CN103782794B - Method for stimulating cordyceps sinensis to generate conidia - Google Patents
Method for stimulating cordyceps sinensis to generate conidia Download PDFInfo
- Publication number
- CN103782794B CN103782794B CN201210433385.1A CN201210433385A CN103782794B CN 103782794 B CN103782794 B CN 103782794B CN 201210433385 A CN201210433385 A CN 201210433385A CN 103782794 B CN103782794 B CN 103782794B
- Authority
- CN
- China
- Prior art keywords
- temperature
- aweto
- mycelium
- eutrophy
- mycelial growth
- 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.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Mushroom Cultivation (AREA)
Abstract
The invention discloses a method for stimulating cordyceps sinensis to generate conidia. The method includes the step of cultivating the cordyceps sinensis under nutritive and environmental stimulation conditions. The method specifically includes: 1), cultivating the cordyceps sinensis on eutrophic solid media under dark conditions to acquire mycelia; 2), transferring the mycelia in the step 1) onto secondary-nutrient solid media to perform at least one type of stimulating cultivation, including chilling cultivation, heating cultivation, temperature-period stimulating cultivation, photoperiod stimulating cultivation, temperature-period and photoperiod stimulating cultivation and oxidation stimulating cultivation, to acquire the conidia. The nitrogen source content of the eutrophic solid media is higher than that of the secondary-nutrient solid media; the colony growth velocity of the cordyceps sinensis in the eutrophic solid media is higher than that of the cordyceps sinensis in the secondary-nutrient solid media.
Description
Technical field
The present invention relates to a kind of conidiiferous method of promotion aweto.
Background technology
Aweto ［ Ophiocordyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones & Spatafora (≡ Cordyceps sinensis (Berk.) Sacc.) ］ be the distinctive famous and precious medicinal fungus in Qinghai-Tibet Platean, modern study shows that Cordyceps sinensis has multiple pharmacological effect, has good application prospect.Although done a large amount of research work to Cordyceps sinensis, in artificial cultivation, also there is obvious technical barrier so far.Address these problems the biological character needing more fully to grasp aweto.Therefore, the innovation of aweto biological study technology, has important theory and practice meaning.
In aweto culture studies, the preparation of conidial research and microbial inoculum is closely related.At present, the main difficulty in the research of aweto conidium is that conidium output is few, the cycle is long, make artificial cultivation obtain conidium, and the preparation of conidiospore suspension is never resolved well.How obtaining a large amount of conidium by cultivating, becoming the bottleneck of aweto biological study gradually.
Summary of the invention
An object of the present invention is to provide a kind of promotion aweto and produce conidial method.
Promotion aweto provided by the present invention produces conidial method, comprises the step of being carried out by aweto cultivating under the condition of nutritive stimulus and environmental stimulus; Described step comprises:
1) aweto is cultivated on eutrophy solid culture medium under dark condition, obtain mycelium;
2) mycelium of described step 1) is transferred on secondary nutritive solid medium and carry out following at least one stimulation cultivation, obtain conidium: cold shock cultivation, heat shock cultivation, temperature cycle stimulate cultivation, photoperiod stimulation cultivation, the stimulation of temperature photoperiod to cultivate and be oxidized stimulation cultivation; The nitrogenous source content of described eutrophy solid culture medium is higher than described nutritive solid medium, and the colony growth speed of described aweto in eutrophy solid culture medium is greater than the colony growth speed of described aweto in secondary nutritive solid medium;
It is described mycelium is first placed in described mycelium can survive but be unfavorable for that the low temperature dark culturing grown is placed in the temperature dark culturing of suitable described mycelial growth again to producing conidium that described cold shock is cultivated;
It is described mycelium is first placed in described mycelium can survive but be unfavorable for that the high temperature dark culturing grown is placed in the temperature dark culturing of described suitable described mycelial growth again to producing conidium that described heat shock is cultivated;
Described temperature cycle stimulates that to cultivate be described mycelium is placed in environment dark culturing that the temperature of described suitable described mycelial growth and described high temperature replaces to producing conidium, or described mycelium is placed in environment dark culturing that the temperature of described suitable described mycelial growth and described low temperature replaces to producing conidium;
The described photoperiod stimulates cultivation to be that the temperature described mycelium being placed in described suitable described mycelial growth is cultured to generation conidium under existing illumination every day has again dark condition;
Stimulate described temperature photoperiod that to cultivate be described mycelium is placed in environment that the temperature of described suitable described mycelial growth and described high temperature replaces to be cultured under existing illumination every day has again dark condition and to produce conidium, or described mycelium is placed in environment that the temperature of described suitable described mycelial growth and low temperature replaces and is cultured under existing illumination every day has again dark condition and produces conidium;
Described oxidation stimulate cultivate be described mycelium is first placed in the environment with oxidative stress cultivate be placed in described suitable described mycelial growth again temperature dark culturing to producing conidium.
In the present invention, described nutritive stimulus refers to carries out Mycelium culture by mycelium at described eutrophy solid culture medium, carries out product spore and cultivate in described nutritive solid medium.
The suitable growth temperature of described aweto can be 10 – 23 DEG C, as 15 – 18 DEG C;
Described low temperature is preference temperature lower than described mycelial growth and described mycelium can be survived but is unfavorable for that the temperature ， grown is as – 40 – 4 DEG C; Described high temperature is preference temperature higher than described mycelial growth and described mycelium can be survived but is unfavorable for the temperature that grows, as 24 – 30 DEG C.Described low temperature concrete Ke Wei – 20-4 DEG C, described high temperature specifically can be 28 – 30 DEG C.
In said method, described cold shock is cultivated can be and described mycelium is first placed in described low temperature dark culturing within 1 day, is placed in the temperature dark culturing of described suitable described mycelial growth again to producing conidium;
Described heat shock is cultivated can be and described mycelium is first placed in described high temperature dark culturing within 1 hour, is placed in the temperature dark culturing of suitable described mycelial growth again to producing conidium;
Described temperature cycle stimulates in cultivation, and the temperature of described suitable described mycelial growth and described high temperature are alternately the temperature in odd number sky is the described temperature being suitable for described mycelial growth, and the temperature in even number sky is described high temperature; Or the temperature in even number sky is the temperature of described suitable described mycelial growth, the temperature in odd number sky is described high temperature;
Described temperature cycle stimulates in cultivation, and the temperature of described suitable described mycelial growth and described low temperature are alternately the temperature in odd number sky is the described temperature being suitable for described mycelial growth, and the temperature in even number sky is described low temperature; Or the temperature in even number sky is the temperature of described suitable described mycelial growth, the temperature in odd number sky is described low temperature;
During described temperature photoperiod stimulates and cultivates, the temperature of the temperature of described suitable described mycelial growth and described high temperature to be alternately the temperature under illumination condition every day be described suitable described mycelial growth, the temperature under every day dark condition is described high temperature;
During described temperature photoperiod stimulates and cultivates, the temperature of the temperature of described suitable described mycelial growth and described low temperature to be alternately the temperature under illumination condition every day be described suitable described mycelial growth, the temperature under every day dark condition is described low temperature;
The described photoperiod stimulates cultivation and described temperature photoperiod to stimulate in cultivation, and existing illumination described every day has again dark condition to can be h light 8 h dark every day 16;
Described oxidation stimulates to cultivate can be and described mycelium is first placed in 20 – 25mM hydrogen peroxide dark culturing and within 5 minutes, is placed in the temperature dark culturing of described suitable described mycelial growth again to producing conidium.
In said method, the cultivation temperature in described step 1) is the temperature of described suitable described mycelial growth.
In said method, the intensity of described illumination can be 4800lux.
In said method, described cold shock cultivation, heat shock are cultivated and oxidation stimulates in cultivation, and the described temperature dark culturing being placed in suitable described mycelial growth more specifically can be 14 days to producing conidial time.Described temperature cycle stimulates cultivation, the described photoperiod stimulates cultivation and the described temperature photoperiod stimulates the incubation time cultivated to be 14 days.
In said method, 1 liter of described eutrophy solid culture medium specifically can be made up of following material: compound nitrogen source 10.0 – 50.0 grams, 200.0 grams, potato, monose or 20.0 grams, disaccharide, yeast extract 1.0 – 2.0 grams, peptone 1.0 – 5.0 grams, dried silkworm chrysalis meal 10.0 – 25.0 grams, agar and water; Described compound nitrogen source is wheat bran, milk powder, dregs of beans or corn flour; Described monose or disaccharide are glucose, sucrose, maltose or lactose.Wherein, as long as the quality of described agar reaches the amount forming solid culture medium, described water is used for culture volume to be settled to 1 liter.
1 liter of described nutritive solid medium specifically can be made up of following material: 200.0 grams, potato, monose or 20.0 grams, disaccharide, agar and water; Described monose or disaccharide are glucose, sucrose, maltose or lactose.Wherein, as long as the quality of described agar reaches the amount forming solid culture medium, described water is used for culture volume to be settled to 1 liter.
Present invention also offers and only promote the conidiiferous method of aweto by nutritive stimulus.
The conidiiferous method of this promotion aweto, comprises the steps:
1) aweto is cultivated on eutrophy solid culture medium under dark condition, obtain mycelium;
2) mycelium of described step 1) to be transferred on time nutritive solid medium dark culturing to producing conidium;
The nutritive element content of described eutrophy solid culture medium is higher than described nutritive solid medium, and the colony growth speed of described aweto in eutrophy solid culture medium is 1.14-1.89 times of the colony growth speed of described aweto in secondary nutritive solid medium.
Described step 1) and step 2) in cultivation temperature all can be the temperature of above-mentioned suitable described mycelial growth, as 10-23 DEG C or 15-18 DEG C.
In above-mentioned all methods, described aweto specifically can be aweto 1621 and Cordyceps sinensis 762; Described aweto 1621 is numbered CGMCC No.6445 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Cordyceps sinensis 762 is numbered CGMCC No.2793 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Described colony growth speed specifically can be the colony growth speed of described aweto in 15 DEG C of dark culturing 70 days.The colony growth speed of described aweto in eutrophy solid culture medium can be the 1.14-1.89 of the colony growth speed of described aweto in secondary nutritive solid medium doubly, as 1.66-1.84 times, 1.66 times or 1.84 times.
Aweto (Ophiocordyceps sinensis) 1621 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on September 4th, 2012.
In above-mentioned all methods, for the ease of transfer and the collection conidium of described aweto, described eutrophy solid culture medium and described nutritive solid media surface all can be covered with conidial semipermeable membrane that can retain described aweto, described aweto is inoculated on described semipermeable membrane, and described semipermeable membrane can retain the conidium of described aweto through component in medium.
Method of the present invention cultivates aweto by nutritive stimulus, or promotes that aweto produces conidium by the double stimuli cultivation of nutritive stimulus and environmental stimulus.Experiment of the present invention proves, adopt method provided by the invention to prepare conidium, its 1621 productive rate is every 45 – 50mg(dry weights) bacterial classification obtains being not less than 2.78 × 10
6individual conidium.Product spore method of the present invention is simple to operate, time saving and energy saving, and very low to the requirement of equipment, reagent, general laboratory condition can meet the demands; On the other hand, aweto conidium and aweto have substantial connection to infecting of Hepialus larva, and obtaining a large amount of conidium will promote the research of the preparation of aweto physiology, Cordyceps sinensis bacteria agent and the semi-artificial cultivation of Cordyceps sinensis.
Preservation explanation
Strain name: aweto
Latin name: Ophiocordyceps sinensis
Strain number: 1621
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on September 4th, 2012
Register on the books numbering in preservation center: CGMCC No.6445
Embodiment
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
All identical for the preparation of the culture dish of solid culture medium flat board in following embodiment, diameter is 9.0cm.
Medium in following embodiment is as follows:
Every 1L eutrophy solid culture medium is made up of following material: 50.0g wheat bran, 200.0g potato, 20.0g glucose, 5.0g fish peptone, 1.0g yeast extract, 25.0g dried silkworm chrysalis meal, 20.0g agar and water.Wherein, with water, medium is settled to 1000mL.
Every 1L nutritive solid medium is made up of following material: 200.0g potato, 20.0g glucose, 20.0g agar and water.Wherein, with water, medium is settled to 1000mL.
Every 1L water Solid agar culture is made up of following material: 20.0g agar and water.Wherein, with water, medium is settled to 1000mL, 121 DEG C of moist heat sterilizations obtain medium half an hour.
Every 1L liquid spawn culture medium is made up of following material: 50.0g wheat bran, 200.0g potato, 20.0g glucose, 5.0g fish peptone, and 1.0g yeast extract, is settled to 1000mL with water.
Every 1L solid spawn medium is made up of following material: 50.0g wheat bran, 200.0g potato, 20.0g glucose, 5.0g fish peptone, and 1.0g yeast extract, 20.0g agar, is settled to 1000mL with water.
Above-mentioned medium pH naturally.
The compound method of above-mentioned eutrophy solid culture medium is as follows: wheat bran, dried silkworm chrysalis meal are boiled 10 minutes, add the peeled potatoes be cut into small pieces and boil half an hour again, then filtered through gauze is used, in filtrate, other compositions are added by above-mentioned formula, be settled to 1000mL with water, 121 DEG C of moist heat sterilizations obtain medium half an hour.
The compound method of secondary nutrient medium is as follows: the peeled potatoes be cut into small pieces is boiled 20 minutes, then uses filtered through gauze, in filtrate, adds other compositions, be settled to 1000mL with water by above-mentioned formula, and 121 DEG C of moist heat sterilizations obtain medium half an hour.
The compound method of liquid spawn culture medium and solid spawn medium is as follows: wheat bran is boiled 10 minutes, add the peeled potatoes be cut into small pieces and boil half an hour again, then filtered through gauze is used, in filtrate, other compositions are added by above-mentioned formula, be settled to 1000mL with water, 121 DEG C of moist heat sterilizations obtain medium half an hour.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Aweto strain liquid in following embodiment is prepared as follows: aweto is inoculated in above-mentioned solid spawn medium, 15 DEG C of dark culturing 90 days, obtains aweto bacterial classification.Dull and stereotyped with aseptic washing aweto bacterial classification, by wash the bacteria suspension that gets off by volume 20% inoculum concentration be inoculated in aforesaid liquid bacterium culture medium, bottling amount is 1/5 of triangular flask volume, then at 18 DEG C, rotating speed 100rpm, carry out shaken cultivation under 24 h dark conditions 14 days, obtain aweto first strain liquid; The inoculum concentration of 10% by volume, is inoculated in new liquid spawn culture medium by Cordyceps sinensis first strain liquid, and other condition of culture is same, obtains aweto second strain liquid.
In following embodiment, conidial elution process is as follows: be transferred to by the mycelium on glassine paper in the triangular flask containing 10 beades, add 0.1% Tween 80 solution, rotating speed 200rpm, thermal agitation 1 hour wash-out conidium, repeat wash-out three times, merge eluent.By eluent successively by 150 orders, 300 orders and 500 order cells sieve, the eluent of the hyphal body that is removed, centrifugal 10 minutes of 10000rpm, obtains conidium precipitation, and with 0.1% Tween 80 Eddy diffusion conidium, blood counting chamber counts.
In following embodiment, blood counting chamber counting method is as follows: be placed in by conidiospore suspension on blood counting chamber, basis of microscopic observation, counts under 400 times of multiplication factors, adds up mitogenetic spore count amount, averages.
In order to understand the present invention better, explain the present invention below for aweto 1621 and Cordyceps sinensis 762, these embodiments are used for understanding instead of restriction the present invention.
Wherein, aweto 1621 Shi Yaoyijian seminar collects in August, 2008 from large Wuxiang, Forage Land In Guoluo Prefecture Maqin County, Qinghai Province, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on September 4th, 2012, it is numbered CGMCC No.6445 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
The biological property of aweto 1621 is as follows:
1, morphological characteristic: on solid spawn medium, bacterium colony is snowy white, and bacterium colony surface is not protruding, has the tenderly white fine and close fine hair shape mycelium of one deck, has gauffer or do not have gauffer.There is dispersion shape fine hair mycelia at edge, and the medium back side is obvious sepia.Its mycelium of microexamination is colourless, has barrier film, branch, wide 2.2 – 5.4 μm (wide 2.2 – 4.3 μm of most of mycelia).Conidiophore is doleiform, colourless, and single life or 2 – 8 cluster, but do not form coremium.Conidium is colourless, and without barrier film, kidney shape or oblong, 5.4 – 14.0 × 3.2 – 5.4 μm (majority is 5.4 – 14.0 × 3.2 – 4.3 μm), single raw or 2 –, 6 a groups (majority is 2 –, 4 a groups), by a lemon shape mucous membrane is surrounded.The mycelia and the conidium that are in growth decline phase gradually become dark brown or black.
2, molecular biological characteristic: according to document ［ Jiang, Y. & Yao, Y.-J.(2006) ITS sequence analysisand ascomatal development of Pseudogymnoascus roseus.Mycotaxon.94:55 – 73. ］ method, strain culture extracts through DNA, pcr amplification nrDNA ITS (ITS) checking order, its ITS sequence is as shown in the sequence 1 in sequence table, and this sequence is consistent with the sequence of Cordyceps sinensis 762.
Cordyceps sinensis 762 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on December 5th, 2008, it is numbered CGMCC No.2793 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center, and this bacterial strain has been published in that China Patent Publication No. is 101760038A, application number is in the Chinese patent application of 200810241008.1.
Embodiment 1, the aweto growing state under nutritive stimulus
Bacterial classification: aweto 1621 and Cordyceps sinensis 762
Aweto is inoculated in solid spawn medium, 15 DEG C of dark culturing 90 days, obtains aweto bacterial classification.Aweto bacterial classification is inoculated in respectively eutrophy solid culture medium and within 70 days, measures colony diameter by right-angled intersection method with time nutritive solid medium 15 DEG C of dark culturing, calculate the average growth rate of bacterium colony.Three repetitions are established in experiment, repeat middle mensuration 10 bacterium colonies at every turn.Result shows that the average growth rate of aweto 1621 in eutrophy solid culture medium is 0.46 millimeter/day, the average growth rate of aweto 1621 in secondary nutritive solid medium is 0.25 millimeter/day, and the average growth rate of aweto 1621 in eutrophy solid culture medium is 1.84 times in time nutritive solid medium; The average growth rate of Cordyceps sinensis 762 in eutrophy solid culture medium is 0.48 millimeter/day, the average growth rate of Cordyceps sinensis 762 in secondary nutritive solid medium is 0.29 millimeter/day, and the average growth rate of Cordyceps sinensis 762 in eutrophy solid culture medium is 1.66 times in time nutritive solid medium.Above-mentioned 1621,762 all produce a large amount of melanin in secondary nutritive solid medium, and strain aging is obvious, and in eutrophy solid culture medium, spawn activity is very strong, cultivates and not yet produces melanin in 70 days.
Embodiment 2, prepare the conidium of aweto
Bacterial classification: aweto 1621 and Cordyceps sinensis 762
Each bacterial classification establishes 9 process, is respectively eutrophy and cultivates contrast, PDA nutritive stimulus process, water agar nutritive stimulus process, thermostimulation process, cold stimulation process, temperature cycle stimulation process, photoperiod stimulation process, temperature photoperiod stimulation process and oxidation stimulation process.
1, eutrophy cultivates contrast
Aweto second strain liquid is inoculated into top to be covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothens with glass spreader, dark quiescent culture 34 days at being placed in 15 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 8.42 × 10
5individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 1.17 × 10
4individual conidium.
2, PDA nutritive stimulus process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, dark quiescent culture 14 days at being placed in 15 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 2.78 × 10
6individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 1.58 × 10
4individual conidium.
3, water agar nutritive stimulus process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on water Solid agar culture flat board, eutrophy solid culture medium flat board switching water Solid agar culture is dull and stereotyped, dark quiescent culture 14 days at being placed in 15 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 4.7 × 10
5individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, then it is dull and stereotyped to be forwarded to water Solid agar culture, and the conidium of generation is considerably less, can not count.
4, thermostimulation process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, darkly at being placed in 28 DEG C to leave standstill after 1 hour, darker quiescent culture 14 days at being placed in 15 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 6.55 × 10
6individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 4.08 × 104 conidiums.
5, cold stimulation process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, darkly at being placed in-20 DEG C to leave standstill after 1 day, darker quiescent culture 14 days at being placed in 15 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 7.19 × 106 conidiums; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 2.0 × 104 conidiums.
6, temperature cycle stimulation process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, be placed in the dark quiescent culture the 14 days: 1st of following temperature cycle, 3, 5, 7, 9, 11, 13 days preference temperatures, 2nd, 4, 6, 8, 10, 12, 14 days low temperature, preference temperature is 15 DEG C, the temperature of low temperature is 4 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 4.12 × 10
6individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 0.73 × 10
4individual conidium.
7, photoperiod stimulation process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, be placed in 15 DEG C of quiescent culture 14 days under following periodicity of illumination: every day 16 h light 8 h dark, wherein the intensity of illumination is 4800lux.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 4.25 × 10
6individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 1.87 × 10
4individual conidium.
8, temperature photoperiod stimulation process
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, to be placed under the following temperature light cycle quiescent culture 14 days: every day 16 h light 8 h dark, temperature during illumination is 15 DEG C, the intensity of illumination is 4800lux, temperature time dark is 4 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 5.04 × 10
6individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of the mycelium (dry weight) of every 50mg obtains 1.98 × 104 conidiums.
9, stimulation process is oxidized
Aweto second strain liquid being inoculated into top is covered with on the eutrophy solid culture medium flat board of sterilizing glassine paper (river, Beijing grand biotechnology Co., Ltd in morning), smoothen with glass spreader, dark quiescent culture 20 days at being placed in 15 DEG C, mycelium covers with glassine paper, the glassine paper covering with bacterium colony is transferred on time nutritive solid culture medium flat plate, eutrophy solid culture medium flat board switching one nutritive solid culture medium flat plate, aseptic 20mM hydrogen peroxide is added in secondary nutritive solid culture medium flat plate, immersion covered with the glassine paper of bacterium colony after 5 minutes, hydrogen peroxide sucking-off is dried up under sterile wind, make that flat board does not have hydrogen peroxide residue, dark quiescent culture 14 days at being placed in 15 DEG C.On wash-out glassine paper, conidium carries out blood counting chamber counting.Three repetitions are established in experiment, in repeating, respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for aweto 1621 at every turn, and respectively inoculation 10 eutrophy solid culture mediums are dull and stereotyped for Cordyceps sinensis 762, these 20 dull and stereotyped inoculum concentrations are all identical, are mycelium (the dry weight)/flat board of 50mg.Result shows that aweto 1621 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 5.96 × 10
6individual conidium; Result shows that Cordyceps sinensis 762 is dull and stereotyped with the inoculum concentration of the mycelium of 50mg (dry weight)/flat board access eutrophy solid culture medium, and the bacterial classification of every 50mg mycelium (dry weight) obtains 2.08 × 10
4individual conidium.
Claims (20)
1. promote aweto conidiiferous method, comprise the step of being carried out by aweto cultivating under the condition of nutritive stimulus and environmental stimulus; Described step comprises:
1) aweto is cultivated on eutrophy solid culture medium under dark condition, obtain mycelium;
2) by described step 1) mycelium transfer to and time nutritive solid medium carry out following at least one and stimulate and cultivate, obtain conidium: cold shock cultivation, heat shock cultivation, temperature cycle stimulate cultivate, the photoperiod stimulate cultivate, the temperature photoperiod stimulates and to cultivate and oxidation stimulates and cultivates; The nitrogenous source content of described eutrophy solid culture medium is higher than described nutritive solid medium;
It is described mycelium is first placed in described mycelium can survive but be unfavorable for the low temperature dark culturing that grows that described cold shock is cultivated, then the temperature dark culturing being placed in suitable described mycelial growth is to producing conidium;
It is described mycelium is first placed in described mycelium can survive but be unfavorable for the high temperature dark culturing that grows that described heat shock is cultivated, then the temperature dark culturing being placed in described suitable described mycelial growth is to producing conidium;
Described temperature cycle stimulates that to cultivate be described mycelium is placed in environment dark culturing that the temperature of described suitable described mycelial growth and described high temperature replaces to producing conidium, or described mycelium is placed in environment dark culturing that the temperature of described suitable described mycelial growth and described low temperature replaces to producing conidium;
The described photoperiod stimulates cultivation to be that the temperature described mycelium being placed in described suitable described mycelial growth is cultured to generation conidium under existing illumination every day has again dark condition;
Stimulate described temperature photoperiod that to cultivate be described mycelium is placed in environment that the temperature of described suitable described mycelial growth and described high temperature replaces to be cultured under existing illumination every day has again dark condition and to produce conidium, or described mycelium is placed in environment that the temperature of described suitable described mycelial growth and low temperature replaces and is cultured under existing illumination every day has again dark condition and produces conidium;
Described oxidation stimulate cultivate be described mycelium is first placed in the environment with oxidative stress cultivate be placed in described suitable described mycelial growth again temperature dark culturing to producing conidium.
2. the conidiiferous method of promotion aweto according to claim 1, is characterized in that: described low temperature is preference temperature lower than described mycelial growth and described mycelium can be survived but is unfavorable for the temperature that grows; Described high temperature is preference temperature higher than described mycelial growth and described mycelium can be survived but is unfavorable for the temperature that grows.
3. the conidiiferous method of promotion aweto according to claim 2, is characterized in that: the temperature of described suitable described mycelial growth is 10 – 23 DEG C; Described low temperature Wei – 40 – 4 DEG C, described high temperature is 24 – 30 DEG C.
4. the conidiiferous method of promotion aweto according to claim 3, is characterized in that: the temperature of described suitable described mycelial growth is 15 – 18 DEG C; Described low temperature Wei – 20 – 4 DEG C, described high temperature is 28 – 30 DEG C.
5., according to the described conidiiferous method of promotion aweto arbitrary in claim 1-4, it is characterized in that:
It is described mycelium is first placed in described low temperature dark culturing within 1 day, to be placed in the temperature dark culturing of described suitable described mycelial growth again to producing conidium that described cold shock is cultivated;
It is described mycelium is first placed in described high temperature dark culturing within 1 hour, to be placed in the temperature dark culturing of described suitable described mycelial growth again to producing conidium that described heat shock is cultivated;
Described temperature cycle stimulates in cultivation, and the temperature of described suitable described mycelial growth and described high temperature are alternately the temperature in odd number sky is the described temperature being suitable for described mycelial growth, and the temperature in even number sky is described high temperature; Or the temperature in even number sky is the temperature of described suitable described mycelial growth, the temperature in odd number sky is described high temperature;
Described temperature cycle stimulates in cultivation, and the temperature of described suitable described mycelial growth and described low temperature are alternately the temperature in odd number sky is the described temperature being suitable for described mycelial growth, and the temperature in even number sky is described low temperature; Or the temperature in even number sky is the temperature of described suitable described mycelial growth, the temperature in odd number sky is described low temperature;
During described temperature photoperiod stimulates and cultivates, the temperature of the temperature of described suitable described mycelial growth and described high temperature to be alternately the temperature under illumination condition every day be described suitable described mycelial growth, the temperature under every day dark condition is described high temperature;
During described temperature photoperiod stimulates and cultivates, the temperature of the temperature of described suitable described mycelial growth and described low temperature to be alternately the temperature under illumination condition every day be described suitable described mycelial growth, the temperature under every day dark condition is described low temperature;
The described photoperiod stimulates cultivation and described temperature photoperiod to stimulate in cultivation, and existing illumination described every day has again dark condition to be h light 8 h dark every day 16;
Described oxidation stimulates cultivation to be described mycelium is first placed in 20 – 25mM hydrogen peroxide dark culturing within 5 minutes, to be placed in the temperature dark culturing of described suitable described mycelial growth again to producing conidium.
6., according to the described conidiiferous method of promotion aweto arbitrary in claim 1-4, it is characterized in that: described step 1) in cultivation temperature be the temperature of described suitable described mycelial growth; The intensity of described illumination is 4800lux.
7., according to the described conidiiferous method of promotion aweto arbitrary in claim 1-4, it is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.14 – 1.89 times of the colony growth speed of described aweto in secondary nutritive solid medium.
8. the conidiiferous method of promotion aweto according to claim 7, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.66 – 1.84 times of the colony growth speed of described aweto in secondary nutritive solid medium.
9. the conidiiferous method of promotion aweto according to claim 8, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.66 times of the colony growth speed of described aweto in secondary nutritive solid medium.
10. the conidiiferous method of promotion aweto according to claim 8, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.84 times of the colony growth speed of described aweto in secondary nutritive solid medium.
11., according to the described conidiiferous method of promotion aweto arbitrary in claim 1-4, is characterized in that: described aweto is aweto 1621 and Cordyceps sinensis 762; Described aweto 1621 is numbered CGMCC No.6445 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Cordyceps sinensis 762 is numbered CGMCC No.2793 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
12. promote the conidiiferous method of aweto, comprise the steps:
1) aweto is cultivated on eutrophy solid culture medium under dark condition, obtain mycelium;
2) by described step 1) mycelium to transfer on time nutritive solid medium dark culturing to producing conidium;
The nitrogenous source content of described eutrophy solid culture medium is higher than described nutritive solid medium, and the colony growth speed of described aweto in eutrophy solid culture medium is greater than the colony growth speed of described aweto in secondary nutritive solid medium.
The conidiiferous method of 13. promotion aweto according to claim 12, is characterized in that: described step 1) and step 2) in cultivation temperature be the temperature of suitable described mycelial growth.
The conidiiferous method of 14. promotion aweto according to claim 13, is characterized in that: described step 1) and step 2) in cultivation temperature be 10 – 23 DEG C.
The conidiiferous method of 15. promotion aweto according to claim 13, is characterized in that: described step 1) and step 2) in cultivation temperature be 15 – 18 DEG C.
16., according to the described conidiiferous method of promotion aweto arbitrary in claim 12-15, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.14 – 1.89 times of the colony growth speed of described aweto in secondary nutritive solid medium.
The conidiiferous method of 17. promotion aweto according to claim 16, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.66 – 1.84 times of the colony growth speed of described aweto in secondary nutritive solid medium.
The conidiiferous method of 18. promotion aweto according to claim 17, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.66 times of the colony growth speed of described aweto in secondary nutritive solid medium.
The conidiiferous method of 19. promotion aweto according to claim 17, is characterized in that: the colony growth speed of described aweto in eutrophy solid culture medium is 1.84 times of the colony growth speed of described aweto in secondary nutritive solid medium.
20., according to the described conidiiferous method of promotion aweto arbitrary in claim 12-15, is characterized in that: described aweto is aweto 1621 and Cordyceps sinensis 762; Described aweto 1621 is numbered CGMCCNo.6445 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center; Described Cordyceps sinensis 762 is numbered CGMCC No.2793 in registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210433385.1A CN103782794B (en) | 2012-11-02 | 2012-11-02 | Method for stimulating cordyceps sinensis to generate conidia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210433385.1A CN103782794B (en) | 2012-11-02 | 2012-11-02 | Method for stimulating cordyceps sinensis to generate conidia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103782794A CN103782794A (en) | 2014-05-14 |
| CN103782794B true CN103782794B (en) | 2015-06-10 |
Family
ID=50659183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210433385.1A Expired - Fee Related CN103782794B (en) | 2012-11-02 | 2012-11-02 | Method for stimulating cordyceps sinensis to generate conidia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103782794B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695390B (en) * | 2016-03-31 | 2017-12-05 | 广东东阳光药业有限公司 | A kind of bacteria residue culture promotees the conidiiferous method of Hirsutella sinensis |
| CN109328944A (en) * | 2018-12-06 | 2019-02-15 | 吉首大学 | The breeding of Yi Zhong Zingiber mioga covers cultivating method with Cortex Eucommiae |
| CN110923151A (en) * | 2019-12-19 | 2020-03-27 | 广东省微生物研究所（广东省微生物分析检测中心） | Cordyceps sinensis anamorph solid culture medium and preparation method thereof |
| CN112616557A (en) * | 2020-12-30 | 2021-04-09 | 东莞市东阳光冬虫夏草研发有限公司 | Culture method and application of high-infectivity cordyceps sinensis strain |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85107991A (en) * | 1985-11-01 | 1987-05-06 | 北京市营养源研究所 | The industrial process of Cordyceps mycelium |
| CN1082310A (en) * | 1992-08-06 | 1994-02-23 | 陈顺志 | The new technology of artificially culturing north Chinese caterpillar fungus |
| KR100239211B1 (en) * | 1997-12-01 | 2000-01-15 | 대한민국 | A cultivation of cordyceps militaris using silkworms |
-
2012
- 2012-11-02 CN CN201210433385.1A patent/CN103782794B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103782794A (en) | 2014-05-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103477994B (en) | Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran | |
| CN103484421B (en) | A kind of chlamydosporic method of liquid fermenting scale up test Gliocladium roseum | |
| CN102119631B (en) | Grifola frondosa strain for producing polysaccharide with composite raw material of rice bran and wheat bran | |
| CN109355204A (en) | A kind of method of fermenting and producing cordyceps sinensis mycelium powder | |
| CN103782794B (en) | Method for stimulating cordyceps sinensis to generate conidia | |
| CN102154407A (en) | Corayceps militaris polysaccharide two-stage fermentation synthesis process | |
| US20210079339A1 (en) | Fermentation production process of paecilomyces hepialis cs-4 | |
| CN105886405A (en) | Dendrobium officinale Kimura et Migo endophytic fungus and applications thereof | |
| CN101195806A (en) | Industrial production method of cordyceps mushroom | |
| CN102283022A (en) | Method for isolating Cordyceps sinensis Sacc. asexual-stage spawn | |
| CN105132331A (en) | Komagataeibacter nataicola and application thereof | |
| CN102747016A (en) | Gellan gum efficient production strain and its application | |
| CN104046569B (en) | Aspergillus tubingensis for high-yield production of glucoamylase, alpha-amylase and acidic protease and application thereof | |
| CN103215311A (en) | Method for producing high-quality aquilaria sinensis material through aspergillus niger conversion | |
| CN101558766A (en) | Trichoderma solid granules for preventing and controlling tobacco soil-borne fungus diseases and preparation method thereof | |
| CN102816701A (en) | Strain used for fermenting rice bran and wheat bran extracts for producing grifolan | |
| CN1095103A (en) | Producing process of Chinese caterpillar fungus hypha fermentation | |
| CN101790937A (en) | Screening and culture preparing method of Russula.alutacea strain | |
| CN104250618B (en) | The aspergillus candidus of a kind of high-yield glucoamylase, alpha amylase and acid protease and its application | |
| CN102021212A (en) | Preparation method of ganoderma polysaccharide | |
| CN100412186C (en) | Tinder fungus and process for deep liquid fermentation preparation of tinder fungus | |
| CN103789212B (en) | One plant height produces spore Cordyceps strain | |
| CN110024623A (en) | L-PROLINE is improving the application in aweto blastopore quantity and hypha biomass | |
| CN103667170B (en) | One prepares the ripe conidial method of China pilose spore | |
| TW201139662A (en) | A formula of culturing medium for Cordyceps spp. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150610 Termination date: 20201102 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |