, by aweto without
Development and production go out a variety of substitute products to the mycelial fermented and cultured of property type, " hundred as made of aweto fermentation mycelium
Enable capsule " it has been used as the important kind new medicine of country.In addition, being invaded by simulating Qinghai-Tibet ecological environment using aweto
Dye bat moth larvae successfully turns out cordyceps sinensis.Therefore, Chinese caterpillar fungus bacterial filament submerged fermentation technology how is improved, mycelia is improved
Biomass is conducive to save cultural hypha time and cost；In addition, infecting bat moth larvae in the way of injection blastopore
Can be shortened the bionical culture cordyceps sinensis period, how to be quickly obtained be largely used to infection blastopore it is artificial to cordyceps sinensis
It cultivates extremely important.
Summary of the invention:
Based on the above issues, the object of the present invention is to provide a kind of L-PROLINEs to improve aweto blastopore number
Application in amount and hypha biomass.L-PROLINE is added in cordyceps sinensis bacterium culture medium can improve cordyceps sinensis in culture solution
Bacterium blastopore quantity and Chinese caterpillar fungus bacterial filament biomass, for cordyceps sinensis, manually the industry of cultivating provides important technology branch
Support.
The present invention is found through experiments that L-PROLINE is added in cordyceps sinensis bacterium culture medium can be improved aweto
The quantity and hypha biomass of blastopore.
Therefore, the first purpose of the invention is to provide L-PROLINEs to improve aweto blastopore quantity and bacterium
Application in silk biomass.
The application is that L-PROLINE is added in the culture medium of inoculation aweto, is then cultivated.
A second object of the present invention is to provide L-PROLINEs to improve aweto blastopore quantity and bacterium in preparation
Application in the biological volume preparation of silk.
Third object of the present invention is to provide a kind of raising aweto blastopore quantity and hypha biomass
Aweto is seeded in the culture medium added with L-PROLINE and cultivates by method, obtain aweto blastopore and
Mycelia.
The culture medium is preferably fluid nutrient medium.
The L-PROLINE, concentration in liquid medium are 2.5mg/L-30g/L.
The aweto is preferably aweto spore liquid.
The fluid nutrient medium is preferably that every 150mL contains 30g potato, 1.5g peptone, 3g maltose, 0.08g sulphur
Sour magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1, surplus is water.
The culture, condition of culture are preferred are as follows: and 12 ± 2 DEG C, 100 revs/min, dark culturing.
How the present invention improves hypha biomass and artificial bionic culture for aweto liquid fermentation in the process
The problem of entomogenous fungi combines cordyceps sinensis how to obtain a large amount of infection blastopores in the process, adds in cordyceps sinensis bacterium culture medium
Add L-PROLINE, induction aweto efficiently generates blastopore, adds the culture medium of L-PROLINE in access cordyceps sinensis
Blastopore quantity is relatively not added with group 1.49~5.26 times of a raising, the blastopore quantity after cultivating 45d after bacterium Spore cultivation 30d
17~52.6 times are improved, dry mycelial weight improves 15.67~66.44% after cultivating 60d.The present invention can more efficient acquisition blastopore
For infecting the bionical culture entomogenous fungi of bat moth in conjunction with cordyceps sinensis, while aweto liquid fermentation can be improved and obtain mycelium
Efficiency, shorten time for manually cultivating of cordyceps sinensis, reduce toxigenic capacity.
Detailed description of the invention:
Fig. 1 is the aweto blastopore that inoculated and cultured of the present invention obtains (fluorescence 28 dyes, 400 ×).
Fig. 2 is aweto Liquid Culture of the present invention.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
Following embodiment inoculating spores liquid is mainly aweto conidium.
The spore liquid is to be prepared by the following method:
The cordyceps sinensis bacteria culture fluid of 45d will be inoculated with through three layers of lens wiping paper or strainer filtering, then at 10 DEG C, 5000 turns/
It is centrifuged 15 minutes under conditions of point, discards part supernatant, blood counting chamber counts spore quantity, obtains concentration spore liquid, concentration
Conidial quantity in spore liquid is 1.5 × 109A/mL is inoculated with mother liquor using concentration spore liquid as inoculation mother liquor with this
It is inoculated with.
Embodiment 1:
The preparation of aweto fluid nutrient medium: potato filtered fluid is cooked in 30g and (weighs 30g peeled potatoes, adds appropriate
Boiling 20min, filtering, take filtered fluid, potato filtered fluid is cooked in as 30g), 1.5g peptone, 3g maltose, 0.08g sulfuric acid
Magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1It is fitted into 500mL triangular flask, and it is (final concentration of that 1.5g L-PROLINE is added
10g/L, L-PROLINE is not added as control), 150mL is settled to water.Through high pressure sterilization, it is pre- to be placed in 12 ± 2 DEG C of culturing room
Cold standby.
Inoculation mother liquor is seeded in the 500mL triangular flask equipped with 150mL aweto fluid nutrient medium, every bottle of culture
Base accesses 1mL and is inoculated with mother liquor to final concentration of 107The conidium of a/mL, wherein containing the raw spore of about 15 buds in every bottle of culture medium
Triangular flask is placed in the shaking table that revolving speed is 100 revs/min, cultivated under 12 ± 2 DEG C, dark condition, cultivate 30d, taken out by son
1mL cultivates bacterium solution, and blood counting chamber counts blastopore quantity and reaches 2.58 × 106A/mL is relatively not added with L-PROLINE group and mentions
It is 5.26 times high；45d is cultivated, 1mL is taken out and cultivates bacterium solution, blood counting chamber counts blastopore quantity and reaches 5.27 × 107
A/mL (Fig. 1) is relatively not added with L-PROLINE group and improves 52.6 times；It cultivates 60d (Fig. 2), three layers of lens wiping paper filtering obtain bacterium
Silk, weighs after freeze-drying, hyphae length 4.95g, is relatively not added with L-PROLINE group and improves 66.44%.
Embodiment 2:
The preparation of aweto fluid nutrient medium: potato filtered fluid is cooked in 30g and (weighs 30g peeled potatoes, adds appropriate
Boiling 20min, filtering, take filtered fluid, potato filtered fluid is cooked in as 30g), 1.5g peptone, 3g maltose, 0.08g sulfuric acid
Magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1It is fitted into 500mL triangular flask, and it is (final concentration of that 3g L-PROLINE is added
20g/L, L-PROLINE is not added as control), 150mL is settled to water.Through high pressure sterilization, it is pre- to be placed in 12 ± 2 DEG C of culturing room
Cold standby.
Inoculation mother liquor is seeded in the 500mL triangular flask equipped with 150mL aweto fluid nutrient medium, every bottle of culture
Base accesses 1mL and is inoculated with mother liquor to final concentration of 107The conidium of a/mL, wherein containing the raw spore of about 15 buds in every bottle of culture medium
Triangular flask is placed in the shaking table that revolving speed is 100 revs/min, cultivated under 12 ± 2 DEG C, dark condition, cultivate 30d, taken out by son
1mL cultivates bacterium solution, and blood counting chamber counts blastopore quantity and reaches 3.02 × 106A/mL is relatively not added with L-PROLINE group and mentions
It is 6.37 times high；45d is cultivated, 1mL is taken out and cultivates bacterium solution, blood counting chamber counts blastopore quantity and reaches 4.83 × 107
A/mL is relatively not added with L-PROLINE group and improves 47 times；It cultivates the 60th day, three layers of lens wiping paper filtering obtain mycelia, freeze-drying
After weigh, hyphae length 4.11g, be relatively not added with L-PROLINE group improve 38.31%.
Embodiment 3:
The preparation of aweto fluid nutrient medium: potato filtered fluid is cooked in 30g and (weighs 30g peeled potatoes, adds appropriate
Boiling 20min, filtering, take filtered fluid, potato filtered fluid is cooked in as 30g), 1.5g peptone, 3g maltose, 0.08g sulfuric acid
Magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1It is fitted into 500mL triangular flask, and 0.38mg L-PROLINE (final concentration is added
For 2.5mg/L, L-PROLINE is not added as control), 150mL is settled to water.Through high pressure sterilization, it is placed in 12 ± 2 DEG C of cultures
The pre- cold standby in room.
Inoculation mother liquor is seeded in the 500mL triangular flask equipped with 150mL aweto fluid nutrient medium, every bottle of culture
Base accesses 1mL and is inoculated with mother liquor to final concentration of 107The conidium of a/mL, wherein containing the raw spore of about 15 buds in every bottle of culture medium
Triangular flask is placed in the shaking table that revolving speed is 100 revs/min, cultivated under 12 ± 2 DEG C, dark condition, cultivate 30d, taken out by son
1mL cultivates bacterium solution, and blood counting chamber counts blastopore quantity and reaches 1.02 × 106A/mL is relatively not added with L-PROLINE group and mentions
It is 1.49 times high；45d is cultivated, 1mL is taken out and cultivates bacterium solution, blood counting chamber counts blastopore quantity and reaches 1.81 × 107
A/mL is relatively not added with L-PROLINE group and improves 17 times；It cultivates the 60th day, three layers of lens wiping paper filtering obtain mycelia, freeze-drying
After weigh, hyphae length 3.44g, be relatively not added with L-PROLINE group improve 15.67%.
Embodiment 4:
The preparation of aweto fluid nutrient medium: potato filtered fluid is cooked in 30g and (weighs 30g peeled potatoes, adds appropriate
Boiling 20min, filtering, take filtered fluid, potato filtered fluid is cooked in as 30g), 1.5g peptone, 3g maltose, 0.08g sulfuric acid
Magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1It is fitted into 500mL triangular flask, and 37.5mg L-PROLINE (final concentration is added
For 250mg/L, L-PROLINE is not added as control), 150mL is settled to water.Through high pressure sterilization, it is placed in 12 ± 2 DEG C of cultures
The pre- cold standby in room.
Inoculation mother liquor is seeded in the 500mL triangular flask equipped with 150mL aweto fluid nutrient medium, every bottle of culture
Base accesses 1mL and is inoculated with mother liquor to final concentration of 107The conidium of a/mL, wherein containing the raw spore of about 15 buds in every bottle of culture medium
Triangular flask is placed in the shaking table that revolving speed is 100 revs/min, cultivated under 12 ± 2 DEG C, dark condition, cultivate 30d, taken out by son
1mL cultivates bacterium solution, and blood counting chamber counts blastopore quantity and reaches 2.02 × 106A/mL is relatively not added with L-PROLINE group and mentions
It is 3.93 times high；45d is cultivated, 1mL is taken out and cultivates bacterium solution, blood counting chamber counts blastopore quantity and reaches 4.73 × 107
A/mL is relatively not added with L-PROLINE group and improves 46 times；It cultivates the 60th day, three layers of lens wiping paper filtering obtain mycelia, freeze-drying
After weigh, hyphae length 3.95g, be relatively not added with L-PROLINE group improve 31.97%.
Embodiment 5:
The preparation of aweto fluid nutrient medium: potato filtered fluid is cooked in 30g and (weighs 30g peeled potatoes, adds appropriate
Boiling 20min, filtering, take filtered fluid, potato filtered fluid is cooked in as 30g), 1.5g peptone, 3g maltose, 0.08g sulfuric acid
Magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1It is fitted into 500mL triangular flask, and it is (final concentration of that 4.5g L-PROLINE is added
30g/L, L-PROLINE is not added as control), 150mL is settled to water.Through high pressure sterilization, it is pre- to be placed in 12 ± 2 DEG C of culturing room
Cold standby.
Inoculation mother liquor is seeded in the 500mL triangular flask equipped with 150mL aweto fluid nutrient medium, every bottle of culture
Base accesses 1mL and is inoculated with mother liquor to final concentration of 107The conidium of a/mL, wherein containing the raw spore of about 15 buds in every bottle of culture medium
Triangular flask is placed in the shaking table that revolving speed is 100 revs/min, cultivated under 12 ± 2 DEG C, dark condition, cultivate 30d, taken out by son
1mL cultivates bacterium solution, and blood counting chamber counts blastopore quantity and reaches 2.01 × 106A/mL is relatively not added with L-PROLINE group and mentions
It is 3.90 times high；45d is cultivated, 1mL is taken out and cultivates bacterium solution, blood counting chamber counts blastopore quantity and reaches 3.83 × 107
A/mL is relatively not added with L-PROLINE group and improves 37 times；It cultivates the 60th day, three layers of lens wiping paper filtering obtain mycelia, freeze-drying
After weigh, hyphae length 3.85g, be relatively not added with L-PROLINE group improve 28.63%.
Embodiment 6:
The preparation of aweto fluid nutrient medium: potato filtered fluid is cooked in 30g and (weighs 30g peeled potatoes, adds appropriate
Boiling 20min, filtering, take filtered fluid, potato filtered fluid is cooked in as 30g), 1.5g peptone, 3g maltose, 0.08g sulfuric acid
Magnesium, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1It is fitted into 500mL triangular flask, and 3.75mg L-PROLINE (final concentration is added
For 25mg/L, L-PROLINE is not added as control), 150mL is settled to water.Through high pressure sterilization, it is placed in 12 ± 2 DEG C of culturing room
Pre- cold standby.
Inoculation mother liquor is seeded in the 500mL triangular flask equipped with 150mL aweto fluid nutrient medium, every bottle of culture
Base accesses 1mL and is inoculated with mother liquor to final concentration of 107The conidium of a/mL, wherein containing the raw spore of about 15 buds in every bottle of culture medium
Triangular flask is placed in the shaking table that revolving speed is 100 revs/min, cultivated under 12 ± 2 DEG C, dark condition, cultivate 30d, taken out by son
1mL cultivates bacterium solution, and blood counting chamber counts blastopore quantity and reaches 1.90 × 106A/mL is relatively not added with L-PROLINE group and mentions
It is 3.63 times high；45d is cultivated, 1mL is taken out and cultivates bacterium solution, blood counting chamber counts blastopore quantity and reaches 4.23 × 107
A/mL is relatively not added with L-PROLINE group and improves 41 times；It cultivates the 60th day, three layers of lens wiping paper filtering obtain mycelia, freeze-drying
After weigh, hyphae length 3.70g, be relatively not added with L-PROLINE group improve 23.62%.
Claims (9)
1.L- proline is improving the application in aweto blastopore quantity and hypha biomass.
2. application according to claim 1, which is characterized in that the application is the culture medium in inoculation aweto
Middle addition L-PROLINE, is then cultivated.
3.L- proline improves the application in aweto blastopore quantity and hypha biomass preparation in preparation.
4. a kind of method for improving aweto blastopore quantity and hypha biomass, which is characterized in that by cordyceps sinensis
Bacterium is seeded in the culture medium added with L-PROLINE and cultivates, and obtains aweto blastopore and mycelia.
5. according to the method described in claim 4, it is characterized in that, the culture medium is fluid nutrient medium.
6. according to the method described in claim 5, it is characterized in that, the L-PROLINE, in liquid medium dense
Degree is 2.5mg/L-30g/L.
7. according to the method described in claim 4, it is characterized in that, the aweto is aweto spore liquid.
8. according to the method described in claim 5, it is characterized in that, the fluid nutrient medium is that every 150mL contains 30g soil
Beans, 1.5g peptone, 3g maltose, 0.08g magnesium sulfate, 0.25g potassium dihydrogen phosphate, 3mg vitamin B1, surplus is water.
9. according to the method described in claim 4, it is characterized in that, the culture, condition of culture are as follows: 12 ± 2 DEG C, 100
Rev/min, dark culturing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111172095A (en) * | 2020-02-24 | 2020-05-19 | 广东省生物资源应用研究所 | Application of methyl farnesyl ester in promoting cordyceps sinensis to produce budding spores |
| CN112772294A (en) * | 2021-01-05 | 2021-05-11 | 中国科学院动物研究所 | Separation method and application of cordyceps sinensis strain |
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