CN101195804A - Huperzia serrata endogenetic epiphyte and uses thereof - Google Patents
Huperzia serrata endogenetic epiphyte and uses thereof Download PDFInfo
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- CN101195804A CN101195804A CNA2006101191497A CN200610119149A CN101195804A CN 101195804 A CN101195804 A CN 101195804A CN A2006101191497 A CNA2006101191497 A CN A2006101191497A CN 200610119149 A CN200610119149 A CN 200610119149A CN 101195804 A CN101195804 A CN 101195804A
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- 241001090156 Huperzia serrata Species 0.000 title claims abstract description 32
- ZRJBHWIHUMBLCN-YQEJDHNASA-N huperzine A Chemical compound N1C(=O)C=CC2=C1C[C@H]1\C(=C/C)[C@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-YQEJDHNASA-N 0.000 claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 3
- 241001248531 Euchloe <genus> Species 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 230000001788 irregular Effects 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 239000008188 pellet Substances 0.000 claims description 2
- 238000012807 shake-flask culturing Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 2
- 230000032696 parturition Effects 0.000 claims 1
- ZRJBHWIHUMBLCN-UHFFFAOYSA-N Shuangyiping Natural products N1C(=O)C=CC2=C1CC1C(=CC)C2(N)CC(C)=C1 ZRJBHWIHUMBLCN-UHFFFAOYSA-N 0.000 abstract description 16
- ZRJBHWIHUMBLCN-BMIGLBTASA-N rac-huperzine A Natural products N1C(=O)C=CC2=C1C[C@@H]1C(=CC)[C@@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-BMIGLBTASA-N 0.000 abstract description 16
- 150000001875 compounds Chemical class 0.000 abstract description 6
- 241000233866 Fungi Species 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 208000024827 Alzheimer disease Diseases 0.000 abstract description 3
- 206010036631 Presenile dementia Diseases 0.000 abstract description 3
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 abstract description 2
- ZRJBHWIHUMBLCN-SEQYCRGISA-N Huperzine A Natural products N1C(=O)C=CC2=C1C[C@H]1/C(=C/C)[C@]2(N)CC(C)=C1 ZRJBHWIHUMBLCN-SEQYCRGISA-N 0.000 abstract 2
- 244000005700 microbiome Species 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- APLHEOBEIBHCHW-YQEJDHNASA-N Selagine Natural products O=C1NC2=C([C@]3(N)/C(=C/C)/[C@@H](CC(C)=C3)C2)C=C1 APLHEOBEIBHCHW-YQEJDHNASA-N 0.000 description 14
- 239000001965 potato dextrose agar Substances 0.000 description 10
- 229930013930 alkaloid Natural products 0.000 description 5
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 150000003797 alkaloid derivatives Chemical class 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 244000050510 Cunninghamia lanceolata Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000022120 Jeavons syndrome Diseases 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
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Abstract
The invention discloses endophytic fungi from huperzia serrata. The invention is obtained through being separated from the living body of pteridophyta huperzia serrata by adopting endophytic fungi separation and purification technology, and is identified through biosystematics to be acremonium endophytium. The preservation number of the strain is CCTCC M 206118. The invention generates Huperzine A analogous compound through the strain liquid fermentation of the endophytic fungi from huperzia serrata, and is significant micro organism for searching the Huperzine A new resource of the active component for curing the presenile dementia.
Description
Technical field
The present invention relates to microbial technology field, particularly Huperzia serrata endogenetic epiphyte and application thereof.
Background technology:
Selagine is as the specifics of a new generation treatment presenile dementia, and market outlook are considerable, and it is clinical that its derivative ZT-1 is just entering the III phase at the authentication registration of European EMEA, and product development is also actively being carried out.At present, selagine is mainly derived from Herba Lycopodii serrati (Huperzia serrata) herb.Though the Herba Lycopodii serrati resource area is wide, distribute and be scattered about like the stars, the resource updates cycle is long, can be fewer for the natural resource of commercial exploitation.Through a large amount of exploitations for many years, the Herba Lycopodii serrati resource of provinces such as Chinese Zhejiang, Jiangxi, Fujian is close to exhaustion.The resource of provinces such as Hunan, Guangxi, Sichuan also will face exhausted danger in two, three years.These areas are as the forfeiture gradually of Herba Lycopodii serrati compartmentation advantage kind status, thereby can not satisfy the demand of market to selagine by the Herba Lycopodii serrati natural resource of directly gathering.On the other hand, the chemosynthesis of selagine is low because of yield, cost is expensive, also is difficult to realize industrialization.Investigators attempt to obtain selagine by biotechnological means, and therefore, this has become the problem that people pay close attention in recent years.
Summary of the invention:
Technical problem to be solved by this invention is to design and a kind ofly can produces the selagine analogue by microbial fermentation.
The invention provides a kind of Huperzia serrata endogenetic epiphyte, give birth to branch top spore mould (Acremoniumendophytium) in the called after.
Huperzia serrata endogenetic epiphyte of the present invention adopts the endogenetic fungus separating and purifying technology to separate from pteridophyte Herba Lycopodii serrati (huperzia serrata) plant living body and obtains, and gives birth to branch top spore mould (Acremonium endophytium) in microbial taxonomy is accredited as.The preservation of this bacterial strain, preservation date are on November 13rd, 2006, and preserving number is CCTCC M 206118, and depositary institution is Chinese typical culture collection center, address: Luojia Mountain, Wuhan, Hubei Province Wuhan University, postcode: 430072.
The solid culture of Huperzia serrata endogenetic epiphyte of the present invention is characterized as:
28 ℃ of cultivations on potato dextrose agar (PDA) substratum, initial aerial hyphae is colourless, gradual change white; The bacterium colony circle, white, the fine hair shape, the edge is irregular, and the back side is khaki.
The liquid culture of Huperzia serrata endogenetic epiphyte of the present invention is characterized as:
(1) substratum PDA, shake-flask culture 7 days, 28 ± 1 ℃ of culture temperature;
(2) fermentation culture feature: cultivated 1-2 days, and do not see obvious growth phenomenon; Cultivated the 3rd day, and had a little white hypha to occur; Cultivated the 3rd day, and formed diameter 1-2mm white hypha ball; Cultivate the 4th day bacterium spherical diameter and increase to 2-4mm; Cultivated the 5th day, bacterium nodule number amount increases; Cultivated the 6th day, fermented liquid is sticky; Cultivated the 7th day, mycelium pellet is assembled.
The morphological specificity of Huperzia serrata endogenetic epiphyte of the present invention is:
1, asexual generation, the mycelia branch, level and smooth, have every, thick 1.2-1.5 μ m; In the product spore zone of young bacterium colony, the sporophore that the mycelia adnation is short, long 10-13 μ m; Conidium is avette to fusiformis, and is colourless, 3.53-5.44 * 1.16-1.32 μ m, and chain is concatenated.
2, sexual generation, do not find
The 18S rDNA base sequence of Huperzia serrata endogenetic epiphyte of the present invention is:
TGCATTATACAGCGAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTGCCTTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCGACTTCGGAAGGGGTGTATTTATTAGATACAAAACCAATGCCCTTCGGGGCTCTTTGGTGATTCATGATAACTATACGAATCGCACGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTCTTGGCCAAACATGGTTGCAACGGGTAACGGAGGGTTAGGGCTCGACCCCGGAGAAGGAGCCTGAGAAACGGCTACTACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACTCGGGGAGGTAGTGACAATAAATACTGATACAGGGCCCTTTCGGGCCTTGTAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAACCTTGGGCCTGGCTGGCCGGTCCGCCTCACCGCGTGCACTGGTCCGGCCGGGCCTTTCCCTCTGTGGAACCCCATACCCTTCACTGGGCGTGGCGGGGAAACAGGACATTTACTTTGAAAAAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATAAAATAGGACGCGCGGTTCTATTTTGTTGGTTTATAGGACCGCCGTAATGATTAATAGGGACAGTCGGGGGCATCAGTATTCAAATGTCAGAGGTGAAATTCTTGGATCATTTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGGTGTTATTCATGACCCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGACACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCACGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGGTGACTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGCATTGCTCCGGCAGTGCGCTGGCTTCTTAGAGGGACTTTCGGCTCAAGCCGAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACGGGGTCAGCGAGTTCCTTGGCCGAAAGGCCCGGGTAATCTTGTTAGCCCCCGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCGTCCGGACTGGCCCAGGGAGGTGGGCAACTACCACCCAGGGCCGGATATAGCTGTCCAAATTCGAG。
Another object of the present invention has provided the application of snake-stone China fir endogenetic fungus.
Huperzia serrata endogenetic epiphyte of the present invention can get the selagine similar compound through fermentation, and its processing step is as follows:
Actication of culture → seed culture → fermentation culture → tunning cytoclasis → total alkaloids extraction → HPLC and LC-MS analysis → selagine similar compound
Wherein, fermentation raw material is the potato culture (PDA) of improvement, and fermentation mode is that liquid shaking bottle is cultivated; Actication of culture adopts the test tube slant to cultivate, and substratum is potato glucose solid medium (solid PDA); Seed culture is potato glucose liquid nutrient medium (liquid PDA); Fermention medium is improvement potato glucose liquid nutrient medium (liquid PDA); Incubation time: strain inclined plane is cultivated activation 72 hours, and the seed shaking table was cultivated 72 hours, fermentation culture 7 days; Culture temperature: the test tube slant culture temperature is 28 ± 1 ℃, and seed shaking table culture temperature is 28 ± 1 ℃, and the fermentation culture temperature is 28 ± 1 ℃; Fermented product extracts: fermentation finishes, and collects tunning, and smudge cells with conventional extracting method extract total alkaloids, adopts HPLC to separate and obtains the selagine similar compound.
Huperzia serrata endogenetic epiphyte of the present invention can produce the selagine similar compound by the strain liquid fermentation, is the important microbe of seeking treatment presenile dementia activeconstituents selagine new resources, and bigger using value is arranged.
Description of drawings:
Fig. 1 is the colonial morphology of Huperzia serrata endogenetic epiphyte of the present invention
Fig. 2 is Huperzia serrata endogenetic epiphyte of the present invention observed mycelia and spore shape under 100 power microscopes
Fig. 3 is the mycelia form of Huperzia serrata endogenetic epiphyte of the present invention under electron microscope
Fig. 4 is Huperzia serrata endogenetic epiphyte of the present invention observed spore shape under electron microscope
Fig. 5 is the contrast of Huperzia serrata endogenetic epiphyte alkaloid component color atlas of the present invention (a) and selagine standard substance color atlas (b)
Fig. 6 is the contrast of Huperzia serrata endogenetic epiphyte alkaloid target components uv absorption spectra of the present invention (left side) and selagine standard substance uv absorption spectra (right side), both carry out matching ratio with the Match program on the Agilent chem workstation, matching attribute reaches 994.568.
Fig. 7 be Huperzia serrata endogenetic epiphyte alkaloid target components scanning of the mass spectrum figure of the present invention (on) with selagine standard substance mass spectrum scintigram (descending), both quasi-molecular ion peaks are respectively 243.10 and 243.11, can assert tentatively that both are same components.
Embodiment:
Example 1:
(1) gets Huperzia serrata endogenetic epiphyte bacterial classification of the present invention, under aseptic condition,, insert sterilized solid PDA substratum test tube, in 28 ± 1 ℃ of activation culture 72 hours with inoculating needle picking a small amount of mycelia;
(2) get bacterial classification after the activation culture, under the aseptic condition, switching is gone in the sterilized liquid PDA seed culture medium, and 28 ± 1 ℃ of following 140rpm shaking tables were cultivated 72 hours, seed;
(3) the improvement PDA liquid nutrient medium for preparing by 20% charging ratio, divides in the triangular flask of the 500ml that packs into, and sterilising treatment is cooled off standby.Under the aseptic condition, the inoculum size according to 10% inserts the 2F09P03B seed, and 28 ± 1 ℃ of following 140rpm shaking tables were cultivated 7 days;
(4) fermentation finishes, collect culture, transfer about pH value to 4.0 smudge cells, standing over night, transfer about pH value to 10.0, centrifugal collection supernatant liquor is transferred pH value to 9.0, in chloroform extraction 5 times of 1/2 ratio, collect extraction liquid, 80 ℃ are reclaimed chloroform, obtain the selagine analogue with the dissolve with methanol residue.
(5) the HPLC chromatographic condition is as follows: chromatographic column---Kromasil C
18Reversed-phase column (4.6 * 250mm * 5 μ m), moving phase---CH
3CN-KH
2PO
4(0.02mol/L) 10: 90, flow velocity---1.0mL/min detected wavelength 310nm.
(6) LC-MS detects chromatographic condition: chromatographic column---BDS Hypersil C
18Reversed-phase column (2.1mm * 100mm * 3 μ m), moving phase---H
2O (1%formic acid)-methanol, gradient elution---[time (min): D (methanol)]=[0.00: 5.0]-[10.00: 80.0]-[15.00: 5.0], flow velocity---0.2mL/min, sample size---0.5 μ L.The MS detection parameters---shealth flow rate (arb)-60, aux gas flow rate (arb)-5, I spray voltage (kv)-5.00, capillary temp (℃)-275.00, (v)-31.00, tubelens offset (v)-10.00 for capillary voltage.
(7) analyze through above HPLC and LC-MS, show that Huperzia serrata endogenetic epiphyte bacterial classification liquid fermentation energy of the present invention enough produces the selagine similar compound.
Claims (6)
1. Huperzia serrata endogenetic epiphyte is characterized in that giving birth to the mould Acremoniumendophytium of branch top spore in its called after, and its preservation registration number is CCTCC M 206118.
2. a kind of Huperzia serrata endogenetic epiphyte according to claim 1, the microscopic morphology that it is characterized in that it is the mycelia branch, and is level and smooth, has every, thick 1.2-1.5 μ m; In the product spore zone of young bacterium colony, the sporophore that the mycelia adnation is short, long 10-13 μ m; Conidium is avette to fusiformis, and is colourless, 3.53-5.44 * 1.16-1.32 μ m, and chain is concatenated.
3. a kind of Huperzia serrata endogenetic epiphyte according to claim 1 is characterized in that its solid culture is 28 ℃ of cultivations on the PDA substratum, and initial aerial hyphae is colourless, gradual change white; The bacterium colony circle, white, the fine hair shape, the edge is irregular, and the back side is khaki.
4. a kind of Huperzia serrata endogenetic epiphyte according to claim 1 is characterized in that its liquid culture is
(1) substratum PDA, shake-flask culture 7 days, 28 ± 1 ℃ of culture temperature; Or
(2) fermentation culture feature: cultivated 1-2 days, and do not see obvious growth phenomenon; Cultivated the 3rd day, and had a little white hypha to occur; Cultivated the 3rd day, and formed diameter 1-2mm white hypha ball; Cultivate the 4th day bacterium spherical diameter and increase to 2-4mm; Cultivated the 5th day, bacterium nodule number amount increases; Cultivated the 6th day, fermented liquid is sticky; Cultivated the 7th day, mycelium pellet is assembled.
5. a kind of Huperzia serrata endogenetic epiphyte according to claim 1 is characterized in that its genome 18SrDNA base sequence is:
TGCATTATACAGCGAAACTGCGAATGGCTCATTATATAAGTTATCGTTTATTTGATAGTGCCTTACTACTTGGATAACCGTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCGACTTCGGAAGGGGTGTATTTATTAGATACAAAACCAATGCCCTTCGGGGCTCTTTGGTGATTCATGATAACTATACGAATCGCACGGCCTTGCGCCGGCGATGGTTCATTCAAATTTCTTCCCTATCAACTTTCGATGTTTGGGTCTTGGCCAAACATGGTTGCAACGGGTAACGGAGGGTTAGGGCTCGACCCCGGAGAAGGAGCCTGAGAAACGGCTACTACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCCGACTCGGGGAGGTAGTGACAATAAATACTGATACAGGGCCCTTTCGGGCCTTGTAATTGGAATGAGTACAATTTAAATCCCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGTGGTTAAAAAGCTCGTAGTTGAACCTTGGGCCTGGCTGGCCGGTCCGCCTCACCGCGTGCACTGGTCCGGCCGGGCCTTTCCCTCTGTGGAACCCCATACCCTTCACTGGGCGTGGCGGGGAAACAGGACATTTACTTTGAAAAAATTAGAGTGCTCCAGGCAGGCCTATGCTCGAATACATTAGCATGGAATAATAAAATAGGACGCGCGGTTCTATTTTGTTGGTTTATAGGACCGCCGTAATGATTAATAGGGACAGTCGGGGGCATCAGTATTCAAATGTCAGAGGTGAAATTCTTGGATCATTTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGATGTTTTCATTAATCAGGAACGAAAGTTAGGGGATCGAAGACGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACGGTGTTATTCATGACCCGTTCGGCACCTTACGAGAAATCAAAGTGCTTGGGCTCCAGGGGGAGTATGGTCGCAAGGCTGACACTTAAAGAAATTGACGGAAGGGCACCACCAGGGGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCACGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGGTGACTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCCGCATTGCTCCGGCAGTGCGCTGGCTTCTTAGAGGGACTTTCGGCTCAAGCCGAAGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACGGGGTCAGCGAGTTCCTTGGCCGAAAGGCCCGGGTAATCTTGTTAGCCCCCGTCGTGCTGGGGATAGAGCATTGCAATTATTGCTCTTCAACGAGGAATCCCTAGTAAGCGCAAGTCATCAGCTTGCGTTGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCGTCCGGACTGGCCCAGGGAGGTGGGCAACTACCACCCAGGGCCGGATATAGCTGTCCAAATTCGAG。
6. the application of Huperzia serrata endogenetic epiphyte as claimed in claim 1 in preparation selagine analogue is characterized in that this preparation method comprises the following steps:
(1) gets Huperzia serrata endogenetic epiphyte bacterial classification of the present invention, under aseptic condition,, insert sterilized solid PDA substratum test tube, in 28 ± 1 ℃ of activation culture 72 hours with inoculating needle picking a small amount of mycelia;
(2) get bacterial classification after the activation culture, under the aseptic condition, switching is gone in the sterilized liquid PDA seed culture medium, and 28 ± 1 ℃ of following 140rpm shaking tables were cultivated 72 hours, seed;
(3) the improvement PDA liquid nutrient medium for preparing by 20% charging ratio, divides in the triangular flask of the 500ml that packs into, and sterilising treatment is cooled off standby.Under the aseptic condition, the inoculum size according to 10% inserts seed, and 28 ± 1 ℃ of following 140rpm shaking tables were cultivated 7 days;
(4) fermentation finishes, collect culture, transfer about pH value to 4.0 smudge cells, standing over night, transfer about pH value to 10.0, centrifugal collection supernatant liquor is transferred pH value to 9.0, in chloroform extraction 5 times of 1/2 ratio, collect extraction liquid, 80 ℃ are reclaimed chloroform, obtain the selagine analogue with the dissolve with methanol residue.
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CN101942393B (en) * | 2009-12-30 | 2012-12-26 | 朱笃 | Huperzia serrata endophytic fungus shiraia sp. strain for producing huperzine A |
CN102168017A (en) * | 2010-09-29 | 2011-08-31 | 湖南农业大学 | Huperzine A high-producing strain and method for producing huperzine A by fermenting same |
CN102168017B (en) * | 2010-09-29 | 2012-07-11 | 湖南农业大学 | Huperzine A high-producing strain and method for producing huperzine A by fermenting same |
CN102653720A (en) * | 2011-03-02 | 2012-09-05 | 华中农业大学 | Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a |
CN102653720B (en) * | 2011-03-02 | 2013-09-25 | 华中农业大学 | Endophytic fungi ES026 of huperzia serrata capable of generating huperzine a |
CN102907253A (en) * | 2012-07-02 | 2013-02-06 | 恩施清江生物工程有限公司 | Huperzia serrata endophyte and application thereof |
CN103667072A (en) * | 2013-09-27 | 2014-03-26 | 浙江工业大学 | Huperzia serrate endophytic fungus and application of huperzia serrate endophytic fungus in preparation of 8alpha,15alpha-epoxydized huperzine A |
CN103667072B (en) * | 2013-09-27 | 2016-04-13 | 浙江工业大学 | A kind of Huperzia serrata endogenetic epiphyte and the application at preparation 8 α, 15 α-epoxidation selagine thereof |
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