CN105838659B - A kind of conidiiferous culture medium of Hirsutella sinensis solid fermentation and method - Google Patents
A kind of conidiiferous culture medium of Hirsutella sinensis solid fermentation and method Download PDFInfo
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- CN105838659B CN105838659B CN201610214162.4A CN201610214162A CN105838659B CN 105838659 B CN105838659 B CN 105838659B CN 201610214162 A CN201610214162 A CN 201610214162A CN 105838659 B CN105838659 B CN 105838659B
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- 241001248610 Ophiocordyceps sinensis Species 0.000 title claims abstract description 58
- 239000001963 growth medium Substances 0.000 title claims abstract description 49
- 238000000855 fermentation Methods 0.000 title claims abstract description 21
- 230000004151 fermentation Effects 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000007787 solid Substances 0.000 title abstract description 15
- 239000003415 peat Substances 0.000 claims abstract description 25
- 239000002689 soil Substances 0.000 claims abstract description 25
- 235000015097 nutrients Nutrition 0.000 claims abstract description 17
- 241001597008 Nomeidae Species 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims description 13
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000011534 incubation Methods 0.000 claims description 8
- 229930003231 vitamin Natural products 0.000 claims description 8
- 235000013343 vitamin Nutrition 0.000 claims description 8
- 239000011782 vitamin Substances 0.000 claims description 8
- 229940088594 vitamin Drugs 0.000 claims description 8
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 8
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 7
- 235000013824 polyphenols Nutrition 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 6
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 6
- 230000002073 mitogenetic effect Effects 0.000 abstract description 5
- 206010061217 Infestation Diseases 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 3
- 230000001418 larval effect Effects 0.000 abstract description 3
- 239000011159 matrix material Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 230000008859 change Effects 0.000 abstract description 2
- 230000006698 induction Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 14
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 10
- 239000000843 powder Substances 0.000 description 9
- 239000002585 base Substances 0.000 description 8
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 229930003268 Vitamin C Natural products 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 235000019154 vitamin C Nutrition 0.000 description 5
- 239000011718 vitamin C Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 239000000835 fiber Substances 0.000 description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 3
- 229930003451 Vitamin B1 Natural products 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000003958 fumigation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000010977 jade Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000001294 propane Substances 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- 235000010374 vitamin B1 Nutrition 0.000 description 2
- 239000011691 vitamin B1 Substances 0.000 description 2
- 235000019158 vitamin B6 Nutrition 0.000 description 2
- 239000011726 vitamin B6 Substances 0.000 description 2
- 229940011671 vitamin b6 Drugs 0.000 description 2
- 241000190633 Cordyceps Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001149900 Fusconaia subrotunda Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 239000008648 bailing capsule Substances 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009567 fermentative growth Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000009571 larval growth Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 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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- Zoology (AREA)
- 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)
- Mushroom Cultivation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to one kind to be applied to the conidiiferous culture medium of Hirsutella sinensis solid fermentation and method.The present invention is by using peat soil as main matrix, add appropriate basic nutrient solution, prepare the solid fermentation culture medium for being adapted to the growth of hair spore, hair spore can be grown inside culture medium, and conidium separation and Extraction is easy, extensive Hirsutella sinensis solid fermentation production conidium can be achieved；Bat moth larval infestation can also be facilitated, applied to breeding for cordyceps sinensis；Present invention also offers the method that the mitogenetic spore of Hirsutella sinensis solid fermentation production is cultivated in peat soil culture medium simultaneously, pass through appropriate change external condition, Hirsutella sinensis vegetative propagation is stimulated to increase spore amount, and/or further increase conidium derivant induction Hirsutella sinensis vegetative propagation to increase conidium amount.
Description
Technical field
The present invention relates to one kind to be applied to the conidiiferous culture medium of Hirsutella sinensis solid fermentation and method.
Technical background
It is Hirsutella sinensis Hirsutella sinensis X.J.Liu to have proven to Anamorph of Cordyceps Sinensis at present,
(Jiang Yi and Yao one build 2003 by Y.L.Guo, Y.X.Yu＆W.Zeng；Li Zengzhi etc. 2000；Liu Xi Jin etc. is 1989).Hirsutella sinensis
Liquid fermentation has been realized in industrialized production, and develops Bailing capsule, hundred makes the products such as piece (Dong et al.2015；Dong
2015) rainbow etc., but finds that Hirsutella sinensis is slow-growing in produce reality, culture for a long time causes pollution probability to increase, together
When also increase the complexity and risk (Li Shulin etc. 2014) of metabolin.Therefore, simulated high altitude ecology breed cordyceps sinensis into
For urgent need.
Cordyceps sinensis is bred firstly the need of the conidium for obtaining being largely used to infecting bat moth larvae.Due to Chinese quilt
Hair spore strain properties are complicated, only few in number at present relevant to be prepared using Hirsutella sinensis under existing zymotechnique
Conidial report, the method in document are all to carry out conidium (the Li Yu tinkling of pieces of jade 2002 that solid slope culture obtains bacterium colony；
Wang Weis etc. 2002；Wang Can 2012；Li Ling's tinkling of pieces of jade etc. 2014；Zhu Zhi steel 2014).Existing Hirsutella sinensis prepares conidium method base
Originally it is to be carried out in test tube, culture dish or wide-mouth bottle solid slope culture medium, culture medium permeability is poor, and Hirsutella sinensis is difficult to
Grown inside culture medium, and culture medium may not apply to breeding for cordyceps sinensis in actual production, be cultivated
The space limitation of basic stature matter and culture vessel, culture medium separate difficulty, conidium separation with Hirsutella sinensis bacterium colony
Extraction needs bacterium colony peeling off culture medium, is separated carrying out mycelia with conidium, complex steps, it is difficult to realize scale.
The content of the invention
The present invention prepares by using peat soil as main matrix, adding appropriate basic nutrient solution and is adapted to the life of hair spore
Long solid fermentation culture medium, hair spore can be grown inside culture medium, and conidium separation and Extraction is easy, can be real
Now extensive Hirsutella sinensis solid fermentation production conidium；Bat moth larval infestation can also be facilitated, applied to cordyceps sinensis
Breed；Simultaneously present invention also offers method Hirsutella sinensis solid fermentation is cultivated in peat soil culture medium produced mitogenetic spore,
By appropriate change external condition, Hirsutella sinensis vegetative propagation is stimulated to increase spore amount, and/or further increase point
Raw Spore inducing agent induces Hirsutella sinensis vegetative propagation to increase conidium amount.
On the one hand, the invention provides a kind of conidiiferous culture medium of Hirsutella sinensis solid fermentation, it is characterized in that
Including：Peat soil and nutrient solution.After basal nutrient composition is absorbed totally by mycelia in peat soil culture medium, the loose few battalion of culture medium
Foster specific condition can promote the conidial formation of Hirsutella sinensis, thus the growth of Hirsutella sinensis aerial hyphae is typically produced
Raw conidium is more.Peat soil culture medium solid fermentation prepare conidium can not only scale, and easy to use, mycelia
Can also be in its growth inside；The Hirsutella sinensis of the medium culture can not only directly as the strain of bat moth larval infestation,
Also can be established as bat moth larval growth and the habitat of activity for scale, the modern production of cordyceps sinensis artificial culture
Fixed basis.
In certain embodiments, the peat soil is the loose, peat soil of fibre object even thickness.
In certain embodiments, inorganic matter control is that 10~15%, pH controls are 6~7 in the peat soil.
In certain embodiments, the peat soil is the loose, peat soil of fibre object even thickness, and wherein inorganic matter controls
It is 6.5 for 10~15%, pH controls.
In certain embodiments, the culture medium further includes puparium powder.
In certain embodiments, the nutrient solution includes peptone, dusty yeast, sucrose, anhydrous magnesium sulfate, biphosphate
Potassium, vitamin C and water.
In further embodiments, the nutrient solution includes 0.10~0.50g/100mL peptone, 0.1~0.50g/
100mL dusty yeast, 1~2g/100mL sucrose, 0.01~0.05g/100mL anhydrous magnesium sulfate, 0.1~0.50g/
100mL potassium dihydrogen phosphate, 0.1~0.50g/100mL vitamin C and water.The nutrient solution of the formula is ensureing that peat soil is
On the basis of the culture medium permeability of matrix, there is provided Hirsutella sinensis grows into the nutritional ingredient needed for certain phase.
In further embodiments, in the nutrient solution, peptone 0.10g/100mL, 0.20g/100mL,
0.30g/100mL, 0.40g/100mL or 0.50g/100mL, dusty yeast 0.1g/100mL, 0.20g/100mL, 0.30g/
100mL, 0.40g/100mL or 0.50g/100mL, sucrose 1.0g/100mL, 1.2g/100mL, 1.4g/100mL, 1.6g/
100mL, 1.8g/100mL or 2.0g/100mL, anhydrous magnesium sulfate 0.01g/100mL, 0.02g/100mL, 0.03g/100mL,
0.04g/100mL or 0.05g/100mL, potassium dihydrogen phosphate 0.1g/100mL, 0.2g/100mL, 0.3g/100mL, 0.4g/
100mL or 0.5g/100mL, vitamin C 0.1g/100mL, 0.2g/100mL, 0.3g/100mL, 0.4g/100mL or 0.5g/
100mL。
In further embodiments, the puparium powder is culture medium quality 2%.
In certain embodiments, the culture medium further includes the puparium powder of culture medium quality 2%, and the nutrition
Liquid include 0.10~0.50g/100mL peptone, 0.1~0.50g/100mL dusty yeast, 1~2g/100mL sucrose,
0.01~0.05g/100mL anhydrous magnesium sulfate, 0.1~0.50g/100mL potassium dihydrogen phosphate, 0.1~0.50g/100mL
Vitamin C and water.
In certain embodiments, culture medium of the present invention is to mix the peat soil and nutrient solution with certain proportion
After sterilized.
In certain embodiments, wherein, the peat soil:Nutrient solution=100:15~10；Sterilizing is entered at 115~121 DEG C
Row 2~3 hours.The mycelia of Hirsutella sinensis is more suitable for growing under the peat soil and nutrient solution of the ratio.
In certain embodiments, wherein, the puparium powder is added after the peat soil and nutrient solution mixing sterilizing.
In certain embodiments, wherein, the puparium powder is individually sterilizing.
In certain embodiments, wherein, the puparium powder individually to sterilize is to carry out fumigation using expoxy propane,
200g/m3It is stifling 12 hours.
In further embodiments, wherein, the peat soil:Nutrient solution=100:15~10；After preparing 115~
121 DEG C sterilize 2~3 hours；Add the puparium powder individually to sterilize.
In further embodiments, the puparium powder individually to sterilize adds culture medium according to the amount of culture medium quality 2%.
On the other hand, the invention provides one kind to use culture medium of the present invention fermentation Hirsutella sinensis production conidium
Method, including：
1) Hirsutella sinensis liquid spawn is inoculated with to the culture medium；
2) culture medium being vaccinated with step 1) is placed in sterile constant-temperature darkroom and cultivated；With
3) cultivation temperature culture is reduced.
By using culture medium of the present invention, using above-mentioned conidiiferous method so that Hirsutella sinensis produces
Conidium can realize scale, and conveniently.
In certain embodiments, the inoculum concentration of inoculation Hirsutella sinensis liquid spawn is 10~15%V/ in the step 1)
W mycelium, its weight in wet base mycelium weight in wet base are 15~20%.
In certain embodiments, in the step 2) cultivation temperature control range be 18~22 DEG C, incubation time be 30~
35 days, humid control scope was 60~80%.
In certain embodiments, cultivation temperature control range is 4~10 DEG C in the step 3), and incubation time is 35~45
My god, humid control scope is 60~75%.
In certain embodiments, spore is produced using culture medium of the present invention fermentation Hirsutella sinensis of the present invention
In the method for son, wherein：
The inoculum concentration of inoculation Hirsutella sinensis liquid spawn is 10~15%V/W mycelium in step 1), its weight in wet base bacterium
Filament weight in wet base is 15~20%；
Cultivation temperature control range is 18~22 DEG C in step 2), and incubation time is 30~35 days, and humid control scope is
60~80%；With
Cultivation temperature control range is 4~10 DEG C in step 3), and incubation time is 35~45 days, and humid control scope is 60
~75%.
In certain embodiments, spore is produced using culture medium of the present invention fermentation Hirsutella sinensis of the present invention
In the method for son, cultivation temperature control range is 18 DEG C in step 2), and incubation time is 30 days, and humid control scope is 75%；
It it is 4 DEG C with cultivation temperature control range in step 3), incubation time is 40 days, and humid control scope is 65%.
In certain embodiments, it is of the present invention to produce spore using culture medium of the present invention fermentation Hirsutella sinensis
Method be included in cool in step 3) after further add conidium derivant.In the inoculation method, further
Addition conidium derivant, Hirsutella sinensis vegetative propagation can be promoted.
In further embodiments, the conidium derivant is Tea Polyphenols.Tea Polyphenols is natural bacteriostatic agent, as point
The green non-poisonous evil of raw Spore inducing agent.
In further embodiments, the conidium derivant includes Tea Polyphenols, vitamin B1, vitamin B6 and Wei Sheng
Plain H.Vitamin B1, vitamin B6 and biotin can further promote Hirsutella sinensis to produce conidium, mitogenetic so as to provide
The yield of spore.
In further embodiments, the conidium derivant includes 0.04~0.08g/100mL Tea Polyphenols, 0.01
~0.03g/100mL vitamin B1,0.02~0.04g/100mL vitamin B6,0.02~0.04g/100mL vitamin
H and water.
In further embodiments, the conidium derivant includes 0.04g/100mL Tea Polyphenols, 0.01g/
100mL vitamin B1,0.02g/100mL vitamin B6,0.02g/100mL biotin and water.
In further embodiments, the dosage of the conidium derivant is 10mL/500g culture mediums.In the range of this
The amount of conidium derivant can preferably trigger the resistance of Hirsutella sinensis.
Nutrient solution in culture medium of the present invention is to provide nutriment and other for Hirsutella sinensis Fermentative growth
The basic solution of material needed for growth.
Conidium derivant of the present invention can suppress Hirsutella sinensis growth to a certain extent.
Liquid spawn of the present invention, it is by choosing the Hirsutella sinensis test tube bacterial strain to grow fine, and picking
1cm2The fungus block of left and right size is inoculated in liquid shaking bottle, and fermentation completion is inoculated in fermentation tank after smashing to pieces, to obtain substantial amounts of liquid
Strain；It is inoculated with the mycelium weight in wet base location parameter of Hirsutella sinensis liquid spawn：10mL zymotic fluids 3000rpm, 15min centrifugation
Afterwards, mycelium accounts for the volume of zymotic fluid.
Brief description of the drawings
Fig. 1 show the Hirsutella sinensis cultivation box growth figure prepared by the present invention
Specific embodiment mode
The embodiment of the invention discloses one kind to prepare the ripe conidial culture medium of Hirsutella sinensis and method.This area
Technical staff can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements
Apparent to those skilled in the art with changing, they are considered as being included in the present invention.The method of the present invention
Be described by preferred embodiment, related personnel substantially can not depart from present invention, in spirit and scope it is right
Product as described herein and method are modified or 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 prepared by one kind provided by the invention Hirsutella sinensis into
Ripe conidial culture medium and method are described in detail.
Embodiment
1) conidium derivant is prepared：
Conidium derivant is by 0.04g/100mL Tea Polyphenols, 0.01g/100mL vitamin B1,0.02g/100mL
Vitamin B6,0.02g/100mL biotin stir composition, excess water is supplied.
2) peat soil culture medium is prepared
The loose, peat soil of fibre object even thickness is chosen, is soaked and is cleaned with water, by the inorganic matter of its peat soil
It is 6.5 to control as 10~15%, pH controls, and it is standby to carry out 100 DEG C of drying pack sealing in 8 hours.Nutrient solution is by 0.10g/100mL
Peptone, 0.1g/100mL dusty yeast, 1g/100mL sucrose, 0.01g/100mL anhydrous magnesium sulfate, 0.1g/100mL
Potassium dihydrogen phosphate, 0.1g/100mL vitamin C stir composition, excess water is supplied.Peat soil is adjusted with nutrient solution
To 10~15% humidity, prepared according to loading amounts of the 500g per box.Prepare and sterilized 2 hours after 121 DEG C.Puparium powder list
Solely sterilizing (carries out fumigation, 200g/m using expoxy propane3Stifling 12h), according to the amount of culture medium mass ratio 2% during inoculation
Add in culture medium.
Hirsutella sinensis bacterial strain obtains to be separated from fresh wild cordyceps.It is inoculated with Hirsutella sinensis liquid spawn
To the peat soil culture medium prepared, inoculum concentration is 15%V/W mycelium, and its weight in wet base mycelium weight in wet base is 15~20%, will
The culture medium of inoculation is placed in sterile constant-temperature darkroom, at 18 DEG C of temperature, humidity 75%, cultivates hirsutella sinensis fungal, after 30 days,
It is covered with white aerial hyphae inside and outside culture medium；Cultivation temperature is reduced to 4 DEG C, humidity 65%, adds the mitogenetic spore of above-mentioned preparation
Cultivated after sub- derivant, after 40 days, sampled microscopy can be clearly visible the mitogenetic spore of Hirsutella sinensis kidney shape that a large amount of maturations are scattered
Son.
With the solid fermentation mycelia block of the direct picking different parts of transfer needle, and lower weight is counted, washed down and divided with Tween 80 liquid
Raw spore, centrifugation (1000r/min, 15 minutes) is collected, then is counted under the microscope with blood counting chamber, average each gram of solid
Hirsutella sinensis conidium amount contained by culture medium is 4.0 × 109~8.0 × 109, each box peat soil culture medium is containing China
Hair spore conidium is 2.0 × 1012~4.0 × 1012。
The explanation of above example is only intended to help the method and its core concept for understanding the present invention.It should be pointed out that pair
For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out
Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.
Claims (1)
1. one kind fermentation conidiiferous method of Hirsutella sinensis, it is characterized in that including：
1) Hirsutella sinensis liquid spawn is inoculated with to culture medium, wherein the culture medium includes：Peat soil and nutrient solution；
2) culture medium being vaccinated with step 1) is placed in sterile constant-temperature darkroom and cultivated；With
3) cultivation temperature culture is reduced；Further add conidium derivant after cooling, the conidium derivant by
0.04g/100mL Tea Polyphenols, 0.01g/100mL vitamin B1,0.02g/100mL vitamin B6,0.02g/100mL
Biotin and water composition；The dosage of the conidium derivant is 10mL/500g culture mediums；
Wherein, the inoculum concentration of inoculation Hirsutella sinensis liquid spawn is 10~15%V/W mycelium in step 1), its mycelium
Weight in wet base is 15~20%；
In step 2) cultivation temperature control range be 18~22 DEG C, incubation time be 30~35 days, humid control scope be 60~
80%；With
In step 3) cultivation temperature control range be 4~10 DEG C, incubation time be 35~45 days, humid control scope be 60~
75%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610214162.4A CN105838659B (en) | 2016-04-06 | 2016-04-06 | A kind of conidiiferous culture medium of Hirsutella sinensis solid fermentation and method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610214162.4A CN105838659B (en) | 2016-04-06 | 2016-04-06 | A kind of conidiiferous culture medium of Hirsutella sinensis solid fermentation and method |
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| Publication Number | Publication Date |
|---|---|
| CN105838659A CN105838659A (en) | 2016-08-10 |
| CN105838659B true CN105838659B (en) | 2017-12-05 |
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| CN1513976A (en) * | 2003-08-19 | 2004-07-21 | 成都思摩纳米技术有限公司 | Cordyceps vegetative stage solid fermentation technology |
| US20150025132A1 (en) * | 2013-07-16 | 2015-01-22 | Georgia Regents University | Compositions and methods for inhibiting endospores using green tea polyphenols |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1513976A (en) * | 2003-08-19 | 2004-07-21 | 成都思摩纳米技术有限公司 | Cordyceps vegetative stage solid fermentation technology |
| US20150025132A1 (en) * | 2013-07-16 | 2015-01-22 | Georgia Regents University | Compositions and methods for inhibiting endospores using green tea polyphenols |
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| 人工培育冬虫夏草分生孢子的研究;李玲玲等;《食品研究与开发》;20160229;第37卷(第3期);第187-190页，参见摘要第188页左栏第10段及表1 * |
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| 培养基及培养条件对中国被毛孢固体发酵产分生孢子的影响;张宗耀等;《菌物学报》;20160324;第35卷(第10期);第1-10页，参见摘要、第3页左栏第2段-右栏首段、第4页右栏末段-第5页左栏首段、第5页右栏第3段、第7页左栏第2段-右栏首段表1、表3及表8-10 * |
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Denomination of invention: Culture medium and method for producing conidia through solid fermentation of hiresutella sinensis X.J.Liu Y.L. Guo Effective date of registration: 20180315 Granted publication date: 20171205 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: 20171205 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: 20210616 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 medium and method for conidia production from hirsutella sinensis by solid state fermentation Effective date of registration: 20210630 Granted publication date: 20171205 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 |