CN103667170B - One prepares the ripe conidial method of China pilose spore - Google Patents
One prepares the ripe conidial method of China pilose spore Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 68
- 241001248610 Ophiocordyceps sinensis Species 0.000 claims abstract description 17
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 230000002538 fungal effect Effects 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 235000015099 wheat brans Nutrition 0.000 claims description 19
- 235000007164 Oryza sativa Nutrition 0.000 claims description 10
- 235000009566 rice Nutrition 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
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- 239000008103 glucose Substances 0.000 claims description 9
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- 239000000843 powder Substances 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 4
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- 240000007594 Oryza sativa Species 0.000 claims 2
- 241000190633 Cordyceps Species 0.000 abstract description 17
- 230000035800 maturation Effects 0.000 abstract description 13
- 238000011161 development Methods 0.000 abstract description 5
- 230000018109 developmental process Effects 0.000 abstract description 5
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- 230000028070 sporulation Effects 0.000 description 2
- 241000130660 Hepialidae Species 0.000 description 1
- 241000143459 Hirsutella Species 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The present invention relates to microorganism field, disclose one and prepare the ripe conidial method of China pilose spore.China pilose spore bacterium colony is inoculated on substratum by the method for the invention, mycelium culture 35-55 days is carried out under the condition of 14-20 DEG C of temperature and 65-90% relative humidity, then go to induced meristem spore 20-40 days under the condition of 10-15 DEG C of temperature and 65-90% relative humidity, obtain the ripe conidium of hirsutella sinensis fungal.China pilose spore is inoculated in specific substratum by the present invention, and control the condition such as temperature and humidity of China pilose spore at Different growth phases, prepare the conidium that a large amount of maturation is scattered, the method of the invention improves the ripe conidial quantity of China pilose spore, is conducive to the industrialized development of artificial cultivation of cordyceps.
Description
This application claims and submitted on 09 20th, 2012 the right of priority that Patent Office of the People's Republic of China, application number are 201210355665.5, denomination of invention be the Chinese patent application of " one prepares the conidial method of China pilose spore maturation " to, its full content combines in this application by reference.
Technical field
The present invention relates to microorganism field, relate to one specifically and prepare the ripe conidial method of China pilose spore.
Background technology
Cordyceps sinensis is that Cordyceps fungus (Cordyceps sinensis) infects and colonizes in the complex body of stroma (sporophore) and the larva polypide that Hepialidae insect larvae grows, belong to edible fungi, be a kind of traditional famous and precious tonic Chinese medicine material of China, have the multiple efficacies such as immunity moderation system function, antitumor, antifatigue.
Wild Cordyceps sinensis only grows in the Alpine vegetation such as high mountain steppe, plateau grassy marshland of Qinghai-Tibet height above sea level 3000-5000 rice, and make its production declining owing to excessively excavating in recent years, its price has gone up more than 1000 times compared with before more than 20 years.Therefore, the study on the industrialization that China scientific research personnel is carrying out utilizing Cordyceps fungus to infect always bat moth larvae artificial cultivation of cordyceps carrys out alternative wild cordyceps, tries hard to allow expensive Cordyceps sinensis come into common people house.Cordyceps sinensis will complete industrialization, first must find its correct bacterial classification, then utilizes Cordyceps fungus to infect bat moth larvae, and then nurtures Cordyceps sinensis.Through years of researches, Cordyceps fungus is accredited as China pilose spore, is also called Hirsutella hepiali Chen et Shen.
Artificial cultivation of cordyceps mainly adopts the mycelium of China pilose spore to infect bat moth larvae, but the infection rate that this routine infects method is not high, the success ratio of cultivating Cordyceps sinensis is affected, hinders the industrialized development of Cordyceps sinensis.Along with the continuous exploration to artificial cultivation of cordyceps technical field, there are some researches show and adopt the conidium of China pilose spore maturation can significantly improve infection rate to infect bat moth larvae.Therefore, the ripe conidial process of artificial preparation hirsutella sinensis fungal just becomes the important step affecting artificial cultivation of cordyceps success ratio.
Only has the relevant China pilose spore that utilizes few in number at present to prepare conidial report, as document " biological factors is to the sporogenic research estranged of Cordyceps sinensis fungi " (Li Yuling, " edible mushrooms " the 5th phase in 2002), it discuss China pilose spore and produce conidial research under different culture media, different light and differing temps; And for example document " the dynamic spore output amount of Cordyceps fungus and anti-different ability " (Wang Wei, Yang Bo, Cai Shiliang etc., " herbal medicine " the 33rd volume the 1st phase in 2002), it discusses the research of Cordyceps fungus sporulation quantity in PDA, Cha Shi, sabouraud medium in the text, but the conidium quantity utilizing the method in above-mentioned document to obtain is not high, only reach 10 respectively
5with 10
7the spore count of the order of magnitude, and and undeclared gained conidium whether maturation is scattered.Therefore, a kind of conidium preparation method that can improve the ripe conidium quantity of China pilose spore is provided, is conducive to the development of artificial cultivation of cordyceps industrialization.
Summary of the invention
In view of this, the object of the present invention is to provide one to prepare the ripe conidial method of China pilose spore, make the method can improve the ripe conidial quantity of China pilose spore.
To achieve these goals, the invention provides following technical scheme:
One prepares the ripe conidial method of China pilose spore, China pilose spore bacterium colony is inoculated on substratum, mycelium culture 35-55 days is carried out under the condition of 14-20 DEG C of temperature and 65-90% relative humidity, then induced meristem spore 20-40 days under the condition of 10-15 DEG C of temperature and 65-90% relative humidity is gone to, obtain the ripe conidium of hirsutella sinensis fungal, described substratum is the wheat bran liquid of 1-5% by 60-65mL/100mL mass percent, the peptone of 0.5-1.5g/100mL, the yeast powder of 0.5-1.5g/100mL, the glucose of 2-4g/100mL, the rice of 30-40g/100mL and the egg liquid the stirred composition of 25-35mL/100mL, excess water is supplied.
The China pilose spore that the method for the invention adopts can by commercially available acquisition, or utilize this area routine techniques means to obtain from the separation of wild Cordyceps sinensis, the China pilose spore bacterium colony inoculated is cultivated by the art ordinary method and is obtained, utilize the numerous colony inoculation of expansion to be conducive to mycelia and conidial growth, this is well known in the art.
Substratum of the present invention preferably by 65mL/100mL mass percent be 2% wheat bran liquid, the peptone of 1g/100mL, the yeast powder of 1g/100mL, the glucose of 3g/100mL, the rice of 35g/100mL and 30mL/100mL the egg liquid stirred form, excess water is supplied.Wherein, described mass percent is the wheat bran liquid of 1-5% can be add 1-5g wheat bran in every 100mL water to soak the filtrate of then filtering gained, also can be the mixed solution adding 1-5g wheat bran in the every 100mL water stirred, and as preferred, described mass percent is that the wheat bran liquid of 1-5% is obtained by following methods:
Add 1-5g wheat bran in every 100mL water, the filtrate of filtering gained after boiling 30min is the wheat bran liquid that mass percent is 1-5%.
Conidium is that microorganism is for a kind of emergency reaction under specified conditions, under can be understood as adverse environment, the one that microorganism produces to not make the kind of self be exterminated is more stable, the vegetative propagule that tolerance is stronger, therefore specific substratum and culture condition is needed mutually to coordinate just to prepare ripe conidium, its institute adaptable substratum and culture condition scope narrower, and above-mentioned cultivation condition cannot be adjusted to a suitable scope by general limited number of time test at all, more cannot obtain and be applicable to the ripe conidial substratum of preparation.Therefore, prior art is rare prepares the ripe conidial method of China pilose spore, and document " biological factors is to the sporogenic research estranged of Cordyceps sinensis fungi " (Li Yuling, " edible mushrooms " the 5th phase in 2002) and " the dynamic spore output amount of Cordyceps fungus and anti-different ability " (Wang Wei, Yang Bo, Cai Shiliang etc., " herbal medicine " the 33rd volume the 1st phase in 2002) in the conidium quantity prepared of method not high, and whether undeclared conidium ripe.
For the problems referred to above, applicant is on the basis furtherd investigate China pilose spore growth and development characteristics, through performing creative labour, provide one and prepare the ripe conidial method of China pilose spore, the method for the invention is divided into mycelium culture and conidium to induce two stages.
Conidium is positioned on conidiophore, and conidiophore and conidial growth depend on the extent of growth of mycelia.The too dense meeting of mycelia consumes too much substratum nutrition, and conidium under-nutrition would not grow; And mycelia is too sparse, be then not easy to grow conidial degree.Therefore, the time of mycelium culture of the present invention is 35-55 days, is preferably 40-50 days, is more preferably 45 days.Meanwhile, the temperature of described mycelium culture is preferably 17-19 DEG C, is more preferably 18 DEG C, and the relative humidity of described mycelium culture is preferably 80-85%.
At induced meristem Spore Stages, optimum culture condition of the present invention, reduce temperature, the conidial generation of low temperature stimulation, the time as preferred induced meristem spore of the present invention is preferably 25-35 days, is more preferably 30 days.Meanwhile, the temperature of described induced meristem spore is preferably 12-14 DEG C, is more preferably 13 DEG C, and the relative humidity of described induced meristem spore is preferably 80-85%.
Being not particularly limited illumination in the method for the invention, can be that astigmatism is irradiated, and also can be that 12h dark and 12h illumination replace, also can be complete lucifuge, the present invention is complete lucifuge preferably.
Utilize conidium prepared by the method for the invention, rinse microscopy through water and obviously can see the China pilose spore bacterial classification kidney shape conidium (see figure 1) that a large amount of maturation is scattered.Meanwhile, product spore simultaneous test is carried out with two sections of documents (see background technology) of prior art.Result shows, all higher than conidium quantity prepared by the existing method of employing on the conidium the number no matter conidium that the present invention obtains is scattered at conidium sum or maturation.
The ripe conidium of the China pilose spore prepared through the present invention is through aseptic water washing, filtration, and namely collecting precipitation can be used for infecting of bat moth larvae after making bacteria suspension with sterilized water again.In addition, after aseptic water washing, carry out density gradient centrifugation, collecting precipitation part can as fungi preservation.
From above technical scheme, China pilose spore is inoculated in specific substratum by the present invention, and control the condition such as humidity and temperature of China pilose spore at Different growth phases, prepare the conidium that a large amount of maturation is scattered, the method of the invention improves the ripe conidial quantity of China pilose spore, is conducive to the industrialized development of artificial cultivation of cordyceps.
Accompanying drawing explanation
Figure 1 shows that the ripe conidial microscopy figure of China pilose spore prepared by the present invention.
Embodiment
The embodiment of the invention discloses one and prepare the ripe conidial method of China pilose spore.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope product as herein described and method are changed or suitably change with combination, realize and apply the technology of the present invention.
In order to understand the present invention further, below in conjunction with embodiment, being prepared by the ripe conidial method of China pilose spore to one provided by the invention and being described in detail.
Embodiment 1: the method for the invention prepares the ripe conidium of China pilose spore
Culture medium prescription: (mass percent is 2%) wheat bran liquid 65ml, peptone 1g, yeast powder 1g, glucose 3g, rice 35g, the egg liquid 30ml that mixes thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, grow under being statically placed in the culture condition of lucifuge, temperature 18 DEG C, relative humidity 85%, after 45d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in induced meristem spore under the inductive condition of lucifuge, temperature 13 DEG C, relative humidity 85% again, after induction 30d, namely obtain ripe conidium.Obtain suspension through aseptic water washing medium slant, add up and microscopy with cell counting to suspension, microscopy obviously can see China pilose spore bacterial classification kidney shape conidium (Fig. 1) that a large amount of maturation is scattered, and in statistics, conidium adds up to 1 × 10
10, ripe conidium quantity is 9 × 10
9.
Embodiment 2: produce spore simultaneous test
Reference literature " biological factors is to the sporogenic research estranged of Cordyceps sinensis fungi " (Li Yuling, " edible mushrooms " the 5th phase in 2002) and " the dynamic spore output amount of Cordyceps fungus and anti-different ability " (Wang Wei, Yang Bo, Cai Shiliang etc., " herbal medicine " the 33rd volume the 1st phase in 2002) in best practice in identical culture environment, prepare conidium with the method for embodiment 1, China pilose spore bacterium colony is same bacterial classification and bacterium colony is in same growth period, inoculum size and culture volume are consistent, finally with the aseptic water washing statistics also microscopy with amount, all the other undeclared conditions of document are identical with embodiment 1.
Wherein, the best practice (hereinafter referred to as control methods 1) of document " biological factors is to the sporogenic research estranged of Cordyceps sinensis fungi " is:
Wheat bran medium (formula is shown in document), culture condition is 20 DEG C, lucifuge.
The best practice (hereinafter referred to as control methods 2) of document " the dynamic spore output amount of Cordyceps fungus and anti-different ability " is:
PDA substratum, culture condition is 22.5 DEG C.
Microscopy result shows, obvious sporophore bundle is there is in control methods 1 and control methods 2, and sporophore end all hangs and has two lune prematurity conidiums, and the display of the microscopy result of the embodiment of the present invention 1, obviously visible a large amount of maturation be scattered, the conidium of kidney shape.
Conidium statistics is in table 1.
Table 1 sporulation quantity statistics
| Conidium sum | Ripe conidium number | |
| Control methods 1 | 1×10 5 | 1×10 4 |
| Control methods 2 | 1×10 7 | 1×10 6 |
| Embodiment 1 | 1×10 10 | 9×10 9 |
As shown in Table 1, the last prepared conidium sum of the method for the invention and ripe conidium number, all higher than spore count prepared by two kinds of control methodss, show that the method for the invention enough improves the ripe conidial quantity of China pilose spore.
Embodiment 3: the method for the invention prepares the ripe conidium of China pilose spore
Culture medium prescription: (mass percent is 5%) wheat bran liquid 60ml, peptone 0.5g, yeast powder 0.5g, glucose 2g, rice 40g, the egg liquid 35ml that mixes thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, grow under being statically placed in the culture condition of astigmatism, temperature 17 DEG C, relative humidity 85%, after 40d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in induced meristem spore under the inductive condition of astigmatism, temperature 12 DEG C, relative humidity 80% again, after induction 25d, namely obtain ripe conidium.Obtain suspension through aseptic water washing medium slant, add up and microscopy with cell counting to suspension, microscopy obviously can see the China pilose spore bacterial classification kidney shape conidium that a large amount of maturation is scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium of preparing according to method and the present embodiment of embodiment 2 contrasts, and the conidium sum of wherein control methods 1 reaches 10
5the order of magnitude, ripe conidium number reaches 10
4the order of magnitude; The conidium sum of control methods 2 reaches 10
7the order of magnitude, ripe conidium number reaches 10
6the order of magnitude.
Embodiment 4: the method for the invention prepares the ripe conidium of China pilose spore
Culture medium prescription: (mass percent is 1%) wheat bran liquid 65ml, peptone 1.5g, yeast powder 1.5g, glucose 4g, rice 30g, the egg liquid 25ml that mixes thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, grow under being statically placed in the culture condition of the dark 12h illumination of 12h, temperature 19 DEG C, relative humidity 80%, after 50d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in induced meristem spore under the inductive condition of lucifuge, temperature 14 DEG C, relative humidity 85% again, after induction 35d, namely obtain ripe conidium.Obtain suspension through aseptic water washing medium slant, add up and microscopy with cell counting to suspension, microscopy obviously can see the China pilose spore bacterial classification kidney shape conidium that a large amount of maturation is scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium of preparing according to method and the present embodiment of embodiment 2 contrasts, and the conidium sum of wherein control methods 1 reaches 10
5the order of magnitude, ripe conidium number reaches 10
4the order of magnitude; The conidium sum of control methods 2 reaches 10
7the order of magnitude, ripe conidium number reaches 10
6the order of magnitude.
Embodiment 5: the method for the invention prepares the ripe conidium of China pilose spore
Culture medium prescription: (mass percent is 3%) wheat bran liquid 65ml, peptone 1g, yeast powder 1g, glucose 3g, rice 35g, the egg liquid 30ml that mixes thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, grow under being statically placed in the culture condition of lucifuge, temperature 14 DEG C, relative humidity 65%, after 55d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in induced meristem spore under the inductive condition of lucifuge, temperature 15 DEG C, relative humidity 90% again, after induction 40d, namely obtain ripe conidium.Obtain suspension through aseptic water washing medium slant, add up and microscopy with cell counting to suspension, microscopy obviously can see the China pilose spore bacterial classification kidney shape conidium that a large amount of maturation is scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium of preparing according to method and the present embodiment of embodiment 2 contrasts, and the conidium sum of wherein control methods 1 reaches 10
5the order of magnitude, ripe conidium number reaches 10
4the order of magnitude; The conidium sum of control methods 2 reaches 10
7the order of magnitude, ripe conidium number reaches 10
6the order of magnitude.
Embodiment 6: the method for the invention prepares the ripe conidium of China pilose spore
Culture medium prescription: (mass percent is 4%) wheat bran liquid 65ml, peptone 1g, yeast powder 1g, glucose 3g, rice 35g, the egg liquid 30ml that mixes thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, grow under being statically placed in the culture condition of lucifuge, temperature 20 DEG C, relative humidity 90%, after 35d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in induced meristem spore under the inductive condition of lucifuge, temperature 10 DEG C, relative humidity 65% again, after induction 20d, namely obtain ripe conidium.Obtain suspension through aseptic water washing medium slant, add up and microscopy with cell counting to suspension, microscopy obviously can see the China pilose spore bacterial classification kidney shape conidium that a large amount of maturation is scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium of preparing according to method and the present embodiment of embodiment 2 contrasts, and the conidium sum of wherein control methods 1 reaches 10
5the order of magnitude, ripe conidium number reaches 10
4the order of magnitude; The conidium sum of control methods 2 reaches 10
7the order of magnitude, ripe conidium number reaches 10
6the order of magnitude.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (9)
1. prepare the ripe conidial method of China pilose spore for one kind, it is characterized in that, China pilose spore bacterium colony is inoculated on substratum, mycelium culture 35-55 days is carried out under the condition of 14-20 DEG C of temperature and 65-90% relative humidity, then induced meristem spore 20-40 days under the condition of 10-15 DEG C of temperature and 65-90% relative humidity is gone to, obtain the ripe conidium of hirsutella sinensis fungal, described substratum is the wheat bran liquid of 1-5% by 60-65mL/100mL mass percent, the peptone of 0.5-1.5g/100mL, the yeast powder of 0.5-1.5g/100mL, the glucose of 2-4g/100mL, the rice of 30-40g/100mL and the egg liquid the stirred composition of 25-35mL/100mL, excess water is supplied.
2. method according to claim 1, it is characterized in that, described substratum by 65mL/100mL mass percent be 2% wheat bran liquid, the peptone of 1g/100mL, the yeast powder of 1g/100mL, the glucose of 3g/100mL, the rice of 35g/100mL and 30mL/100mL the egg liquid stirred form, excess water is supplied.
3. method according to claim 1, it is characterized in that, the temperature of described mycelium culture is 17-19 DEG C.
4. method according to claim 1, it is characterized in that, the relative humidity of described mycelium culture is 80-85%.
5. method according to claim 1, it is characterized in that, the temperature of described induced meristem spore is 12-14 DEG C.
6. method according to claim 1, it is characterized in that, the relative humidity of described induced meristem spore is 80-85%.
7. method according to claim 1, it is characterized in that, the time of described mycelium culture is 40-50 days.
8. method according to claim 1, it is characterized in that, the time of described induced meristem spore is 25-35 days.
9. method according to claim 1, it is characterized in that, described mass percent is that the wheat bran liquid of 1-5% is prepared by following methods:
Add 1-5g wheat bran in every 100mL water, the filtrate of filtering gained after boiling 30min is the wheat bran liquid that mass percent is 1-5%.
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| CN112088717A (en) * | 2020-10-14 | 2020-12-18 | 青海大学 | Method for cultivating cordyceps sinensis in field |
| CN112342145A (en) * | 2020-11-04 | 2021-02-09 | 青海久实虫草生物科技有限公司 | Rejuvenation method of hirsutella hepiali Chen et Shen fungi |
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| CN1274006A (en) * | 2000-06-09 | 2000-11-22 | 车振明 | Method for artificially cultivating cordyceps |
| CN102676631A (en) * | 2011-03-08 | 2012-09-19 | 中国科学院微生物研究所 | Fabrication method of cover glass for conidiospore of Ophio cordyceps sinensis and dynamic counting method of produced conidiospore |
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