CN101126069B - Cordyceps sinensis asexual propagation method - Google Patents
Cordyceps sinensis asexual propagation method Download PDFInfo
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- CN101126069B CN101126069B CN2007100168163A CN200710016816A CN101126069B CN 101126069 B CN101126069 B CN 101126069B CN 2007100168163 A CN2007100168163 A CN 2007100168163A CN 200710016816 A CN200710016816 A CN 200710016816A CN 101126069 B CN101126069 B CN 101126069B
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Abstract
The invention relates to a cloning method of a Chinese hirsutella, which is characterized in that: a bacteria tissue is cut into small pieces by operation knife in a steam deinfected glass bottle, inoculated on the surface of a medium in the glass bottle; the glass bottle is covered with an air permeable cover, and placed in a culativation region until the bacteria tissue is ripe. The invention makes use of the monosporous splitting breeding principle of fungus hirsutella, and successfully prepares qualified bacteria tissue with sexual characteristics of hirsutella that can be a substitute of natural Cordyceps aweto by cloning method.
Description
Technical field
The present invention relates to a kind of asexual reproduction method, particularly a kind of China pilose spore asexual reproduction method.Described China pilose spore (Hirsutella Sinensis Liu, Guo, Yu et Zeng) different name is a Cordyceps sinensis fungi Cephalosporium bacterial classification.
Background technology
The process of growth of the Cordyceps sinensis of nature growth, be actually the bat moth larvae and be used as being infected of fungi in the process on the feed by the hair spore, utilized the bat moth larvae to make the nutrition basal growth by the hair spore, be consumed totally, form the set of thalline at the head of larva until larva body internal protein, be called stroma, the top produces spore, collapses after the maturation to send out again, and infects again, go round and begin again, finish by all processes of hair spore sexual propagation.On the stricti jurise, Cordyceps sinensis is that insect larvae and fungi are by the combination of hair spore neither worm neither be careless.
The sexual propagation rule of natural imitation circle Cordyceps sinensis is cultivated by the hair spore fully, also has sizable difficulty at present technically, and is subjected to the restriction of production cost.Production field has only the mycelium of fermentation as the Cordyceps sinensis substitute at present.And the mycelium of fermentation still is with natural cordyceps very big difference to be arranged all aspect the pharmaceutical use at growthhabit, cell tissue, active component content.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned prior art, and provide a kind of China pilose spore asexual reproduction method, technical problem to be solved is: the first, utilize fungi by hair spore monospore fissiparity rule, the employing asexual reproduction method is cultivated a thalline tissue that obtains as the quilt hair spore sexualise of natural cordyceps substitute.The second, by insect protein, animal proteinum, marine organisms albumen and vegetable-protein are combined according to specified proportion, and by unique working method, prepare and be fit to by the hair spore low-cost polynary substratum of high-quality of growth fast.Three, provide a kind of spawn culture special-purpose ventilative bottle cap, not only can realize culturing the interior free water of bottle and discharge in order, and effectively prevent the mould intrusion with the steam form.
In order to solve the problems of the technologies described above, the present invention has adopted following technical scheme:
A kind of China pilose spore asexual reproduction method is characterized in that: with scalpel the thalline tissue is shredded in the vial behind steam sterilizing; Be seeded in the media surface in the vial; Cover ventilative bottle cap in glass bottle opening; The vial that is stamped ventilative bottle cap is placed on the culture zone, until maturation;
Wherein, 110～121 ℃ of the sterilising temps of substratum and surgical knife tool, sterilization time 25～30 minutes; 300,000 grades of culture zone purifying air degree, 10～18 ℃ of room temps, indoor humidity RH40%～55%;
Wherein, described substratum is: contain silkworm chrysalis 7～9kg in every 100kg substratum, the peeling scallop muscle 3～5kg that shells, fresh beef 1.5～2.5kg, fresh ox bone 4～6kg, Semen arachidis hypogaeae 0.4～0.6kg, fresh milk 4～6kg, soy peptone 0.8～1.2kg, agar 1.0～1.4kg, potassium primary phosphate 40～60g, sal epsom 15～25g, surplus is a water.
The control of ambient moisture is a gordian technique of the present invention, and humidity is higher than RH55% and grows mould easily, is lower than RH40% and then causes substratum moisture excessively to volatilize.
Asexual reproduction method of the present invention cultivate by a hair spore, make thalline with the Cordyceps sinensis of natural condition growths and fermentation method and compare: the comparison of (1), visual inspection dry product: the thalline that obtains with fermentation technique is a canescence state tissue, and proportion is lighter.The thalline that obtains with the inventive method is circular, hard, the rat injustice, and yellowish brown, proportion is bigger, and is basic identical with the histocyte form of natural cs stroma.(2), through China Academy Of Traditional Chinese Medicine Traditional Chinese Medicine Research Institute's check, active constituent content: thalline＞natural cs of the present invention＞fermented hypha.Detect through Institute of Microorganism, Academia Sinica, hirsutella sinensis fungal of the present invention body tissue proterties is stable, possesses the sexual propagation feature.(3), transferred species is observed: will grow into being inverted on the vial that substratum is housed by hair spore thalline even bottle about 4～5 months, bottleneck is connected sealing with the bottleneck junction as aseptically process.After one month, five times of magnifying glasses are observed, the distribute cell tissue of few round dot white of media surface, and media surface was covered with white translucent Φ 2～3mm circle in 2～3 months, intermediate recess and association down shape, have and erect granulation state cell tissue, the speed of growth is accelerated subsequently.There is few part can bear 1～2 and the false stroma of natural cs stroma homomorphosis, but growing way a little less than, high about 20～30mm, slightly about Φ 2mm, growth time is short, very easily fall shape, fall behind the shape with the thalline tissue growth together, no vestige, extremely crisp after drying after getting, after growing to 12～13 months, by white, translucent yellowing, light brown, maturation.There is not significant difference with vegetative thalli morphology.Observation shows that vegetative propagation is still had the sexual reproduction memory character by the hair spore.
The positively effect of substratum of the present invention and preparation method thereof is: (1), facts have proved for many years, substratum of the present invention can be satisfied by the quick merisis of hair spore cell required necessary amino acid and other nutritive elements.Every 100ml substratum cocoa is produced 5g by hair spore thalline.(2), adopt the preparation method of substratum of the present invention, guaranteed to make the small molecules solution of substratum for can directly being absorbed, shortened enzyme resolving time, improved by a hair spore speed of growth.(3), substratum with short production cycle, low cost of manufacture, good economy performance is fit to industrialization big area utilization and extention.
Wherein, especially outstanding as the proteic scallop muscle effect of marine organisms, if adopt other albumen to replace, can't realize purpose of the present invention.In addition, the proportioning of various components also is a key of the present invention, is the data that obtain through experiment for many years.Processing step among the preparation method, processing condition also are gordian techniquies of the present invention, also search out through exploring accumulation for many years, if change to some extent will directly influence the use properties of substratum.The usage ratio of ox bone is conventional high, and this is to study, test discovery for many years, adopts this ratio to promote that obviously by the histiocytic merisis of hair spore, this also is a key point of the present invention.
The positively effect of ventilative bottle cap of the present invention is: (1), permeability cell are arranged on the bottle cap side, and by pair lid filter paper is installed in the permeability cell outer end, not only can guarantee to culture the interior free water of bottle and discharge in order, and effectively prevent fungal infection with the steam form; On the other hand, because bottle cap top accessibility designs, breed bottle vertically multilayer is put, and has effectively utilized the space, has saved floor space.(2), in sterilization process, above-mentioned permeability cell can play supercharging and depressurization rapidly, thereby shortens sterilization time.(3), filling substratum, especially in seeded process, can shorten and duration of contact of outside air, minimizing fungal infection chance, easy to operate.(4) even the bottle cap of should breathing freely is contaminated, it is reusable to clean back replacing filter paper, has reduced material consumption, has saved production cost.(5), permeability cell is provided with sufficient length, also is difficult to infect a bacterial classification in the bottle even mould spores sees through filter paper.(6), ventilative tube chamber vertical section is long-pending through the science calculation Design, make the free water throughput be controlled in the reasonable range, can not cause too much, the too fast dehydration of substratum,, influence the normal growth of bacterial classification perhaps because the too small bottle interior ponding that causes of throughput because of throughput is excessive.(7), bottle cap closed section lower edge is reserved with to be used to closely cooperate and inserts the section of probeing in the bottleneck, the vaporize water that has produced when having avoided sterilization is produced the problem that the bacterium of mildewing grows carrier from the slit flow between bottleneck and the bottle cap screw thread.
Description of drawings
Fig. 1 is the structural representation of the ventilative bottle cap of the present invention.
Fig. 2 is by hair spore surface growth state picture.
Fig. 3 is by the fresh and alive thalline yardstick of hair spore picture.
Fig. 4 is by the fresh and alive thalline height of hair spore picture.
Fig. 5 is by hair spore normal growth state picture.
Embodiment
Further specify the present invention below in conjunction with drawings and Examples.
One, China pilose spore vegetative propagation
At first from natural cordyceps, isolate the thalline tissue, perhaps get the thalline tissue that utilizes present method breeding.With scalpel the thalline tissue is shredded in the vial after sterilization, thin more good more.Thalline tissue in small, broken bits is seeded in media surface in the vial, covers ventilative bottle cap in glass bottle opening; The vial that is stamped ventilative bottle cap is placed on the culture zone, until maturation.
Wherein, 110～121 ℃ of the sterilising temps of substratum and surgical knife tool, sterilization time 25～30 minutes.Naturally cooling was put district's temperature below 18 ℃ more than 12 hours behind the medium sterilization.
300,000 grades of culture zone purifying air degree, 10～18 ℃ of room temps, indoor humidity RH40%～55%;
Inoculum size is: every bottle of 20mm
3About.Inoculation bottle is 50ml and two kinds of loading amounts of 100ml PDA substratum.
Be divided into three phases vegetative period.Fs is about 30 days, and five times of magnifying glasses are observed, and generates oyster white, translucent cells tissue in the contact surface slit of PDA media surface and bacterial classification, the surperficial association adularescent of cell tissue down, local growth uprightly, branch, granulation shape tissue.
Subordinate phase is about 120 days fast growing period, and cell tissue division causes upwards protuberance of extruding, and there have granulation shape tissue to divide to be the fastest, and bump surface is and is uneven, and cell tissue is still white, translucent, and the about 10～15mm of thickness thickens individually to more than the 25mm.Microscopic examination has the softgel shell areola at top surface, has conidiogenous cell to form in the chamber.Sections observation, cell tissue are little yellow, translucent.Denseer medicine fragrance is arranged, and there is small holes inside, and hole is arranged around the hole wall adularescent down, vessel cell.
Phase III is the ripe collection phase, and thalline superficial cell tissue is by the translucent flavescence gradually of white, yellowish brown, and the substratum residue is few, and cracking, drying shrinkage, but does not have utility value.Former bacterial classification chip becomes dark brown, and lignifying does not have the regeneration sign, and with newly-generated cell tissue together.Gathering the back thalline has adhesion part substratum, blows off with 0.2MPa pressurized air, concentrates drying, vacuum packaging, primary products.
Fig. 2 shows that finished product is by the tissue morphology of hair spore.
Fig. 3 shows the ratio of thalline and substratum.
Fig. 4 shows the actual height of thalline.
Fig. 5 shows by the normal growth state of hair spore.
Two, medium preparation example
Example 1
According to preparation 100kg substratum,
(1), the peeling scallop muscle 4kg that will shell soaked 48 hours with clear water, changed water desalination more than 6 times again, ground pulping at last, made the A component;
(2), Semen arachidis hypogaeae 0.5kg was soaked in water 10 hours, defibrination is made the B component then;
(3), fresh beef 2kg defibrination is made the C component;
(4), the D component is made in silkworm chrysalis 8kg defibrination, peeling;
(5), A, B, C, D component are mixed, add water and add to 80kg, boil, slow fire boiled 30 minutes, and precipitation, filtering bits are made the E component;
(6), boiled 30 minutes after fresh ox bone 5kg boiled with pressure kettle, precipitation, filtering bits are made the F component;
(7), with E component and F component mixing, add fresh milk 5kg, soy peptone 1kg, agar 1.2kg, potassium primary phosphate 50g, sal epsom 20g, and add water and add to 100kg, bottling endured out in the lump, sterilization, usefulness is inoculated in cooling fully, and wherein sterilising temp is 115 ℃, sterilization time 30 minutes.
Example 2
According to preparation 100kg substratum,
(1), the peeling scallop muscle 3kg that will shell soaked 36 hours with clear water, changed water desalination more than 6 times again, ground pulping at last, made the A component;
(2), Semen arachidis hypogaeae 0.6kg was soaked in water 8 hours, defibrination is made the B component then;
(3), fresh beef 1.5kg defibrination is made the C component;
(4), the D component is made in silkworm chrysalis 9kg defibrination, peeling;
(5), A, B, C, D component are mixed, add water and add to 85kg, boil, slow fire boiled 20 minutes, and precipitation, filtering bits are made the E component;
(6), boiled 20 minutes after fresh ox bone 4kg boiled with pressure kettle, precipitation, filtering bits are made the F component;
(7), with E component and F component mixing, add fresh milk 6kg, soy peptone 0.8kg, agar 1.4kg, potassium primary phosphate 40g, sal epsom 15g, and add water and add to 100kg, bottling endured out in the lump, sterilization, usefulness is inoculated in cooling fully, and wherein sterilising temp is 110 ℃, sterilization time 28 minutes.
Example 3
According to preparation 100kg substratum,
(1), the peeling scallop muscle 5kg that will shell soaked 60 hours with clear water, changed water desalination more than 6 times again, ground pulping at last, made the A component;
(2), Semen arachidis hypogaeae 0.4kg was soaked in water 12 hours, defibrination is made the B component then;
(3), fresh beef 2.5kg defibrination is made the C component;
(4), the D component is made in silkworm chrysalis 7kg defibrination, peeling;
(5), A, B, C, D component are mixed, add water and add to 75kg, boil, slow fire boiled 40 minutes, and precipitation, filtering bits are made the E component;
(6), boiled 40 minutes after fresh ox bone 6kg boiled with pressure kettle, precipitation, filtering bits are made the F component;
(7), with E component and F component mixing, add fresh milk 4kg, soy peptone 1.2kg, agar 1.0kg, potassium primary phosphate 60g, sal epsom 25g, and add water and add to 100kg, bottling endured out in the lump, sterilization, usefulness is inoculated in cooling fully, and wherein sterilising temp is 121 ℃, sterilization time 25 minutes.
It is pure water that used water requires, and used various raw materials must guarantee fresh, does not have rotten sign.In addition, the dead pupa after the pupa of living is better than reeling off raw silk from cocoons, dead pupa is better than dried pupa.
Prepared substratum pH value is between 5.5～6.5.
Summer, the nutritive medium of preparation storage period generally can not be above 6 hours.
Three, ventilative bottle cap embodiment
As shown in Figure 1, present embodiment comprises that inwall has the linkage section 1 of the screw thread that cooperates with bottleneck, also has closed section 2 in linkage section 1 upper end, and one section permeability cell 4 is arranged in the side of closed section 2, and filter paper 5 is fixed on the collar extension end of described permeability cell 4 by pair lid 6.
The tube chamber vertical section of permeability cell 4 is circular.Diameter is 9 millimeters or 12 millimeters, corresponding to 50ml and two kinds of loading amounts of 100ml PDA substratum.
The length of permeability cell 4 is generally 16 millimeters.
Claims (3)
1. a China pilose spore asexual reproduction method is characterized in that: with scalpel the thalline tissue is shredded in the vial behind steam sterilizing; Be seeded in the media surface in the vial; Cover ventilative bottle cap in glass bottle opening; The vial that is stamped ventilative bottle cap is placed on the culture zone, until maturation;
Wherein, 110～121 ℃ of the sterilising temps of surgical knife tool, sterilization time 25～30 minutes; 300,000 grades of culture zone purifying air degree, 10～18 ℃ of room temps, indoor humidity RH 40%～55%;
The preparation method of described substratum is:
According to preparation 100kg substratum,
(1), the peeling scallop muscle 4kg that will shell soaked 48 hours with clear water, changed water desalination more than 6 times again, ground pulping at last, made the A component;
(2), Semen arachidis hypogaeae 0.5kg was soaked in water 10 hours, defibrination is made the B component then;
(3), fresh beef 2kg defibrination is made the C component;
(4), the D component is made in silkworm chrysalis 8kg defibrination, peeling;
(5), A, B, C, D component are mixed, add water and add to 80kg, boil, slow fire boiled 30 minutes, and precipitation, filtering bits are made the E component;
(6), boiled 30 minutes after fresh ox bone 5kg boiled with pressure kettle, precipitation, filtering bits are made the F component;
(7), with E component and F component mixing, add fresh milk 5kg, soy peptone 1kg, agar 1.2kg, potassium primary phosphate 50g, sal epsom 20g, and add water and add to 100kg, bottling endured out in the lump, sterilization, usefulness is inoculated in cooling fully, and wherein sterilising temp is 115 ℃, sterilization time 30 minutes;
Described ventilative bottle cap comprises that inwall has the linkage section of the screw thread that cooperates with bottleneck (1), also has closed section (2) in linkage section (1) upper end, in the side of closed section (2) one section permeability cell (4) is arranged, filter paper (5) is fixed on the collar extension end of described permeability cell (4) by secondary lid (6).
2. China pilose spore asexual reproduction method as claimed in claim 1 is characterized in that: closed section (2) lower end is insinuated into below the inwall of linkage section (1) upper surface, and this section of probeing into (3) external diameter matches with a supporting breed bottle internal diameter of the bottleneck size.
3. China pilose spore asexual reproduction method as claimed in claim 1 is characterized in that: the tube chamber vertical section of permeability cell (4) is for circular, and diameter is 9 millimeters or 12 millimeters; The length of permeability cell (4) is the 14-18 millimeter.
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| CN2007100168163A CN101126069B (en) | 2007-07-05 | 2007-07-05 | Cordyceps sinensis asexual propagation method |
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| CN2007100168163A CN101126069B (en) | 2007-07-05 | 2007-07-05 | Cordyceps sinensis asexual propagation method |
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| CN101126069B true CN101126069B (en) | 2010-12-15 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1274006A (en) * | 2000-06-09 | 2000-11-22 | 车振明 | Method for artificially cultivating cordyceps |
| CN1445360A (en) * | 2002-03-20 | 2003-10-01 | 中国科学院沈阳应用生态研究所 | Method of artificial planting northerly Chinese caterpillar fungus for increasing content of physiologically active substance |
| CN1670181A (en) * | 2005-04-07 | 2005-09-21 | 雷明 | Culture medium for improving fermentation yield of aweto mycelium and method for preparing same |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1274006A (en) * | 2000-06-09 | 2000-11-22 | 车振明 | Method for artificially cultivating cordyceps |
| CN1445360A (en) * | 2002-03-20 | 2003-10-01 | 中国科学院沈阳应用生态研究所 | Method of artificial planting northerly Chinese caterpillar fungus for increasing content of physiologically active substance |
| CN1670181A (en) * | 2005-04-07 | 2005-09-21 | 雷明 | Culture medium for improving fermentation yield of aweto mycelium and method for preparing same |
Non-Patent Citations (3)
| Title |
|---|
| JP特开2004-242507A 2004.09.02 |
| 李增智，等，.确证冬虫夏草无性型的分子生物学证据I.中国被毛孢与冬虫夏草之间的关系.菌物系统19 1.2000,19(1),60-64. |
| 李增智等.确证冬虫夏草无性型的分子生物学证据I.中国被毛孢与冬虫夏草之间的关系.菌物系统19 1.2000,19(1),60-64. * |
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