CN1262638C - Chinese caterpillar fungus and its separating method - Google Patents
Chinese caterpillar fungus and its separating method Download PDFInfo
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- CN1262638C CN1262638C CN 200410025774 CN200410025774A CN1262638C CN 1262638 C CN1262638 C CN 1262638C CN 200410025774 CN200410025774 CN 200410025774 CN 200410025774 A CN200410025774 A CN 200410025774A CN 1262638 C CN1262638 C CN 1262638C
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Abstract
The present invention discloses a Chinese caterpillar fungus and a separating method thereof. In the present invention, single cyst fungi are separated and filtered out of the fruiting bodies of wild Cordyceps Ophioglossoides (Ehrenb.) Link. The method for separating the caterpillar fungus in the present invention is split into steps as follows: the selection of the fruiting bodies of Cordyceps Ophioglossoides (Ehrenb.) Link, sterilization, inoculation, cultivation, etc.
Description
Technical field
The present invention relates to a kind of medicinal fungi, especially, relate to pure bacterial strain of a kind of Goldenthread Cordyceps (Cordycepsophioglossoides) and isolating method thereof.
Background technology
Because the scarcity of wild resource and the deterioration of growing environment, many famous and precious medicinal fungis are endangered, how to save these valuable medicine resources, satisfy the drug development needs and seem particularly important, utilize microbial technique to separate active bacterial strain, adopt modern fermentation technique to carry out large-scale medicinal material and produce necessary.
Goldenthread Cordyceps is a kind of rare Chinese herbal medicine, and the use among the people of China's southern area is comparatively general, has a long history.Its sporophore includes abundant alkaloid, the general name ergotine, and pharmaceutical use is high aspect the treatment gynaecopathia, is mainly used in diseases such as treatment menoxenia, dysmenorrhoea, amenorrhoea, and postpartum hemorrhage, promotion uterine involution in postpartum are also had unusual effect.
Summary of the invention
The objective of the invention is to obtain single bacterial strain from a kind of wild sporophore separation of famous and precious medicinal Cordyceps fungus, and according to its morphological specificity and it being identified, thereby make that adopting modern fermentation technique to carry out large-scale medicinal material production becomes possibility based on the phylogenetic analysis of 18S rDNA sequence.
In order to realize purpose of the present invention, the technical solution used in the present invention is as follows: the isolated Cordyceps fungus of the present invention, it is characterized in that, it is the pure bacterial strain of this fungi that separation and purification obtains on Goldenthread Cordyceps (Cordyceps ophioglossoides) sporophore, and its 18S rDNA has the sequence of SEQ ID No.1.Be kept at China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China common micro-organisms center, preservation is numbered: CGMCC No.1146.
The present invention also provides a kind of method of separating the described medicinal Cordyceps fungus of claim 1, it is characterized in that, be divided into following steps: (1) selects the Goldenthread Cordyceps sporophore: select that ascoma is big, handle short, eight or nine fens sophisticated Goldenthread Cordyceps sporophores, cut and cover two base portions; (2) sterilization: being 0.1% mercuric chloride water logging bubble with concentration in sterile cupboard, is 75% cotton ball soaked in alcohol wiping cap and stem with concentration again, carries out surface sterilization; 3) inoculation: during inoculation, sporophore is cut, sprout lid and stem intersection, picking one little block organization at it; Move and receive on the potato sucrose basic medium PSA; 4) cultivate: cultivated 7～10 days under 20 ℃～30 ℃ temperature, wait to organize to produce white fine hair shape mycelia, tube expansion promptly obtains the Goldenthread Cordyceps bacterial classification.
The present invention has following technique effect: Goldenthread Cordyceps is the traditional macro fungi with pharmaceutical use of China, have only the utilization to wild resource at present, so that falls sharply in the medicine source.Utilization of the present invention separation and purification from the wild plant obtains pure Cordyceps cphioglossoides (Ehrh.Ex. Fr.) Link strain, and identify by traditional morphological method, fermentation process and modern gene means, for utilizing this bacterium to carry out that large scale fermentation is produced this medicinal material, the scarcity of alleviating wild resource provides the foundation and may.
" China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation, preservation date are on May 14th, 2004 to Cordyceps cphioglossoides (Ehrh.Ex. Fr.) Link of the present invention, and deposit number is: CGMCC No.1146 in specified depositary institution of Patent Office of the People's Republic of China.
Embodiment
Describe the present invention below in detail.The separation method of Cordyceps fungus of the present invention is as follows
The wild plant of Goldenthread Cordyceps of the present invention colonizes on the sporophore of false truffle (Elaphomycesgranulatus Fr) of underground growth, the ascoma sphere, and diameter is 1～4.5 centimetre, brown is hard; Stroma root shape is born in basic beyond the region of objective existence, and upright, head has the long handle multi-branched, and height does not wait, and general 2～15 centimetres, club-like, brown.Ascogonidium is facile hydrolysis not, and thecaspore is linear.Separate the morphological specificity of bacterial strain on solid plate that obtains, bacterium colony is grown thickly, white thread fungus filament.Bacterium liquid become to mix during liquid culture, produces spherical mycelium, and first mycelium be white, after fade to brown.
The present invention adopts the separate tissue culture method, select that ascoma is big, handle is short, eight or nine fens sophisticated wild kinds, cut and cover two base portions, in sterile cupboard, be 0.1% mercuric chloride water logging 10～20 minutes with concentration of volume percent, be 75% cotton ball soaked in alcohol wiping cap and stem 2 times with concentration of volume percent again, carry out surface sterilization.During inoculation, will plant sporophore and cut, sprout lid and stem intersection, picking one little block organization; Move and receive on the potato sucrose basic medium (PSA).Cultivated 7～10 days under 20 ℃～30 ℃ temperature, wait to organize to produce white fine hair shape mycelia, tube expansion promptly obtains the Goldenthread Cordyceps bacterial classification.
Carry out the evaluation of Cordyceps fungus bacterial classification below
(1) form of Cordyceps fungus and physicochemical characteristics
Colony's form: on the solid medium, white thread fungus filament, mycelia prosperity are grown thickly, produced to bacterium colony, and liquid oscilaltion produces spherical mycelium when cultivating, and first mycelium be white, after fade to brown.Individual morphology: mycelium has the separation of rule, and barrier film has simply every the hole, and woronin bodies is arranged, the cell walls bilayer, and interior bed thickness is and transparent, outer thin and intensive, and thecaspore is linear.
Physicochemical characteristics: to disaccharide utilize effect better than monose, can utilize starch as carbon source.Fixedly nitrogen utilizes effect relatively poor to inorganic nitrogen-sourced.Produce a kind of microbiotic-antifongin (ophiocordin) in the process of growth.
The cultural characteristic and the morphological specificity of this bacterial strain see Table 1.
| The solid culture feature | Bacterium colony | Grow thickly |
| Mycelium | Have | |
| The mycelium shape | Thread, radial | |
| The mycelium color | White | |
| Chromogenesis | Do not have | |
| The liquid culture feature | Muddy | Have |
| Mycelium | Have | |
| The mycelium shape | Be combined into spherical | |
| Chromogenesis | Just for becoming brown after the white | |
| Personal feature | Spore | Have |
| The spore feature | Wire | |
| Mycelium has or not at interval | Barrier film is arranged, porose on the barrier film | |
| The cell walls form | Bilayer, interior bed thickness is and transparent, and skin is thin and intensive | |
| Woronin bodies | Have |
Table 1
(2) phylogenetic analysis of 18S rDNA sequence
Measure the sequence of Goldenthread Cordyceps fungi (CGMCC No.1146) 18S rDNA part
Table 2 is the accession number of the 18S rDNA sequence of the part correlation bacterial strain that accesses according to the Cordyceps fungus sequence of the present invention from GeneBank.
| The sequence accession number | Similarity (%) | |
| Cordyceps sphingum | AY245673.1 | 99 |
| Cordyceps militaris | AY245671.1 | 99 |
| Paecilomyces canadensis | AY245666.1 | 99 |
| Cordyceps sinensis | AY245670.1 | 99 |
| Cordyceps heteropoda | AY245656.1 | 99 |
| Cordyceps brongniartii | AY245653.1 | 99 |
| Phytocordyceps ninchukispora | AY245668.1 | 99 |
| Cordyceps japonica | AY245669.1 | 99 |
| Cordyceps isarioides | AY245657.1 | 99 |
| Cordyceps subsessilis | AY245665.1 | 99 |
| Cordyceps memorabilis | AY245658.1 | 99 |
| Cordyceps agriota | AY245651.1 | 99 |
Table 2
In conjunction with above-mentioned morphological specificity and 18S rDNA sequential analysis, can determine that this bacterium is Ascomycetes, Spheeriales, Clavicipitaceae, Cordyceps sinensis fungus, really be Cordyceps cphioglossoides (Ehrh.Ex. Fr.) Link thereby determine to separate the bacterial strain that obtains.The sequence SEQ ID No.1 table of Cordyceps cphioglossoides (Ehrh.Ex. Fr.) Link 18S rDNA part:
＜110〉Zhejiang University
＜120〉a kind of Cordyceps fungus and separation method thereof
<160>1
<170>PatentIn version 3.1
<210>1
<211>1519
<212>DNA
<213>Cordyceps sp.
<400>1
aaagattaag ccatgcatgt ctaagtataa gcaattatac agcgaaactg cgaatggctc 60
attatataag ttatcgttta tttgatagta ccttactact tggataaccg tggtaattct 120
agagctaata catgctaaaa atcccgactt cggaagggat gtatttatta gattaaaaac 180
caatgccctc tgggctctct ggtgattcat gataacttct cgaatcgcat ggccttgcgc 240
cggcgatggt tcattcaaat ttcttcccta tcaactttcg atgtttgggt agtggccaaa 300
catggttgca acgggtaacg gagggttagg gctcgacccc ggagaaggag cctgagaaac 360
ggctactaca tccaaggaag gcagcaggcg cgcaaattac ccaatcccga cacggggagg 420
tagtgacaat aaatactgat acagggctct tttgggtctt gtaattggaa tgagtacaat 480
ttaaatccct taacgaggaa caattggagg gcaagtctgg tgccagcagc cgcggtaatt 540
ccagctccaa tagcgtatat taaagttgtt gtggttaaaa agctcgtagt tgaaccttgg 600
gcctggctgg ccggtccgcc tcaccgcgtg tactggtccg gccgggcctt tccctctgtg 660
gaaccccatg cccttcactg ggtgtggcgg ggaaacagga cttttacttt gaaaaaatta 720
gagtgctcca ggcaggccta tgctcgaata cattagcatg gaataatgaa ataggacgtg 780
cggttctatt ttgttggttt ctaggaccgc cgtaatgatt aatagggaca gtcgggggca 840
tcagtattca attgtcagag gtgaaattct tggatttatt gaagactaac tactgcgaaa 900
gcatttgcca aggatgtttt cattaatcag gaacgaaagt taggggatcg aagacgatca 960
gataccgtcg tagtcttaac cataaactat gccgactagg gatcggacga tgttattttt 1020
tgactcgttc ggcaccttac gagaaatcaa agtgcttggg ctccaggggg agtatggtcg 1080
caaggctgaa acttaaagaa attgacggaa gggcaccacc aggggtggag cctgcggctt 1140
aatttgactc aacacgggga aactcaccag gtccagacac aatgaggatt gacagattga 1200
gagctctttc ttgattttgt gggtggtggt gcatggccgt tcttagttgg tggagtgatt 1260
tgtctgctta attgcgataa cgaacgagac cttaacctgc taaatagccc gtattgcttt 1320
ggcagtacgc tggcttctta gagggactat cggctcaagc cgatggaagt ttgaggcaat 1380
aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact gacggagcca 1440
gcgagtactc ccttggccgg aaggcccggg taatcttgtt aaactccgtc gtgctgggga 1500
agagcattgc aattattgc 1519
Claims (1)
1, a kind of Cordyceps fungus is characterized in that, it is the pure bacterial strain of this fungi that separation and purification obtains on Goldenthread Cordyceps (Cordycepsophioglossoides) sporophore, and its 18S rDNA has the sequence of SEQ ID No.1; Be kept at China Committee for Culture Collection of Microorganisms of specified depositary institution of Patent Office of the People's Republic of China common micro-organisms center, preservation is numbered: CGMCC No.1146.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410025774 CN1262638C (en) | 2004-06-30 | 2004-06-30 | Chinese caterpillar fungus and its separating method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410025774 CN1262638C (en) | 2004-06-30 | 2004-06-30 | Chinese caterpillar fungus and its separating method |
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| CN1594541A CN1594541A (en) | 2005-03-16 |
| CN1262638C true CN1262638C (en) | 2006-07-05 |
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| CN 200410025774 Expired - Fee Related CN1262638C (en) | 2004-06-30 | 2004-06-30 | Chinese caterpillar fungus and its separating method |
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| CN114231574A (en) * | 2021-11-25 | 2022-03-25 | 浙江大学 | Culture medium and method for producing protein kinase C inhibitor balanol |
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| CN1302101C (en) * | 2005-06-22 | 2007-02-28 | 浙江大学 | Method for producing C. ophioglossouides using liquid submerged culture |
| CN101230380B (en) * | 2007-12-26 | 2010-06-23 | 广东省微生物研究所 | Characteristic sequences and method for discriminating pivot-end cordyceps sinensis |
| CN102440141B (en) * | 2010-10-08 | 2012-12-26 | 镇江市食用菌研究所 | Method for separating Cordyceps militaris strains |
| CN102229595B (en) * | 2011-04-30 | 2013-06-12 | 浙江大学 | Sesquiterpenoids in Cordyceps Ophioglossoides and use thereof |
| CN102523927A (en) * | 2012-01-17 | 2012-07-04 | 辽宁仙榆湾北冬虫夏草（集团）有限公司 | Method for culturing cordyceps militaris in natural environment |
| CN103232995A (en) * | 2012-12-03 | 2013-08-07 | 中国食品发酵工业研究院 | Cordyceps sinensis original powder high-purity genome DNA extraction method |
| CN103262797B (en) * | 2013-06-04 | 2014-10-29 | 神舟太空产品高科技成果推广中心集团有限公司 | Space-induced efficient cordyceps militaris and application thereof as well as preparation method of capsule preparation of space-induced efficient cordyceps militaris |
| CN104611234B (en) * | 2015-01-12 | 2017-12-26 | 北京林业大学 | A kind of separation method of the raw medicinal fungi of wood |
| CN107723308B (en) * | 2017-09-14 | 2020-04-24 | 浙江大学 | Biosynthesis method and gene cluster of compound balanol |
| CN108913607B (en) * | 2018-07-19 | 2021-06-15 | 安徽大学 | Preparation method and application of wild russula filiformis mycelium with efficient dye removal function |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114231574A (en) * | 2021-11-25 | 2022-03-25 | 浙江大学 | Culture medium and method for producing protein kinase C inhibitor balanol |
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