CN105483073B - A kind of very fast conidial method of acquisition - Google Patents
A kind of very fast conidial method of acquisition Download PDFInfo
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- CN105483073B CN105483073B CN201610013554.4A CN201610013554A CN105483073B CN 105483073 B CN105483073 B CN 105483073B CN 201610013554 A CN201610013554 A CN 201610013554A CN 105483073 B CN105483073 B CN 105483073B
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 241001248610 Ophiocordyceps sinensis Species 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 7
- 235000011187 glycerol Nutrition 0.000 claims description 7
- 230000003020 moisturizing effect Effects 0.000 claims description 7
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 239000004744 fabric Substances 0.000 claims description 2
- 241000330899 Hepialus Species 0.000 claims 1
- 239000000853 adhesive Substances 0.000 claims 1
- 230000001070 adhesive effect Effects 0.000 claims 1
- 235000013372 meat Nutrition 0.000 claims 1
- 238000010422 painting Methods 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000006872 improvement Effects 0.000 abstract description 3
- 230000006698 induction Effects 0.000 abstract description 3
- 230000000087 stabilizing effect Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 description 6
- 238000002791 soaking Methods 0.000 description 5
- 229920000136 polysorbate Polymers 0.000 description 4
- 241000190633 Cordyceps Species 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000002073 mitogenetic effect Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 244000283482 Alloteropsis cimicina Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000017898 Digitaria ciliaris Nutrition 0.000 description 1
- 241000130660 Hepialidae Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000005574 cross-species transmission Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of very fast conidial method of acquisition.This method is a kind of technology of the microcycle conidiation of improvement, take that Hirsutella sinensis ascospore is sloughed off using bat moth larvae body surface, bat moth larvae worm or bat moth adult wing is carrier, vegetative stage is not suffered from, directly induction produces conidium, not only cultivation cycle is short, cost is low but also process stabilizing, yield are high, and stable conidium source is provided successfully to infect bat moth larvae.
Description
Technical field
The invention belongs to bio-science field, more particularly to a kind of very fast conidial method of acquisition.
Background technology
Cordyceps sinensis [Ophiocordyceps sinensis (Berk.) G.H.Sunget et al.] is China Qinghai-Tibet height
Traditional rare traditional Chinese medicine of former special product, it be the stroma that aweto is colonized on Hepialidae bat moth category insect larvae and
The complex of larva corpse.In recent years, with the popularization that cordyceps sinensis nutrition and medical value recognize, people's living standard in addition
Improve constantly, the demand of cordyceps sinensis is constantly increased, causes unauthorized and excessive mining constantly to aggravate, the destruction Of resources are increasingly severe, cause
Cordyceps Resources are made to fall sharply, artificial cultivation of cordyceps is imperative.Due to prior art exist larva contamination rate it is low or dye
The problems such as survival rate is low after bacterium, artificial cultivation of cordyceps large-scale development is govern always, years of researches confirm, Chinese quilt
Hair spore (H.sinensis) is the phorozoon of aweto, and conidium is as particularly important in the Hirsutella sinensis history of life
One link, infected in cordyceps sinensis and very crucial effect is played in forming process, there are some researches show use Chinese hair
Spore conidium can significantly improve infection rate to infect bat moth larvae.
At present, the conventional conidial method of acquisition Hirsutella sinensis has：Solid fermentation induces and microcycle conidiation.
Solid fermentation induction acquisition conidial cycle is longer, generally 60~90 days, causes the dye in incubation
The increase of bacterium probability, and yield is unstable, is limited to Hirsutella sinensis bacterial strain type and the quality of solid medium.
After microcycle conidiation is the spore germination of fungi, without (or only atomic weak) vegetative stage, directly
Conidial fructification and spore are produced from spore.This method produces the short identification for being used for aweto phorozoon of spore cycle, Mo Ming
With grade in periodical《Fungus system》Article " microcycle conidiation of cordyceps sinensis and its point of phorozoon that 4th phase in 2001 delivers
From " in disclose on slide plus a drop sabouraud medium, ascospore point is connected on culture medium and carries out microcycle conidiation,
Conidium is produced after 5 days, it is identified according to conidial microscopic features, author thinks that this method is easy to be reliable,
With good practicality.Xiao Yanyan etc. is in periodical《Agricultural University Of Anhui's journal》Article " the winter that the 1st phases of volume 38 in 2011 deliver
Hirsutella sinensis conidium is accessed into liquid in the morphological observation of worm summer grass seed cystospore and its phorozoon in incubation "
Body culture medium, after carrying out microcycle conidiation, the bacterial strain being separated to is accredited as Chinese hair by binding molecule biology techniques means
Spore.It can be seen that traditional microcycle conidiation is accredited as mesh to cultivate a small amount of conidium as aweto phorozoon
, conidial yield is not pursued, in-depth study is not done to the ambient temperature and humidity and nutritional condition of microcycle conidiation.
The content of the invention
In view of this, it is an object of the invention to provide a kind of method of the microcycle conidiation of improvement, this method is not only
Cultivation cycle is short, cost is low, and process stabilizing, yield are high, and stable conidium is provided successfully to infect bat moth larvae
Source.
To achieve these goals, the present invention provides following technical scheme：
A kind of very fast conidial method of acquisition, bat moth children is uniformly applied to by Hirsutella sinensis ascospore solution
Polypide table, bat moth larvae worm slough off or the surface of bat moth adult wing, is respectively placed in and is lined with the culture dish of wet filter paper, in temperature
12~18 DEG C of degree, relative humidity 60~90%, 24~96h is cultivated under the gnotobasis of lucifuge, produces the mitogenetic spore of Hirsutella sinensis
Son.Bat moth larvae body surface, bat moth larvae worm are sloughed off or bat moth adult wing surface elutes, collects conidium.
In some embodiments, Hirsutella sinensis ascospore solution of the present invention, comprising with moisturizing and adhesion work
Solvent, preferably 2~5% glycerine water solutions.
In some embodiments, Hirsutella sinensis ascospore solution of the present invention, the concentration of spore for 4500~
5500/ml, preferably 5000/ml.
It is of the present invention that Hirsutella sinensis ascospore solution is uniformly applied to bat moth larvae in some embodiments
Body surface, bat moth larvae worm slough off or the surface of bat moth adult wing, dauber are compliant tool, do not damage larva, flexible work
Tool can be writing brush, cloth, cotton swab etc., preferably writing brush.
It is of the present invention that Hirsutella sinensis ascospore solution is uniformly applied to bat moth larvae in some embodiments
Body surface, bat moth larvae worm slough off or the surface of bat moth adult wing, and the applying amount of each carrier is 15~25 μ l, and preferably 20
μl。
It is of the present invention that Hirsutella sinensis ascospore solution is uniformly applied to bat moth larvae in some embodiments
Body surface, bat moth larvae worm slough off or the surface of bat moth adult wing, bat moth larvae are preferably the larva of the work in 3~5 ages.
In some embodiments, the culture dish of the present invention for being lined with wet filter paper, filter paper humid control is fully wetting, full
It is full of but does not spill over moisture.Check daily, carried out adding water process according to the water regime of filter paper.
In some embodiments, culture environment of the present invention, preferably 14~16 DEG C of temperature, relative humidity 65~
85%, the gnotobasis of lucifuge.
In some embodiments, it is of the present invention to bat moth larvae body surface, bat moth larvae worm sloughs off or bat moth adult
Wing surface is eluted, and eluent is preferably the 0.003mol/l KH containing 0.1% Tween 802PO4Buffer solution.
Conidium can produce blue-fluorescence under fluorescence microscope, fluorescence microscope can be used to monitor production spore feelings in real time
Condition, it is collected when conidium amount is larger, 24~96h preferably after smearing produces a large amount of conidiums.
The ascospore of the present invention can be the ascospore of wild fresh cordyceps sinensis ejection, can also gather from artificial
The ascospore for the fresh cordyceps sinensis ejection cultivated
Because microcycle conidiation produces, conidial quantity is few, and technique is used for the mirror of Anamorph of Cordyceps Sinensis
Fixed, Hirsutella sinensis growing environment is special, and the nutrition and environmental factor to artificial induction's microcycle conidiation require very strict, Shen
Ask someone on the basis of to Hirsutella sinensis growth characteristics for a long time further investigation, by performing creative labour, there is provided Yi Zhongneng
The method of enough microcycle conidiations for obtaining a large amount of conidial improvement.Technical scheme passes through the length fresh worm summer in winter
The direct induced meristem spore of ascospore of grass ejection, vegetative stage is not undergone, the production spore cycle is short, avoids in strain point
The problems such as from being changed with microbiological contamination present in very long incubation or strain character.With bat moth larvae body surface, bat
Moth larvae worm sloughs off or bat moth adult wing is culture medium, under the control of specific environmental condition, can produce substantial amounts of conidium,
There is breakthrough progress obtaining Hirsutella sinensis conidium field.
Brief description of the drawings
Accompanying drawing 1：Polypide and conidium under fluorescence microscope
Legend：The conidium of 1 bat moth larvae body surface bristle 2
Embodiment
The embodiment of the invention discloses a kind of very fast conidial method of acquisition, those skilled in the art can use for reference this
Literary content, it is suitably modified technological parameter realization.In particular, all similar replacements and change are to art technology
It is it will be apparent that they are considered as being included in the present invention for personnel.The method of the present invention is by preferably implementing
Example is described, and related personnel can substantially not depart from present invention, the method for the invention entered in spirit and scope
Row change is suitably changed with combining, to realize and using the technology of the present invention.
For a further understanding of the present invention, with reference to embodiment to a kind of very fast acquisition conidium provided by the invention
Method be described in detail.
Filter paper used in the present invention passes through advance 121 DEG C of high temperature, high pressure 30min sterilization treatments.
Embodiment 1
The ascospore of fresh collection is taken, it is molten to be diluted to the ascospore that concentration is 4500/ml with 2% glycerine water solution
Liquid；Ascospore liquid is uniformly applied to the healthy bat moth larvae body surface in 50 3 ages with the fine, soft fur pen sterilized in advance, every
The applying amount of worm is 20 μ l, and the bat moth larvae after smearing is respectively placed in the clean culture dish with cover for being lined with filter paper moisturizing
It is interior.Filter paper humid control is fully soaking, in the range of being completely full of but not spilling over moisture.Check daily, according to the moisture shape of filter paper
Condition carries out adding water process.Cultivated at 14 DEG C of temperature, the clean area environment of relative humidity 65%, lucifuge.Use fluorescence
Microscope monitors production spore situation (as shown in Figure 1) in real time, stops cultivating in 24h, with the 0.003mol/l containing 0.1% Tween 80
KH2PO4Buffer solution elutes conidium, and larva puts back to former space and continues to raise, is filtered to remove a little miscellaneous in conidium liquid
Matter, it is 8.14 × 10 to count conidium number under the microscope5Individual conidium.Conidium is colourless under microscope, kidney shape, single
Born of the same parents are smooth.
Embodiment 2
The ascospore of fresh collection is taken, it is molten to be diluted to the ascospore that concentration is 5000/ml with 3% glycerine water solution
Liquid；Ascospore liquid is uniformly applied to the healthy bat moth larvae body surface in 50 4 ages with the fine, soft fur pen sterilized in advance, every
The applying amount of worm is 20 μ l, and the bat moth larvae after smearing is respectively placed in the clean culture dish with cover for being lined with filter paper moisturizing
It is interior.Filter paper humid control is fully soaking, in the range of being completely full of but not spilling over moisture.Check daily, according to the moisture shape of filter paper
Condition carries out adding water process.Cultivated at 15 DEG C of temperature, the clean area environment of relative humidity 70%, lucifuge.Use fluorescence
Microscope monitors production spore situation in real time, stops cultivating in 54h, with the 0.003mol/l KH containing 0.1% Tween 802PO4Buffer solution
Conidium is eluted, larva puts back to former space and continues to raise, a little impurity being filtered to remove in conidium liquid, under the microscope
It is 9.23 × 10 to count conidium number5Individual conidium.
Embodiment 3
The ascospore of fresh collection is taken, the son that ascospore concentration is 5500/ml is diluted to 4% glycerine water solution
Cystospore solution；Ascospore liquid is uniformly applied to the healthy bat moth larvae in 50 5 ages with the fine, soft fur pen sterilized in advance
Body surface, the applying amount per cestode is 25 μ l, and the bat moth larvae after smearing is respectively placed in the clean band for being lined with filter paper moisturizing
In lid culture dish.Filter paper humid control is fully soaking, in the range of being completely full of but not spilling over moisture.Check daily, according to filter paper
Water regime carry out adding water process.Cultivated at 16 DEG C of temperature, the clean area environment of relative humidity 75%, lucifuge.
Monitor production spore situation in real time using fluorescence microscope, stop cultivating in 48h, with the 0.003mol/l containing 0.1% Tween 80
KH2PO4Buffer solution elutes conidium, and larva puts back to former space and continues to raise, is filtered to remove a little miscellaneous in conidium liquid
Matter, it is 1.05 × 10 to count conidium number under the microscope6Individual conidium.
Embodiment 4
The ascospore of fresh collection is taken, it is molten to be diluted to the ascospore that concentration is 5000/ml with 5% glycerine water solution
Liquid；Ascospore liquid is uniformly applied to 50 bat moth larvae worms with the fine, soft fur pen sterilized in advance and sloughs off surface, what each worm sloughed off
Applying amount is 20 μ l, and the worm after smearing is sloughed off and is respectively placed in clean be lined with the culture dish of filter paper moisturizing.Filter paper humid control
Fully soaking, in the range of being completely full of but not spilling over moisture.Check daily, according to the water regime of filter paper add at water
Reason.Cultivated at 18 DEG C of temperature, the clean area environment of relative humidity 90%, lucifuge.Monitored in real time using fluorescence microscope
Spore situation is produced, stops cultivating in 78h, with the 0.003mol/l KH containing 0.1% Tween 802PO4Buffer solution elutes conidium, mistake
A little impurity in conidium liquid is filtered out, it is 7.42 × 10 to count conidium number under the microscope5Individual conidium.
Embodiment 5
The ascospore of fresh collection is taken, the son that ascospore concentration is 5000/ml is diluted to 3% glycerine water solution
Cystospore solution；Ascospore liquid is uniformly applied to the wing surface of 50 bat moth adults with the fine, soft fur pen sterilized in advance, often
Individual wing surface smear amount is 20 μ l, and the worm wing after smearing is respectively placed in into clean be lined with the culture dish of filter paper moisturizing.Filter paper
Humid control is fully soaking, in the range of being completely full of but not spilling over moisture.Check, mended according to the water regime of filter paper daily
Add water process.Cultivated at 12 DEG C of temperature, the clean area environment of relative humidity 80%, lucifuge.It is real using fluorescence microscope
When monitoring production spore situation, in 72h stop cultivate, with the 0.003mol/l KH containing 0.1% Tween 802PO4Buffer solution elution is mitogenetic
Spore, a little impurity being filtered to remove in conidium liquid, it is 6.56 × 10 to count conidium number under the microscope5It is individual mitogenetic
Spore.
Claims (8)
1. one kind obtains conidial method, it is characterised in that with bat moth larvae body surface, bat moth larvae worm sloughs off or bat
Moth adult wing is carrier, and Hirsutella sinensis ascospore does not suffer from vegetative stage, directly produces conidium；
It the described method comprises the following steps：
1) by Hirsutella sinensis ascospore solution is uniformly applied to bat moth larvae body surface, bat moth larvae worm sloughs off or bat moth
The surface of adult wing；
2) the bat moth larvae for being coated with Hirsutella sinensis ascospore, bat moth larvae worm are sloughed off or bat moth adult wing is distinguished
It is placed in and is lined with the culture dish of wet filter paper, 12~18 DEG C of keeping temperature, relative humidity 60~90%, under the gnotobasis of lucifuge
Cultivate 24~96h；
3) bat moth larvae body surface, bat moth larvae worm are sloughed off or bat moth adult wing surface elutes, collect conidium；
Hirsutella sinensis ascospore solution wherein described in step 1), the concentration of ascospore is 4500~5500/ml.
2. according to the method for claim 1, it is characterised in that the Hirsutella sinensis ascospore in the step 1) is molten
Liquid, include the solvent with moisturizing and adhesive attraction.
3. according to the method for claim 2, it is characterised in that the solvent is 2~5% glycerine water solutions.
4. according to the method for claim 1, it is characterised in that in the step 1) that Hirsutella sinensis ascospore is molten
Liquid is uniformly applied to bat moth larvae body surface, bat moth larvae worm sloughs off or the surface of bat moth adult wing, and dauber is flexibility
Instrument.
5. according to the method for claim 4, it is characterised in that the compliant tool is writing brush, cloth or cotton swab.
6. according to the method for claim 1, it is characterised in that in described step 1) by Hirsutella sinensis ascospore
Solution is uniformly applied to bat moth larvae body surface, bat moth larvae worm sloughs off or the surface of bat moth adult wing, the painting of each carrier
The amount of smearing is 15~25 μ l.
7. according to the method for claim 1, it is characterised in that the wet filter paper in described step 2) is meat on guarantee filter paper
Eye can water breakthrough, tilt anhydrous flowing.
8. according to the method for claim 1, it is characterised in that in described step 3) to bat moth larvae body surface, bat
Hepialus larva worm is sloughed off or bat moth adult wing surface is eluted, and eluent is the 0.003mol/l containing 0.1% Tween 80
KH2PO4Buffer solution.
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| CN201610013554.4A CN105483073B (en) | 2016-01-07 | 2016-01-07 | A kind of very fast conidial method of acquisition |
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| CN201610013554.4A CN105483073B (en) | 2016-01-07 | 2016-01-07 | A kind of very fast conidial method of acquisition |
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Denomination of invention: Method for quick acquisition of conidia Effective date of registration: 20180315 Granted publication date: 20171110 Pledgee: Bank of Bohai, Limited by Share Ltd, Yichang branch|Bank of China, Limited by Share Ltd, Three Gorges Branch|Bank of Hubei Limited by Share Ltd Yichang Nanhu sub branch|Bank of Hankou, Limited by Share Ltd, Yichang branch Pledgor: Dongyangguang Pharmaceutical Co., Ltd., Guangdong|YICHANG SHANCHENG SHUIDU CORDYCEPS SINENSIS CO., LTD. Registration number: 2018420000009 |
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Date of cancellation: 20210603 Granted publication date: 20171110 Pledgee: Bank of Bohai Limited by Share Ltd. Yichang branch|Bank of China Limited by Share Ltd. Three Gorges Branch|Bank of Hubei Limited by Share Ltd. Yichang Nanhu sub branch|Bank of Hankou Limited by Share Ltd. Yichang branch Pledgor: SUNSHINE LAKE PHARMA Co.,Ltd.|YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Registration number: 2018420000009 |
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Effective date of registration: 20210615 Address after: 443300 baotawan village, Lucheng, Yidu City, Yichang City, Hubei Province Patentee after: YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Address before: 523808 No. 1 Industrial North Road, Songshan Industrial Park, Songshan, Guangdong, Dongguan, Hubei Patentee before: SUNSHINE LAKE PHARMA Co.,Ltd. Patentee before: YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. |
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Denomination of invention: A method of obtaining conidia quickly Effective date of registration: 20210630 Granted publication date: 20171110 Pledgee: Bank of Bohai Limited by Share Ltd. Yichang branch|Bank of China Limited by Share Ltd. Three Gorges Branch|Bank of Hubei Limited by Share Ltd. Yichang Nanhu sub branch|Bank of Hankou Limited by Share Ltd. Yichang branch Pledgor: YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Registration number: Y2021420000062 |
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