CN103667170A - Method of preparing mature conidia of hirsutella sinensis - Google Patents
Method of preparing mature conidia of hirsutella sinensis Download PDFInfo
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- 230000006698 induction Effects 0.000 claims description 12
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Abstract
The invention relates to the field of microorganisms, and discloses a method of preparing mature conidia of hirsutella sinensis. The method comprises the following steps: inoculating a hirsutella sinensis bacterial colony on a culture medium; carrying out mycelium culture for 35-55 days under a condition that the temperature is 14-20 DEG C and the relative humidity is 65-90%; and then, transferring to a condition that the temperature is 10-15 DEG C and the relative humidity is 65-90% to induce conidia for 20-40 days to obtain the mature conidia of hirsutella sinensis. According to the method provided by the invention, hirsutella sinensis is inoculated on the special culture medium, and conditions such as temperature and humidity of hirsutella sinensis in different growth stages are controlled, so that a lot of mature scattered conidia are prepared. The method provided by the invention improves the quantity of the conidia of hirsutella sinensis, thereby facilitating industrialization development of artificially planting Cordyceps sinensis.
Description
The application requires to submit 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 is the Chinese patent application of " a kind of ripe conidial method of China pilose spore of preparing ", and its full content is by reference in conjunction with in this application.
Technical field
The present invention relates to microorganism field, relate to specifically a kind of ripe conidial method of China pilose spore of preparing.
Background technology
Cordyceps sinensis is that Cordyceps fungus (Cordyceps sinensis) infects and colonizes in the stroma (sporophore) that grows on Hepialidae insect larvae and the complex body of larva polypide, belong to edible fungi, be a kind of traditional famous and precious tonic Chinese medicine material of China, have multiple efficacies such as regulating function of immune system, antitumor, antifatigue.
Wild Cordyceps sinensis is only grown in the high and cold vegetation such as the high mountain steppe, plateau grassy marshland of Qinghai-Tibet height above sea level 3000-5000 rice, owing to excessively excavating in recent years, makes its production declining, and its price has gone up more than 1000 times with comparing before more than 20 years.Therefore, China scientific research personnel utilizes Cordyceps fungus to infect the study on the industrialization that bat moth larvae artificial cultivation of cordyceps substitutes wild cordyceps always, tries hard to allow expensive Cordyceps sinensis can 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 called again Hirsutella hepiali Chen et Shen.
Artificial cultivation of cordyceps is mainly to adopt the mycelium of China pilose spore to infect bat moth larvae, not high but this routine infects the infection rate of method, and the success ratio of cultivating Cordyceps sinensis is affected, and has hindered the industrialized development of Cordyceps sinensis.Along with the continuous exploration to artificial cultivation of cordyceps technical field, there are some researches show that adopting the conidium of China pilose spore maturation to infect bat moth larvae can obviously improve infection rate.Therefore, manually prepare the ripe conidial process of hirsutella sinensis fungal and just become the important step that affects artificial cultivation of cordyceps success ratio.
Only have at present the relevant China pilose spore that utilizes few in number to prepare conidial report, as document " the sporogenic research estranged of different factor pair Cordyceps sinensis fungis " (Li Yuling, < < edible mushrooms > > the 5th phase in 2002), it has been discussed China pilose spore and under different culture media, different light and differing temps, has produced conidial research; Document " the dynamic spore output amount of Cordyceps fungus and anti-different ability " (Wang Wei and for example, Yang Bo, Cai Shiliang etc., < < herbal medicine > > the 33rd the 1st phase of volume in 2002), it has discussed the research of Cordyceps fungus sporulation quantity in PDA, Cha Shi, sabouraud medium in the text, but utilize the resulting conidium quantity of method in above-mentioned document not high, only reach respectively 10
5with 10
7the spore count of the order of magnitude, and and undeclared gained conidium whether maturation is scattered.Therefore, provide a kind of conidium preparation method that can improve the ripe conidium quantity of China pilose spore, be 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 a kind of ripe conidial method of China pilose spore of preparing, make the method can improve the ripe conidial quantity of China pilose spore.
To achieve these goals, the invention provides following technical scheme:
A kind of ripe conidial method of China pilose spore of preparing, China pilose spore bacterium colony is inoculated on substratum, under the condition of 14-20 ℃ of temperature and 65-90% relative humidity, carry out mycelium culture 35-55 days, then go under the condition of 10-15 ℃ of temperature and 65-90% relative humidity and induce conidium 20-40 days, obtain the ripe conidium of hirsutella sinensis fungal, the wheat bran liquid that described substratum is 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 egg liquid stirring of the rice of 30-40g/100mL and 25-35mL/100mL forms, excess water is supplied.
The China pilose spore that the method for the invention adopts can be by commercially available acquisition, or utilize routine techniques means separation from wild Cordyceps sinensis in this area to obtain, the China pilose spore bacterium colony of inoculating can be cultivated and be obtained by the art ordinary method, utilize the numerous colony inoculation of expansion to be conducive to mycelia and conidial growth, this is being known in the art.
The egg liquid stirring of the wheat bran liquid that medium optimization of the present invention is 2% by 65mL/100mL mass percent, the peptone of 1g/100mL, the glucose of the yeast powder of 1g/100mL, 3g/100mL, the rice of 35g/100mL and 30mL/100mL forms, and excess water is supplied.Wherein, described mass percent is that the wheat bran liquid of 1-5% can be in every 100mL water, to add 1-5g wheat bran to soak the filtrate of then filtering gained, also can be in the every 100mL water stirring, to add the mixed solution of 1-5g wheat bran, and as preferred, the wheat bran liquid that described mass percent is 1-5% is obtained by following methods:
In every 100mL water, add 1-5g wheat bran, boil the filtrate of filtering gained after 30min and be the wheat bran liquid that mass percent is 1-5%.
Conidium is that microorganism is for a kind of emergency reaction under specified conditions, can be understood as under adverse environment, microorganism for the kind that does not make self be exterminated and produce a kind of more stable, the vegetative propagule that tolerance is stronger, therefore need specific substratum and culture condition mutually to coordinate just can prepare ripe conidium, its adaptable substratum of institute and culture condition scope are narrower, and the test of general limited number of time cannot be adjusted to above-mentioned cultivation condition a suitable scope at all, more cannot obtain and be applicable to the ripe conidial substratum of preparation.Therefore, the rare ripe conidial method of China pilose spore of preparing of prior art, and document " the sporogenic research estranged of different factor pair Cordyceps sinensis fungis " (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., conidium quantity prepared by the method < < herbal medicine > > the 33rd the 1st phase of volume in 2002) is not high, and whether undeclared conidium is ripe.
For the problems referred to above, applicant is on the basis that China pilose spore growth and development characteristics is furtherd investigate, through performing creative labour, a kind of ripe conidial method of China pilose spore of preparing is provided, the method for the invention is divided into mycelium culture and conidium two stages of induction.
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 just can not grow; And mycelia is too sparse, be 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, more preferably 45 days.Meanwhile, the temperature of described mycelium culture is preferably 17-19 ℃, and more preferably 18 ℃, the relative humidity of described mycelium culture is preferably 80-85%.
At induction Conidial Stage, optimum culture condition of the present invention, reduces temperature, and the conidial generation of low temperature stimulation, induces conidial time to be preferably 25-35 days as preferred the present invention, more preferably 30 days.Meanwhile, the conidial temperature of described induction is preferably 12-14 ℃, and more preferably 13 ℃, the conidial relative humidity of described induction is preferably 80-85%.
In the method for the invention, illumination being not particularly limited, can be that astigmatism is irradiated, and can be also that 12h dark and 12h illumination replace, and can be also complete lucifuge, the preferred lucifuge completely of the present invention.
The conidium that utilizes the method for the invention to prepare, rinses microscopy through water and can obviously see the China pilose spore bacterial classification kidney shape conidium (see figure 1) that a large amount of maturations are scattered.Meanwhile, produce spore simultaneous test with two pieces of documents (seeing background technology) of prior art.Result demonstration, the conidium the quantity no matter conidium that the present invention obtains all prepares higher than the existing method of employing on conidium sum or the ripe conidium number being scattered.
The ripe conidium of China pilose spore of preparing through the present invention is through aseptic water washing, filtration, and 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 partly can be used as bacterial classification and preserves.
From above technical scheme, the present invention is inoculated in China pilose spore in specific substratum, and control China pilose spore in the conditions such as humidity and temperature of different growth phases, prepare the conidium that a large amount of maturations are scattered, the method of the invention has improved 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 the prepared China pilose spore of the present invention.
Embodiment
The embodiment of the invention discloses a kind of ripe conidial method of China pilose spore of preparing.Those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, 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 be within not departing from content of the present invention, spirit and scope to product as herein described and method is changed or suitably change and combination, realize and apply the technology of the present invention.
In order further to understand the present invention, below in conjunction with embodiment, a kind of ripe conidial method of China pilose spore of preparing provided by the invention is elaborated.
Embodiment 1: the method for the invention is prepared 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 mixing thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, be statically placed under the culture condition of lucifuge, 18 ℃ of temperature, relative humidity 85% and grow, after 45d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in again under the inductive condition of lucifuge, 13 ℃ of temperature, relative humidity 85% and induce conidium, after induction 30d, obtain ripe conidium.Through aseptic water washing medium slant, obtain suspension, with cell counting, suspension is added up and microscopy, microscopy can obviously be seen the China pilose spore bacterial classification kidney shape conidium (Fig. 1) that a large amount of maturations are 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 " the sporogenic research estranged of different factor pair Cordyceps sinensis fungis " (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 the 1st phase of volume in 2002) best practice in is prepared conidium with the method for embodiment 1 in identical culture environment, China pilose spore bacterium colony is that same bacterial classification and bacterium colony are in same growth period, inoculum size and culture volume are consistent, finally with aseptic water washing statistics 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 " the sporogenic research estranged of different factor pair Cordyceps sinensis fungis " is:
Wheat bran substratum (formula is shown in document), culture condition is 20 ℃, 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 ℃.
Microscopy result shows, in control methods 1 and control methods 2, there is obvious sporophore bundle, and sporophore end all hangs two lune prematurity conidiums, and the microscopy result of the embodiment of the present invention 1 shows, obvious visible a large amount of maturations are 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 spore count that the last prepared conidium sum of the method for the invention and ripe conidium number are all prepared higher than two kinds of control methodss, shows that the method for the invention enough improves the ripe conidial quantity of China pilose spore.
Embodiment 3: the method for the invention is prepared 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 mixing thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, be statically placed under the culture condition of astigmatism, 17 ℃ of temperature, relative humidity 85% and grow, after 40d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in again under the inductive condition of astigmatism, 12 ℃ of temperature, relative humidity 80% and induce conidium, after induction 25d, obtain ripe conidium.Through aseptic water washing medium slant, obtain suspension, with cell counting, suspension is added up and microscopy, microscopy can obviously be seen the China pilose spore bacterial classification kidney shape conidium that a large amount of maturations are scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium contrast of preparing according to the method for embodiment 2 and the present embodiment, wherein the conidium sum of 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 is prepared 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 mixing thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, be statically placed under the culture condition of the dark 12h illumination of 12h, 19 ℃ of temperature, relative humidity 80% and grow, after 50d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in again under the inductive condition of lucifuge, 14 ℃ of temperature, relative humidity 85% and induce conidium, after induction 35d, obtain ripe conidium.Through aseptic water washing medium slant, obtain suspension, with cell counting, suspension is added up and microscopy, microscopy can obviously be seen the China pilose spore bacterial classification kidney shape conidium that a large amount of maturations are scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium contrast of preparing according to the method for embodiment 2 and the present embodiment, wherein the conidium sum of 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 is prepared 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 mixing thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, be statically placed under the culture condition of lucifuge, 14 ℃ of temperature, relative humidity 65% and grow, after 55d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in again under the inductive condition of lucifuge, 15 ℃ of temperature, relative humidity 90% and induce conidium, after induction 40d, obtain ripe conidium.Through aseptic water washing medium slant, obtain suspension, with cell counting, suspension is added up and microscopy, microscopy can obviously be seen the China pilose spore bacterial classification kidney shape conidium that a large amount of maturations are scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium contrast of preparing according to the method for embodiment 2 and the present embodiment, wherein the conidium sum of 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 is prepared 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 mixing thoroughly, water complements to 100mL;
China pilose spore bacterium colony is inoculated in the medium slant after above-mentioned sterilizing, be statically placed under the culture condition of lucifuge, 20 ℃ of temperature, relative humidity 90% and grow, after 35d, substratum growth is thorough, surface is covered with aerial hyphae, be statically placed in again under the inductive condition of lucifuge, 10 ℃ of temperature, relative humidity 65% and induce conidium, after induction 20d, obtain ripe conidium.Through aseptic water washing medium slant, obtain suspension, with cell counting, suspension is added up and microscopy, microscopy can obviously be seen the China pilose spore bacterial classification kidney shape conidium that a large amount of maturations are scattered, and in statistics, conidium sum reaches 10
10the order of magnitude, ripe conidium quantity is 10
9the order of magnitude.
The conidium contrast of preparing according to the method for embodiment 2 and the present embodiment, wherein the conidium sum of 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 is just for helping to understand method of the present invention and core concept thereof.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 improvement and modification 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, under the condition of 14-20 ℃ of temperature and 65-90% relative humidity, carry out mycelium culture 35-55 days, then go under the condition of 10-15 ℃ of temperature and 65-90% relative humidity and induce conidium 20-40 days, obtain the ripe conidium of hirsutella sinensis fungal, the wheat bran liquid that described substratum is 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 egg liquid stirring of the rice of 30-40g/100mL and 25-35mL/100mL forms, excess water is supplied.
2. method according to claim 1, it is characterized in that, the egg liquid stirring of the wheat bran liquid that described substratum is 2% by 65mL/100mL mass percent, the peptone of 1g/100mL, the glucose of the yeast powder of 1g/100mL, 3g/100mL, the rice of 35g/100mL and 30mL/100mL forms, and excess water is supplied.
3. method according to claim 1, is characterized in that, the temperature of described mycelium culture is 17-19 ℃.
4. method according to claim 1, is characterized in that, the relative humidity of described mycelium culture is 80-85%.
5. method according to claim 1, is characterized in that, the conidial temperature of described induction is 12-14 ℃.
6. method according to claim 1, is characterized in that, the conidial relative humidity of described induction is 80-85%.
7. method according to claim 1, is characterized in that, the time of described mycelium culture is 40-50 days.
8. method according to claim 1, is characterized in that, conidial time of described induction is 25-35 days.
9. method according to claim 1, is characterized in that, the wheat bran liquid that described mass percent is 1-5% is prepared by following methods:
In every 100mL water, add 1-5g wheat bran, boil the filtrate of filtering gained after 30min and be the wheat bran liquid that mass percent is 1-5%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998404A (en) * | 2018-09-13 | 2018-12-14 | 安徽农业大学 | A kind of conidial method of induction anthrax-bacilus generation |
| 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 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998404A (en) * | 2018-09-13 | 2018-12-14 | 安徽农业大学 | A kind of conidial method of induction anthrax-bacilus generation |
| CN108998404B (en) * | 2018-09-13 | 2022-02-25 | 安徽农业大学 | Method for inducing anthrax bacteria to produce conidia |
| 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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| CN103667170B (en) | 2015-10-14 |
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