CN103250566A - Cordyceps militaris optimal tissue isolation phase screening method - Google Patents
Cordyceps militaris optimal tissue isolation phase screening method Download PDFInfo
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Abstract
The invention discloses a cordyceps militaris optimal tissue isolation phase screening method. Mother seed isolation is conducted on tissues of cordyceps militaris fruit bodies in five different growth and development phases, namely a primordium phase, a phase when the fruit bodies grow for 15 days, a phase when the fruit bodies grow for 30 days, a phase when the fruit bodies grow for 45 days, and a phase when the fruit bodies grow for 60 days so that optimal tissue isolation materials can be screened. According to the screening method, mother seed mycelium obtained through tissue isolation when cordyceps militaris grows to the primordium phase is the highest in growth speed, the best in growth trend, the most rapid in color transformation and the best in color transforming effect. The most appropriate phase for the master seed tissue isolation is the primordium phase of the cordyceps militaris. A reason is related to cell viability of the mycelium of the cordyceps militaris in different growth phases. The mycelium in the primordium phase is the lowest in cell differentiation degree and the highest in cell viability. The mother seeds obtained through the tissue isolation in the primordium phase are the best in viability and growth trend.
Description
Technical field
The present invention relates to a kind of northern Chinese caterpillar fungus, particularly a kind of northern Chinese caterpillar fungus the best is organized the separation period screening technique.
Background technology
Northern Chinese caterpillar Fungus claims northern Chinese caterpillar fungus, Cordyceps militaris again, is under the jurisdiction of Ascomycota, cup fungi subphylum, excrement shell Gammaproteobacteria, Hypocreales, Clavicipitaceae Cordyceps.Found Cordyceps sinensis fungus kind more than 400 at present in the world, China records 100 kinds.Chinese caterpillar fungus is the type sepecies of Cordyceps sinensis fungus in north, its host is mainly lepidopterous Bombycidae, Notodontidae, Saturniidae etc., it is by stroma (being careless part) and sclerotium (being the corpse part of worm or pupa) complex stroma dimerous single giving birth to or several to be arranged, go out from host's head or joint minister, be orange-yellow.Winter, larva lived in seclusion in the soil, and the mushroom parasitism wherein absorbs nutrition, was full of mycelia in its body at last and dead, and pupa worm corpse is excellent; To summer, on its corpse, bear " seedling " (fruit body), likeness in form grass, gather before and after the Summer Solstice and it mainly is distributed in ground such as Jilin, Liaoning, Shaanxi, Hebei, Liaoning, Hubei, Guangdong.
The north Chinese caterpillar fungus be Cordyceps sinensis belong to the xenogenesis fungi together, both chemical compositions and nutrient component and medicinal efficacy are all quite similar, cordycepin in the northern Chinese caterpillar Fungus, cordycepic acid, Cordyceps sinensis polysaccharide and protein content are apparently higher than natural cordyceps, its content is respectively 29 times, 27 times and 32 times of Cordyceps sinensis, and amino acid content is approaching substantially, edible and medical value can compare favourably with natural cordyceps, is the desirable substitute of natural cordyceps.Since the nineties in 20th century, medicineization, pharmacology, toxicity and toxicity as the northern Chinese caterpillar fungus of medicine and health product have been carried out a large amount of research and safety evaluation, proved that tame northern Chinese caterpillar fungus has no side effect, it has nourishing and fit keeping function, invigorate the lung and the kidney, calm, relieving asthma, anti-arrhythmia cordis, antifatigue, inhibition ulcer cell, treatment pulmonary tuberculosis, antifatigue, antitumor, suppress special efficacy such as cancer cell, and can strengthen body's immunity, can compare favourably with genseng, pilose antler, it is in great shortage to add resource, gather difficulty, the reputation that " national treasure " therefore, arranged.
Northern Chinese caterpillar Fungus and Cordyceps sinensis are two kinds of high Cordyceps Militaris of the widest medical value of the modal distribution of China, abroad also northern Chinese caterpillar Fungus are studied.The sixties in 20th century, Canada, the U.S., Germany etc. have been studied Chinese caterpillar fungus, and Japan has set up " northern Chinese caterpillar Fungus research association " in 1900 and exchanged mutually with the Chinese medicine expert, make the research of northern Chinese caterpillar fungus obtain development faster.Wild northern Chinese caterpillar Fungus in natural world quantity seldom, and is expensive.North Chinese caterpillar fungus artificial cultivation domestication is succeedd, can commercially produce, but employed bacterial classification obtains with the wild Chinese caterpillar fungus separation mostly in producing, the report that separates about northern Chinese caterpillar fungus tissue seldom, the research that different times tissue separates female kind of growing of northern Chinese caterpillar fungus does not then appear in the newspapers especially.
Summary of the invention
Purpose of the present invention just is to provide that a kind of long speed is the fastest, growing way is best, annesl is the fastest, the annesl effect preferably northern Chinese caterpillar fungus the best organize the separation period screening technique.
To achieve these goals, the technical solution used in the present invention is such: northern Chinese caterpillar fungus the best of the present invention is organized the separation period screening technique, select five different growth and development stages of Cordyceps militaris fruiting body for use, be that original hase, sporophore growth 15 days, sporophore growth 30 days, sporophore growth 45 days, 60 days tissue of sporophore growth carry out mother and plant and separate, in order to screen the optimum parting material of organizing, these screening technique concrete steps are as follows:
(1) chooses medium and preparation formula: potato 200g, agar 20g, glucose 20g, peptone 5g, aluzyme 5g, K
2HPO
40.5g, pH nature, water 1000ml, its compound method is: each component is good with electronic balance weighing in will filling a prescription earlier, then the potato that weighs up is cut into strip, put into boiling water and boiled 20-30 minute, during constantly stir with glass bar, in case potato is boiled Jiao; With well-done potato filtered through gauze twice, get filtrate again; Then filtrate is put into electromagnetic oven top heating edge agar is added wherein, and constantly stir with glass bar, at last other composition is added wherein successively; After treating that various components are fully dissolved, be settled to 1000ml, with the test tube packing of 180mm * 18mm, every test tube packing 8ml, add a cover tampon, and cover which floor newspaper, put sterilization in the high-pressure sterilizing pot, kept 30 minutes for 121 ℃, take out the pendulum inclined-plane then, cooling, stand-by behind the steriling test;
(2) tissue separates, north Chinese caterpillar fungus mother plants choosing and sampling of parting material: the precondition that parting material is chosen is not contaminated in the cultivation, choose the good Chinese caterpillar fungus that grows as organizing the material that separates the period of growing five differences respectively, concrete grammar is as follows, original hase period choose the culture bottle that original hase is more, be evenly distributed, fruit body period to choose the Chinese caterpillar fungus form good, the fruit body of robust growth, it is indoor stand-by that the culture bottle of choosing is put into sterile working; Tissue separates inoculation and cultivates: the medium test tube that will inoculate used tool before the inoculation and set the inclined-plane is put in the superclean bench, then the uviol lamp in transfer room and the superclean bench is opened, shone after 30 minutes the dark place reason 20 minutes, in superclean bench, use cotton ball soaked in alcohol wiping both hands and culture bottle appearance, then at the other film that seals of opening culture bottle of alcolhol burner flame, original hase directly is linked into medium central authorities with inoculation hoe picking one fritter, parting material for other periods, with one little section of sterile scissors clip cordyceps militaris sporocarp upper end, be linked on the medium with the inoculation hoe then, the Chinese caterpillar fungus in each period connects 10 respectively, place 23 ℃ of cultivations down in the constant incubator then, regularly observe every day, the record different times organize field planting sprouting and the mycelial growth situation of piece on medium;
(3) annesl, the mycelial test tube that covers with that cultivation is finished places temperature to be 20 ℃ to have the environment of scattered light irradiation to carry out annesl, and regularly observe every day, the annesl speed of record different times, the annesl rate of observing different times after seven days;
(4) (2), (3) step triplicate.
Compared with prior art, the invention has the advantages that: northern Chinese caterpillar fungus different times tissue separates the mycelial growth situation significant difference that obtains, and has obtained that comparatively desirable northern Chinese caterpillar fungus is female to be planted the separation period of organizing.North is organized and to be separated the mother who obtains to plant mycelial growth fastest in the former base period of Chinese caterpillar fungus, be 3.18mm/d, plant mycelial growth speed with other four mothers that period, tissue separated to obtain utmost point significant difference is arranged, the mycelium growing way that this period, separation obtained also obviously is better than separating the mothers that obtain other four periods and plants mycelium, mycelial growth is neat, pure white, dense, annesl is effective, is the separation period of organizing of northern Chinese caterpillar fungus the best.
Embodiment
The invention will be further described below in conjunction with accompanying drawing.
Embodiment:
Northern Chinese caterpillar fungus the best of the present invention is organized the separation period screening technique, select five different growth and development stages of Cordyceps militaris fruiting body for use, be that original hase, sporophore growth 15 days, sporophore growth 30 days, sporophore growth 45 days, 60 days tissue of sporophore growth carry out mother and plant and separate, in order to screen the optimum parting material of organizing, these screening technique concrete steps are as follows:
(1) chooses medium and preparation formula: potato 200g, agar 20g, glucose 20g, peptone 5g, aluzyme 5g, K
2HPO
40.5g, pH nature, water 1000ml, its compound method is: each component is good with electronic balance weighing in will filling a prescription earlier, then the potato that weighs up is cut into strip, put into boiling water and boiled 20-30 minute, during constantly stir with glass bar, in case potato is boiled Jiao; With well-done potato filtered through gauze twice, get filtrate again; Then filtrate is put into electromagnetic oven top heating edge agar is added wherein, and constantly stir with glass bar, at last other composition is added wherein successively; After treating that various components are fully dissolved, be settled to 1000ml, with the test tube packing of 180mm * 18mm, every test tube packing 8ml, add a cover tampon, and cover which floor newspaper, put sterilization in the high-pressure sterilizing pot, kept 30 minutes for 121 ℃, take out the pendulum inclined-plane then, cooling, stand-by behind the steriling test;
(2) tissue separates, north Chinese caterpillar fungus mother plants choosing and sampling of parting material: the precondition that parting material is chosen is not contaminated in the cultivation, choose the good Chinese caterpillar fungus that grows as organizing the material that separates the period of growing five differences respectively, concrete grammar is as follows, original hase period choose the culture bottle that original hase is more, be evenly distributed, fruit body period to choose the Chinese caterpillar fungus form good, the fruit body of robust growth, it is indoor stand-by that the culture bottle of choosing is put into sterile working; Tissue separates inoculation and cultivates: the medium test tube that will inoculate used tool before the inoculation and set the inclined-plane is put in the superclean bench, then the uviol lamp in transfer room and the superclean bench is opened, shone after 30 minutes the dark place reason 20 minutes, in superclean bench, use cotton ball soaked in alcohol wiping both hands and culture bottle appearance, then at the other film that seals of opening culture bottle of alcolhol burner flame, original hase directly is linked into medium central authorities with inoculation hoe picking one fritter, parting material for other periods, with one little section of sterile scissors clip cordyceps militaris sporocarp upper end, be linked on the medium with the inoculation hoe then, the Chinese caterpillar fungus in each period connects 10 respectively, place 23 ℃ of cultivations down in the constant incubator then, regularly observe every day, the record different times organize field planting sprouting and the mycelial growth situation of piece on medium;
(3) annesl, the mycelial test tube that covers with that cultivation is finished places temperature to be 20 ℃ to have the environment of scattered light irradiation to carry out annesl, and regularly observe every day, the annesl speed of record different times, the annesl rate of observing different times after seven days;
(4) (2), (3) step triplicate.
Draw following form by above-mentioned steps,
Table 1 different times tissue separates female mycelial growth situation and mm/d of The result of multiple comparisons unit of planting
In the last table, ++ ++ the expression mycelial growth is neat, pure white, dense; +++expression mycelial growth is uneven, pure white, closeer; ++ the expression mycelial growth uneven, in vain, more sparse; * represents that difference is extremely remarkable, and * represents significant difference.
As can be drawn from Table 1, tissue of former base period of northern Chinese caterpillar fungus separates the mother who obtains and plants the long speed of mycelium comparatively fast, and the mycelium growing way is good, neat, pure white, dense; The sporophore growth mother that tissue separates in the time of 60 days plants that the long speed of mycelium is slower, and growing way is relatively poor, different times tissue is separated female mycelium length speed of planting carry out one-way analysis of variance, draws F=20.045, table look-up F
4,10,0.01=5.994, F＞F
4,10,0.01So difference is extremely remarkable, it is remarkable that the mother that the different growth and development stage tissues separation of northern Chinese caterpillar fungus are described plants mycelial growth speed difference heteropole, carry out multiple ratio after as can be seen from Table 1: the mother that tissue of former base period of northern Chinese caterpillar fungus separates plants four mothers that period, tissue separated of mycelium and other and plants mycelium to grow fast difference extremely remarkable, and growing way is also best.It is also extremely remarkable with 60 days differences that the mother that sporophore growth 15 days tissue separates plants the long speed of mycelium, organized the mother who separates to plant the long fast significant difference of mycelium with 45 days, but with 30 days no significant differences of the long speed of the mycelium that separates of tissue, the mother of sporophore growth tissue separation in 30 days plants mycelium and plants the long fast significant difference of mycelium with the mother that growth obtained in 60 days, but organized the mother who separates to plant the no significant difference of the long speed of mycelium in 45 days with growth, it is the fastest that thereby the mother who draws former base period tissue separation plants the long speed of mycelium, growing way is best, organizes separation period for best mother plants.
The mother that table 2 tissue separates plants the mycelium annesl % of unit as a result
As can be seen from Table 2, annesl after 7 days the mycelia annesl effect in original hase period best, the mycelium annesl effect of 15 days and 30 days is taken second place, the mycelia annesl effect in 45 day period is relatively poor, and the mycelia annesl effect in 60 days (i.e. ripe period) is the poorest.Thereby the mycelia annesl that draws original hase period is fastest, the most suitablely does female the kind separation period of organizing.
To sum up can draw: northern Chinese caterpillar fungus grows to original hase tissue in period and separates the mother obtain to plant the long speed of mycelium the fastest, growing way is best, annesl is the fastest, the annesl effect is best, to the north of Chinese caterpillar fungus be best suited for doing female the kind period that tissue separates when growing to original hase, reason is relevant with the mycelial cell vigor of northern Chinese caterpillar fungus growth different times, the mycelial cell differentiation degree in original hase period is minimum, cell viability is the highest, it is best that the mother who organizes separation to obtain in this period plants vigor, growing way is also best, and the mycelial cell differentiation degree height of the fruit body of growth and maturity, cell is aging, cell viability descends, so that the mother who organizes separation to obtain with the fruit body in this period plants vigor is lower, regeneration capacity is very weak, so growth rate is slow, growing way is poor, and the annesl weak effect is not suitable for female kind of this tissue separation in period.
Claims (1)
1. northern Chinese caterpillar fungus the best is organized the separation period screening technique, it is characterized in that, select five different growth and development stages of Cordyceps militaris fruiting body for use, be that original hase, sporophore growth 15 days, sporophore growth 30 days, sporophore growth 45 days, 60 days tissue of sporophore growth carry out mother and plant and separate, in order to screen the optimum parting material of organizing, these screening technique concrete steps are as follows:
(1) chooses medium and preparation formula: potato 200g, agar 20g, glucose 20g, peptone 5g, aluzyme 5g, K
2HPO
40.5g, pH nature, water 1000ml, its compound method is: each component is good with electronic balance weighing in will filling a prescription earlier, then the potato that weighs up is cut into strip, put into boiling water and boiled 20-30 minute, during constantly stir with glass bar, in case potato is boiled Jiao; With well-done potato filtered through gauze twice, get filtrate again; Then filtrate is put into electromagnetic oven top heating edge agar is added wherein, and constantly stir with glass bar, at last other composition is added wherein successively; After treating that various components are fully dissolved, be settled to 1000ml, with the test tube packing of 180mm * 18mm, every test tube packing 8ml, add a cover tampon, and cover which floor newspaper, put sterilization in the high-pressure sterilizing pot, kept 30 minutes for 121 ℃, take out the pendulum inclined-plane then, cooling, stand-by behind the steriling test;
(2) tissue separates, north Chinese caterpillar fungus mother plants choosing and sampling of parting material: the precondition that parting material is chosen is not contaminated in the cultivation, choose the good Chinese caterpillar fungus that grows as organizing the material that separates the period of growing five differences respectively, concrete grammar is as follows, original hase period choose the culture bottle that original hase is more, be evenly distributed, fruit body period to choose the Chinese caterpillar fungus form good, the fruit body of robust growth, it is indoor stand-by that the culture bottle of choosing is put into sterile working; Tissue separates inoculation and cultivates: the medium test tube that will inoculate used tool before the inoculation and set the inclined-plane is put in the superclean bench, then the uviol lamp in transfer room and the superclean bench is opened, shone after 30 minutes the dark place reason 20 minutes, in superclean bench, use cotton ball soaked in alcohol wiping both hands and culture bottle appearance, then at the other film that seals of opening culture bottle of alcolhol burner flame, original hase directly is linked into medium central authorities with inoculation hoe picking one fritter, parting material for other periods, with one little section of sterile scissors clip cordyceps militaris sporocarp upper end, be linked on the medium with the inoculation hoe then, the Chinese caterpillar fungus in each period connects 10 respectively, place 23 ℃ of cultivations down in the constant incubator then, regularly observe every day, the record different times organize field planting sprouting and the mycelial growth situation of piece on medium;
(3) annesl, the mycelial test tube that covers with that cultivation is finished places temperature to be 20 ℃ to have the environment of scattered light irradiation to carry out annesl, and regularly observe every day, the annesl speed of record different times, the annesl rate of observing different times after seven days;
(4) (2), (3) step triplicate.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103548559A (en) * | 2013-10-18 | 2014-02-05 | 北京市农林科学院 | Strain isolation method capable of effectively obtaining effective inoculant of cordyceps militaris |
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2013
- 2013-05-20 CN CN2013101857686A patent/CN103250566A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20020087312A (en) * | 2001-05-15 | 2002-11-22 | 주식회사 엔에스케이텍 | A process for isolation of cordyceps sinensis |
| CN102138436B (en) * | 2010-12-28 | 2012-08-22 | 湖北圣峰药业有限公司 | Method for culturing selenium-enriched Cordyceps militaris |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103548559A (en) * | 2013-10-18 | 2014-02-05 | 北京市农林科学院 | Strain isolation method capable of effectively obtaining effective inoculant of cordyceps militaris |
| CN103548559B (en) * | 2013-10-18 | 2016-01-20 | 北京市农林科学院 | The strain separating method of the effective inoculum of a kind of effective acquisition Cordyceps militaris |
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