CN103571788B - A kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link. - Google Patents
A kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link. Download PDFInfo
- Publication number
- CN103571788B CN103571788B CN201210282858.2A CN201210282858A CN103571788B CN 103571788 B CN103571788 B CN 103571788B CN 201210282858 A CN201210282858 A CN 201210282858A CN 103571788 B CN103571788 B CN 103571788B
- Authority
- CN
- China
- Prior art keywords
- sand
- collected
- link
- thecasporous
- cordyceps militaris
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 46
- 238000004321 preservation Methods 0.000 title abstract description 18
- 239000004576 sand Substances 0.000 claims abstract description 69
- 210000004215 spores Anatomy 0.000 claims abstract description 69
- 238000001035 drying Methods 0.000 claims abstract description 31
- 230000035800 maturation Effects 0.000 claims abstract description 24
- 230000001954 sterilising Effects 0.000 claims abstract description 24
- 239000002253 acid Substances 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 230000001264 neutralization Effects 0.000 claims description 7
- 238000011033 desalting Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 230000001488 breeding Effects 0.000 claims description 3
- 230000004913 activation Effects 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 description 12
- 238000000926 separation method Methods 0.000 description 10
- 230000001580 bacterial Effects 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 239000000969 carrier Substances 0.000 description 7
- 239000002609 media Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L mgso4 Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 241000446313 Lamella Species 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 230000000717 retained Effects 0.000 description 4
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N CORDYCEPIN Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 description 3
- 206010010254 Concussion Diseases 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 206010013786 Dry skin Diseases 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000000050 nutritive Effects 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000003068 static Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000190633 Cordyceps Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000001016 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229940032362 Superoxide Dismutase Drugs 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide Dismutase Proteins 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000006011 modification reaction Methods 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 235000012015 potatoes Nutrition 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- 230000004763 spore germination Effects 0.000 description 2
- OIRDTQYFTABQOQ-GAWUUDPSSA-N 9-β-D-XYLOFURANOSYL-ADENINE Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@H](O)[C@H]1O OIRDTQYFTABQOQ-GAWUUDPSSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- OIRDTQYFTABQOQ-SXVXDFOESA-N Adenosine Natural products Nc1ncnc2c1ncn2[C@@H]3O[C@@H](CO)[C@H](O)[C@@H]3O OIRDTQYFTABQOQ-SXVXDFOESA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 240000005710 Auricularia polytricha Species 0.000 description 1
- 235000000024 Auricularia polytricha Nutrition 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 210000003298 Dental Enamel Anatomy 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- DNVPQKQSNYMLRS-APGDWVJJSA-N Ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)/C=C/[C@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-APGDWVJJSA-N 0.000 description 1
- DNVPQKQSNYMLRS-LNHMRCHQSA-N Ergosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3C([C@H]4[C@@](C)([C@H]([C@@H](/C=C/[C@H](C(C)C)C)C)CC4)CC3)=CC=2)CC1 DNVPQKQSNYMLRS-LNHMRCHQSA-N 0.000 description 1
- 240000007842 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 210000003734 Kidney Anatomy 0.000 description 1
- FBPFZTCFMRRESA-BXKVDMCESA-N L-mannitol Chemical compound OC[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)CO FBPFZTCFMRRESA-BXKVDMCESA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L Mercury(II) chloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 210000004080 Milk Anatomy 0.000 description 1
- 241001506047 Tremella Species 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003712 anti-aging Effects 0.000 description 1
- 230000000844 anti-bacterial Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002082 anti-convulsion Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cells Anatomy 0.000 description 1
- 230000000249 desinfective Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 230000001146 hypoxic Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000004301 light adaptation Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920001690 polydopamine Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 230000001568 sexual Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Abstract
The invention provides a kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link., collection method comprises the steps: sand to be put into many spores separated and collected container that can seal, sterilizing, drying; Select Cordyceps militaris (L.) Link. ascoma fully-developed sporophore, be placed with in many spores separated and collected container of sand; Many spores separated and collected of vibrating container, the thecasporous sand of maturation collected the thecaspore containing the maturation of launching out and do not launch out.The sand collected is placed in dry sterile chamber seal, room temperature lucifuge or refrigerator cold-storage are preserved.Collection method provided by the invention is simple to operate, improve thecasporous collection rate, can retain the inherited character of Cordyceps militaris (L.) Link. original parent after the thecaspore activation of preservation, and for screening degeneration-resistant new variety.
Description
Technical field
The invention belongs to cordyceps cultivation technology field, particularly, relate to a kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link..
Background technology
Cordyceps militaris (L.) Link. Cordycepsmilitaris(L.exFr) Link, have another name called Cordyceps militaris (L.) Link., Cordyceps militaris, colonize on the insect pupal cells such as lepidopteran, Coleoptera, Diptera.Growth, in the region, Plain of low altitude area, is mainly distributed in the ground such as northeast, North China, northwest.Modern study shows, Cordyceps militaris (L.) Link. contains the chemical compositions such as cordycepin (3'-Deoxyadenosine), cordycepic acid (PEARLITOL 25C), adenosine, Cordyceps polysaccharide, ergosterol, superoxide-dismutase (SOD), selenium (Se).There is effects such as improving immune function of human body, anticancer, antibacterial, antifatigue, anti-ageing, anticonvulsion, hypoxia tolerance, calmness, establishing-Yang are reinforced the kidney.Also there is the effects such as fat-reducing, weight reducing, beauty treatment simultaneously.
In recent years, a lot of research all shows, tame Cordyceps militaris (L.) Link. chemical composition and pharmacological action similar to Cordyceps sinensis, but price is well below Cordyceps sinensis, and therefore, the Application and Development of Cordyceps militaris (L.) Link. has great potential market.Tissue Culture of Cordyceps militaris will obtain sporophore, and key is that this cordyceps militaris link bacterial strain will have the ability forming sporophore, and the genetic background that different strains possesses is different, so the ability that they possess the sporophore of formation is variant.But, in actual production, even advantage cordyceps militaris link bacterial strain, along with bacterial classification go down to posterity, the increase of number of times of transferring or the prolongation of preservation time, microorganism resource is very easily degenerated.
Spore separation method, is sprout into mycelia with the sexual spore of edible mushrooms or asexual spore, is trained the method for bacterial classification.This bacterial classification vitality is comparatively strong, but variant between spore individuality, and natural differentiation phenomenon is comparatively serious, and variation is large, need could application in production through fruiting experiment.Current spore separation method mainly contains: (one) single spore separation method: be each or often prop up test tube and only get a sporidium, allows it sprout into mycelium to obtain the method for pure strain.Single spore separation produces upper less employing, and technical sophistication, general employing many spores partition method.(2) many spores partition method be many spore inoculatings on same substratum, allow their are sprouted, pangamy to be to obtain a kind of method of edible mushrooms pure strain.Concrete operation method, has following several: 1. kind of mushroom spore launches method: eight or nine points that selection adaptation is strong ripe kind mushrooms, stem aseptic water washing number all over after blot surface-moisture with sterilized gauze or absorbent cotton, filter paper again.In inoculation tank or sterilisable chamber, the lamella of kind of mushroom is hung upside down below glass funnel with iron wire down, and funnel covers on culture dish; Upper end aperture clogs with cotton.Culture dish is placed on one and is covered with on the enamel tray of gauze, and leave standstill 12 ~ 20 hours, the spore on lamella will be scattered in culture dish.Form the Powdered spore print of one deck.Pick outside the agar of a small amount of spore in test tube or streak inoculation on culture dish with inoculating needle.Treat spore germination, when generating bacterium colony, select spore germination early, bacterium colony that growing way is good carries out Tube propagation.Also spore collected by available spore collector.By said procedure, raise bell glass gently, kind of a mushroom handle is inserted on the wire frame of spore collector down, is placed on culture dish centre.Build lens immediately, with gauze, the clock palm is around stoppered.And on gauze a little mercuric chloride or sterilized water.Move into about 20 DEG C thermostat containers to cultivate.2. streak method on pleat: select ripe kind mushroom, directly insert between gusset with inoculating needle, smears the spore got gusset surface sporophore and not yet launch gently, then streak inoculation on substratum.3. hook hangs method: several lamella or a fritter auricle (black fungus, Auricularia polytricha (Mout) Sacc., Tremella) of getting ripe cap, hang on the top of the substratum in triangular flask with aseptic Stainless Steel Wire (or other suspension materials such as iron wire, cotton thread), do not make to touch substratum or surrounding bottle wall.Cultivate under putting optimal temperature, transfer.4. attach method: the lamella of maturation or auricle are got a fritter, is attached on the test tube wall directly over pipe arrangement slant medium with the nutrient agar dissolved or gum arabic, paste etc.Through the cultivation of 6 ~ 12 hours, treat that spore drops on inclined-plane, spore is transplanted in new test tube together with part nutrient agar cultivates immediately.Mother's kind that spore separation obtains, to purificate and rejuvenate further, when mother plants field planting about one week, when mycelia is covered with inclined-plane, select mycelia healthy and strong, grow vigorous without aging, plant test tube without the mother infecting assorted mattress, and then tube expands.Mother's kind that spore separation obtains, must pass through fruiting experiment, after being accredited as good quality strain, just can supply production and application.There are two deficiencies in aforesaid method: the first, complicated operation.The second, can only collect ripe by the spore launched out, and but can not collected by the ripe spore launched out of maturation.
Summary of the invention
The object of this invention is to provide a kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link., to overcome above-mentioned deficiency.
In order to realize the object of the invention, the thecasporous collection method of a kind of Cordyceps militaris (L.) Link. of the present invention, it comprises the steps:
1) sand is put into many spores separated and collected container that can seal, sterilizing, drying;
2) Cordyceps militaris (L.) Link. ascoma fully-developed sporophore puts into many spores separated and collected container of step 1);
3) many spores separated and collected of vibrating container, the thecasporous sand of maturation collected the thecaspore containing the maturation of launching out and do not launch out.
Wherein, described step 1) sand is neutral dry sand, and it obtains by the following method:
A) river sand is crossed 60 mesh sieves, discard macrobead and impurity;
B) sieve with 80 orders again, discard fine sand, collect unsifted sand;
C) irony in sand is removed;
D) with after the salt acid soak sand 24h of 10%-20%, acid of desalting, washes bubble with water to neutral, dries sand.
Further, the irony in magnet removing sand can be used in step c).
Many spores separated and collected container that the present invention uses, the sealable Glass Containers that can have any shape or metal vessel.
Wherein, the laying method that many spores separated and collected container that can seal put into by the sand described in step 1) be by etc. after the sand of quality mixes with distilled water, 1:3-10 is placed in many spores separated and collected container by volume.
Wherein, the sterilizing described in step 1) is 121 DEG C, moist heat sterilization 30min.
Wherein, the drying described in step 1) is 80 DEG C of dry 24-48h.
In the step of above-mentioned sterilizing and drying, many spores separated and collected container all strictly seals and carries out sterilizing and drying.
Wherein, step 2) the abundant sporophore head of the preferred 1-2cm ascoma of Cordyceps militaris (L.) Link. ascoma fully-developed sporophore, put into many spores separated and collected container of step 1), room temperature places 10-30 days, to sporophore complete drying.
Wherein, step 2) described in sporophore put into many spores separated and collected container, the sporophore number put into and the number mass ratio of container sand are 1: 2.5-5g.
Step 2 in Cordyceps militaris (L.) Link. thecaspore collection method provided by the invention) in, room temperature places 10-30 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore on fine sand by nature, have the ascoma of Partial mature not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
Wherein, the vibration described in step 3) is with 80-200r/min speed oscillation 24-48h by many spores separated and collected container.
The thecasporous collection method of a kind of Cordyceps militaris (L.) Link. provided by the invention also comprises takes out the sporophore of drying.
The present invention also provides a kind of Cordyceps militaris (L.) Link. thecasporous store method, and the thecaspore containing the maturation of launching out obtained by aforesaid method and the thecasporous sand of maturation do not launched out are placed in dry sterile chamber sealing preservation.
Further, the present invention preserves the thecasporous storage conditions of Cordyceps militaris (L.) Link. is room temperature lucifuge or 0-6 DEG C of refrigeration.
The invention provides the application of Cordyceps militaris (L.) Link. thecaspore collection method in Cordyceps militaris (L.) Link. breeding.
The present invention utilizes the rubbing effect of fine sand, when the ascoma of the maturation of drying and immature ascoma are subject to the rubbing effect of fine sand thus broken and release thecaspore wherein, falls into sand carrier.By collecting the sand of macroscopic, thus collect the sightless Cordyceps militaris (L.) Link. thecaspore of naked eyes, therefore present method can not only collect the thecaspore of ejection, can also collect the thecaspore do not ejected, thecasporous collection rate reaches 100%, improves thecasporous collection effciency.And the thecaspore adopting catapult technique to collect in prior art is the parton cystospore launched out after ascoma maturation, thecaspore quantity is less than 1% of thecaspore total amounts all in sporophore ascoma.The sporophore of drying is taken out by the present invention further, by thecaspore activation or save backup, finally achieves the object of the thecasporous separation of Cordyceps militaris (L.) Link., collection and preservation.Cordyceps militaris (L.) Link. thecaspore collection method provided by the invention is simple to operate, use manpower and material resources sparingly, improve thecasporous collection rate simultaneously, the Cordyceps militaris (L.) Link. thecaspore store method provided effectively can keep thecasporous biological activity in 2-4, maintain the inherited character of original parent, for seed selection novel bacterial provides effective ways, be with a wide range of applications.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The cultivation of embodiment 1 Cordyccps-militaris-(L.)-link. Sporophore
1, bacterial classification: the strain number adopting autonomous screening to obtain is HX-64 cordyceps militaris link bacterial strain (purchased from Beijing City Agriculture and Forestry Institute Bio-Centers).
2, liquid shaking bottle is with substratum (1L): peeled potatoes 300g(liquor), glucose 10g, peptone 2g, potassium primary phosphate 0.5g, magnesium sulfate 1g, water 1000ml, pH7.2.
3, sporophore produces nutritive medium: the ratio of soya bean 2%, glucose 1%, milk powder 5%, potassium primary phosphate 0.5%, magnesium sulfate 0.05%, VB11 0mg/1000ml is made into nutritive medium, and pH is 5.6 ~ 7.2 in adjustment.
4, PP is adopted to expect the cultivation box (go to the bottom diameter 8cm for high 10cm, upper base diameter 12cm) made.Every box rice 40g and sporophore produce nutritive medium 56g packing mixing, disinfection inoculation.
5, mycelium culture management
Culturing room's optimal temperature is 18 ~ 25 DEG C, and humidity is 70 ~ 85%, and lucifuge cultivates 8 days.Mycelia starts aerlbic culture after being covered with surface.
6, sporophore is cultivated
After by kind, treat that mycelia is covered with whole culture vessel and dark bundle arrives container bottom, start to see light, but avoid sunlight direct projection.If culturing room's light is too dark, fluorescent lamp can be supplemented and shine.After mycelia sees light, see there is orange chromogenesis on culture material surface or surrounding, and occur the orange button of grain of rice shape, namely button becomes sporophore after extending.Sporophore breeding phase, temperature should be 20 ~ 23 DEG C, and more than 25 DEG C of poor growths, humidity should bring up to 80 ~ 85%.The mycelia atrophy more than 27 DEG C can not go out bacterium bundle.In sporophore late stage of culture, strengthen circulation and the exchange of room air gradually, if culturing room's cultivation stage finds that pollution should be removed immediately.
7, gather: adopt rear mean fresh and reach 28.7 grams/cultivation box.
The thecasporous Collection and preservation of embodiment 2 Cordyceps militaris (L.) Link. (1)
(1) preparation of many spores carrier adsorption sand:
River sand 60 orders are sieved, discards macrobead and impurity, then sieve with 80 orders, remove fine sand.Suck irony with magnet, put into container 10% salt acid soak, acid of desalting after 24 hours, wash bubble with water for several times to neutral, sand is dried.
(2) sterilizing of multi spore collection container and drying treatment:
Get after the many spores carrier adsorption sand about 25g handled well mixes with equivalent distilled water and put into 250mL triangular flask, then overall 121 DEG C of moist heat sterilization 30min.Loft drier 80 DEG C of dryings 24 hours are put into until complete drying after sterilizing.
(3) many spores are separated the selection of parent's sporophore:
Choose just not polluted of Later growth in culture bottle in embodiment 1, the sporophore growth cycle is short, bar shaped neat, and even thickness, color Exocarpium Citri Rubrum, ascoma fully-developed culture bottle put into Bechtop after bottle surface sterilization.
(4) collection of many spores:
Choose ascoma fully-developed sporophore, through aseptic technique, the head scissors that ascoma is abundant is cut 1cm, put into many spores separated and collected bottle that sterilizing-drying is crossed, collect 5 for every bottle.
Room temperature places 10 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore on fine sand by nature, have the ascoma of Partial mature not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
After static drying terminates, many spores separated and collected bottle is put into shaking table, carry out concussion 24 hours with the rotating speed of 80r/min.
Due to the rubbing effect of fine sand, the ascoma of dry maturation and immature ascoma receive the rubbing effect of fine sand thus broken and then release thecaspore wherein.Then the sporophore part of drying is taken out, the thecasporous sand of maturation collected the thecaspore containing the maturation of launching out and do not launch out.Finally achieve the object of the thecasporous separation of Cordyceps militaris (L.) Link., collection.The present embodiment can collect the whole thecaspores in sporophore in ascoma, and collection rate is 100%.
(5) preservation of many spores
Small test tube with 160 DEG C of 2 hours dry sterilizations, by collect containing launching out ripe thecaspore and the thecasporous sand of maturation that do not launch out is sub-packed in small test tube, then seal with solid paraffin, prevent moisture from entering, room temperature keeps in Dark Place 2 years.
The thecasporous Collection and preservation of embodiment 3 Cordyceps militaris (L.) Link. (2)
(1) preparation of many spores carrier adsorption sand:
River sand 60 orders are sieved, discards macrobead and impurity, then sieve with 80 orders, remove fine sand.Suck irony with magnet, put into container 15% salt acid soak, acid of desalting after 24 hours, wash bubble with water for several times to neutral, sand is dried.
(2) sterilizing of multi spore collection container and drying treatment:
Get after the many spores carrier adsorption sand about 50g handled well mixes with equivalent distilled water and put into 250mL triangular flask, then overall 121 DEG C of moist heat sterilization 30min.Loft drier 80 DEG C of dryings 48 hours are put into until complete drying after sterilizing.
(3) many spores are separated the selection of parent's sporophore:
To choose in embodiment 1 just not polluted of Later growth in culture bottle, the sporophore growth cycle is short, bar shaped neat, even thickness, color Exocarpium Citri Rubrum, ascoma fully-developed culture bottle put into Bechtop after bottle surface sterilization.
(4) collection of many spores:
Choose ascoma fully-developed sporophore, through aseptic technique, the head scissors that ascoma is abundant is cut 2cm, put into many spores separated and collected bottle that sterilizing-drying is crossed, approximately collect 10 for every bottle.
Room temperature places 30 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore on fine sand by nature, have the ascoma of Partial mature not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
After static drying terminates, many spores separated and collected bottle is put into shaking table, carry out concussion 48 hours with the rotating speed of 200r/min.
Due to the rubbing effect of fine sand, the ascoma of dry maturation and immature ascoma receive the rubbing effect of fine sand thus broken and then release thecaspore wherein.Then the sporophore part of drying is taken out, the thecasporous sand of maturation collected the thecaspore containing the maturation of launching out and do not launch out.Finally achieve the object of the thecasporous separation of Cordyceps militaris (L.) Link., collection.The present embodiment can collect the whole thecaspores in sporophore in ascoma, and collection rate is 100%.
(5) preservation of many spores
Small test tube is with 160 DEG C of 2 hours dry sterilizations, by collect containing launching out ripe thecaspore and the thecasporous sand of maturation that do not launch out is sub-packed in small test tube, then with solid paraffin sealing, prevent moisture from entering, storage conditions is 0 DEG C of refrigeration 4 years.
The thecasporous collection of embodiment 4 Cordyceps militaris (L.) Link. (3)
(1) preparation of many spores carrier adsorption sand:
River sand 60 orders are sieved, discards macrobead and impurity, then sieve with 80 orders, remove fine sand.Suck irony with magnet, put into container 20% salt acid soak, acid of desalting after 24 hours, wash bubble with water for several times to neutral, sand is dried.
(2) sterilizing of multi spore collection container and drying treatment:
Get after the many spores carrier adsorption sand about 50g handled well mixes with equivalent distilled water and put into 500mL triangular flask, then overall 121 DEG C of moist heat sterilization 30min.Loft drier 80 DEG C of dryings 38 hours are put into until complete drying after sterilizing.
(3) many spores are separated the selection of parent's sporophore:
To choose in embodiment 1 just not polluted of Later growth in culture bottle, the sporophore growth cycle is short, bar shaped neat, even thickness, color Exocarpium Citri Rubrum, ascoma fully-developed culture bottle put into Bechtop after bottle surface sterilization.
(4) collection of many spores:
Choose ascoma fully-developed sporophore, through aseptic technique, the head scissors that ascoma is abundant is cut 2cm, put into many spores separated and collected bottle that sterilizing-drying is crossed, approximately collect 8 for every bottle.
Room temperature places 25 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore on fine sand by nature, have the ascoma of Partial mature not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
After static drying terminates, many spores separated and collected bottle is put into shaking table, carry out concussion 40 hours with the rotating speed of 150r/min.
Due to the rubbing effect of fine sand, the ascoma of dry maturation and immature ascoma receive the rubbing effect of fine sand thus broken and then release thecaspore wherein.Then the sporophore part of drying is taken out, the thecasporous sand of maturation collected the thecaspore containing the maturation of launching out and do not launch out.Finally achieve the object of the thecasporous separation of Cordyceps militaris (L.) Link., collection.The present embodiment can collect the whole thecaspores in sporophore in ascoma, and collection rate reaches 100%.
(5) preservation of many spores
Small test tube is with 160 DEG C of 2 hours dry sterilizations, by collect containing launching out ripe thecaspore and the thecasporous sand of maturation that do not launch out is sub-packed in small test tube, then with solid paraffin sealing, prevent moisture from entering, storage conditions is 6 DEG C of refrigerations 3 years.
The thecasporous activation of Cordyceps militaris (L.) Link. of embodiment 5 Collection and conservation and cultivation
The Cordyceps militaris (L.) Link. thecaspore of Example 2-4 Collection and preservation, aseptically opens many spores preservation pipe, gets the part grains of sand on PDA slant medium, transfer once after growing bacterium colony again respectively.Or get the grains of sand in suitable liquid nutrient medium (formula of 1L liquid culture medium is peeled potatoes 300g(liquor), glucose 10g, peptone 2g, potassium primary phosphate 0.5g, magnesium sulfate 1g, water 1000ml) in, directly do fruiting experiment or inclined-plane of transferring again after multiplication culture.
After the Cordyceps militaris (L.) Link. thecaspore activation that embodiment 2-4 preserves, the method recorded according to embodiment 1 carries out the cultivation of Cordyccps-militaris-(L.)-link. Sporophore.Cultivation results finds, the Cordyceps militaris (L.) Link. thecaspore that embodiment 2-4 preserves all can be cultivated and obtain sporophore, adopt rear mean fresh and reach 28.7,28.9,28.8 grams/cultivation box respectively, the output of sporophore and biological character and embodiment 1 cultivate the sporophore that obtains all without significant difference, illustrate that the thecasporous method of Collection and preservation Cordyceps militaris (L.) Link. of the present invention can retain parent's inherited character.The present embodiment result illustrates: Cordyceps militaris (L.) Link. thecaspore fungi preservation 2-4 under room temperature or general refrigerator condition of the collection in above-described embodiment 2-4 still has biological activity, and fruiting body yield and biological transformation ratio are without significant difference, substantially remain the inherited character of original parent.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. the thecasporous collection method of Cordyceps militaris (L.) Link., is characterized in that, comprise the steps:
1) sand is put into many spores separated and collected container that can seal, sterilizing, drying;
2) Cordyceps militaris (L.) Link. ascoma fully-developed, dry sporophore are put into step 1) many spores separated and collected container;
3) many spores separated and collected of vibrating container, the thecasporous sand of maturation collected the thecaspore containing the maturation of launching out and do not launch out; Wherein, step 3) described in vibration be with 80-200r/min speed oscillation 24-48h by many spores separated and collected container.
2. collection method as claimed in claim 1, is characterized in that, described step 1) sand is neutral dry sand, it obtains by the following method:
A) river sand is crossed 60 mesh sieves, discard macrobead and impurity;
B) sieve with 80 orders again, discard fine sand, collect unsifted sand;
C) irony in sand is removed;
D) with after the salt acid soak sand 24h of 10%-20%, acid of desalting, washes bubble with water to neutral, dries sand.
3. collection method as claimed in claim 1, it is characterized in that, step 1) described in the sand laying method of putting into many spores separated and collected container that can seal be by etc. after the sand of quality mixes with distilled water, 1:3-10 is placed in many spores separated and collected container by volume.
4. collection method as claimed in claim 1, is characterized in that, step 1) described in sterilizing be 121 DEG C, moist heat sterilization 30min.
5. collection method as claimed in claim 1, is characterized in that, step 1) described in drying be 80 DEG C of dry 24-48h.
6. collection method as claimed in claim 1, is characterized in that, step 2) described in sporophore put into many spores separated and collected container, the sporophore number put into and the mass ratio of container sand are 1: 2.5-5g.
7. the collection method as described in as arbitrary in claim 1-6, is characterized in that, step 3) in also comprise the sporophore of drying taken out.
8. the arbitrary described application of thecasporous collection method in Cordyceps militaris (L.) Link. breeding of claim 1-6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210282858.2A CN103571788B (en) | 2012-08-09 | 2012-08-09 | A kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210282858.2A CN103571788B (en) | 2012-08-09 | 2012-08-09 | A kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103571788A CN103571788A (en) | 2014-02-12 |
| CN103571788B true CN103571788B (en) | 2015-11-25 |
Family
ID=50044503
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210282858.2A Active CN103571788B (en) | 2012-08-09 | 2012-08-09 | A kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link. |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103571788B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955794B (en) * | 2017-11-27 | 2021-04-13 | 沈阳农业大学 | High-quality preservation method of cordyceps militaris strains |
| CN108739053A (en) * | 2018-06-22 | 2018-11-06 | 辽东学院 | A kind of method of long-term preservation high cordycepin Cordyceps militaris spawn |
| CN109122049A (en) * | 2018-08-03 | 2019-01-04 | 内蒙古农业大学 | A kind of more spore separators of Cordyceps militaris and method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676598A (en) * | 2004-03-29 | 2005-10-05 | 龙泉市兴龙科技开发研究所 | Glassy ganoderm spore powder breeding and collecting technique |
| CN101748073A (en) * | 2008-12-18 | 2010-06-23 | 北京农业生物技术研究中心 | Method for separating and preserving Cordyceps militaris spawn |
| CN101861795A (en) * | 2010-05-10 | 2010-10-20 | 云南农业大学 | Method for promoting propagation of tricholoma matsutake artificially |
-
2012
- 2012-08-09 CN CN201210282858.2A patent/CN103571788B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1676598A (en) * | 2004-03-29 | 2005-10-05 | 龙泉市兴龙科技开发研究所 | Glassy ganoderm spore powder breeding and collecting technique |
| CN101748073A (en) * | 2008-12-18 | 2010-06-23 | 北京农业生物技术研究中心 | Method for separating and preserving Cordyceps militaris spawn |
| CN101861795A (en) * | 2010-05-10 | 2010-10-20 | 云南农业大学 | Method for promoting propagation of tricholoma matsutake artificially |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103571788A (en) | 2014-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101215527A (en) | Method for cultivating silkworm chrysalis Cordyceps sinensis | |
| CN101473808A (en) | Artificial culture, breeding and preservation method of plant root-knot nematode | |
| CN103404364A (en) | Grifola frondosa liquid culture cultivating and high-yield cultivating method | |
| CN102612966B (en) | Method for obtaining dendrobium aphyllum seeds to germinate effective symbiotic fungi | |
| CN107034147A (en) | The selection and cultural method of flat mushroom kind are planted for industrial bottle | |
| WO2014010314A1 (en) | Method for cultivating mushrooms using reusable fiber substrate, and culture medium for cultivation using same | |
| CN112021068B (en) | Rejuvenation method of tremella aurantialba sporocarp strains | |
| CN105660191A (en) | Amauroderma rugosum sporocarp culture method | |
| CN105638467B (en) | A kind of method that roxburgh anoectochilus terminal bud is cultivated with culture bag | |
| CN103843583A (en) | Industrialized production method of green cordyceps militaris | |
| CN101333550B (en) | Method for preparing cyclic dipeptides compounds and use thereof | |
| CN101874453A (en) | Culture method of snow white mushroom strains and sporocarps | |
| CN103571788B (en) | A kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link. | |
| CN102630534B (en) | Method for controlling pine wood nematode diseases by utilizing body surface bacteria of American pine wood nematodes | |
| CN106434349A (en) | Method for collecting mycorrhizal fungi through using tissue culture seedling of sterile blueberry | |
| CN104686196B (en) | Method for preserving and separating toadstool strain through sporocarp dried in shade | |
| CN103891526A (en) | Method for cultivating bud-thinning-free pleurotus eryngii cultivated in bags | |
| CN101748073B (en) | Method for separating and preserving Cordyceps militaris spawn | |
| CN110140591A (en) | Hickory chick superior strain and its application | |
| CN100574614C (en) | A kind of calla lily germplasm resources in vitro conservation method | |
| CN109661972A (en) | A kind of selection of high temperature resistant mushroom | |
| JP2017038541A (en) | Cultivation method of boletus | |
| CN103320329B (en) | Apocynum venetum melampsora propagation method | |
| CN104737775A (en) | Organic pleurotus eryngii production process | |
| CN107484461A (en) | A kind of method that the pocket orchid seed germination rate for improving cryopreservation is handled using sodium hypochlorite |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 100195 Beijing city Haidian District West Fourth Ring Road No. 15 East Building Room 307 Patentee after: Beijing Zhongyi Yuanda Agriculture Development Co. Ltd. Address before: 100195 Beijing city Haidian District West Fourth Ring Road No. 15 East Building Room 307 Patentee before: BEIJING ZHONGYI YUANDA MECHANICAL AND ELECTRICAL EQUIPMENT CO., LTD. |