CN102660466A - Endophytic fungi for improving content of main active ingredients of schisandra chinensis through fermentation method - Google Patents
Endophytic fungi for improving content of main active ingredients of schisandra chinensis through fermentation method Download PDFInfo
- Publication number
- CN102660466A CN102660466A CN2012101261692A CN201210126169A CN102660466A CN 102660466 A CN102660466 A CN 102660466A CN 2012101261692 A CN2012101261692 A CN 2012101261692A CN 201210126169 A CN201210126169 A CN 201210126169A CN 102660466 A CN102660466 A CN 102660466A
- Authority
- CN
- China
- Prior art keywords
- schisandra chinensis
- main active
- endophytic fungi
- aspergillus
- active ingredients
- 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.)
- Granted
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to an endophytic fungi for improving content of main active ingredients of schisandra chinensis through a fermentation method. The endophytic fungi is Aspergilluspenicillioides WT4 which is preserved in the China Center for Type Culture Collection (CCTCC) with preservation numbers of CCTCC No: M2012043 in Wuhan University in Wuhan city, and the preservation date is February 29<th>, 2012. The WT4 in the endophytic fungi can remarkably improve the main active ingredients of the schisandra chinensis, such as schizandrin, schisantherin, deoxyschizandrin and schisandrin b. Therefore, in the raw material production with the purpose of obtaining the schizandrin, the schisantherin, the deoxyschizandrin and the schisandrin b, people can select schisandra chinensis endophytic fungi WT4 to ferment the schisandra chinensis to improve the yield and save medicinal resources.
Description
Technical field
The present invention relates to a strain endogenetic fungus.
Background technology
Schisandra chinensis [Schisandra chinensis (Turcz.) Baill.], the dry mature fruit of magnoliaceae schisandra is not only to have eaten but also the medicine functional health-care food.The activeconstituents of Schisandra chinensis is to be the lignan component of representative with schisandrin, Wuweizi ester A, deoxyschizandrin, Wuweizisu B etc.; That this constituents has is significantly anticancer, anti-inflammatory, calmness; Anxiety; The protection liver cell, anti-chemical damage, effect such as antibiotic are widely used in medicine and food service industry at present.Because the Schisandra chinensis demand goes up, area of woods reduces and excavating blindly gradually, makes the Schisandra chinensis resource face test, and Schisandra chinensis is the medicinal plant in imminent danger of three grades of focused protections of country at present.
Fermentation technique can change or strengthen some activity of food, and some activeconstituents in the protection food is saved resource, for the acquisition of food, pharmaceutical active ingredient provides novel method.Correlative study about the Schisandra chinensis fermentation does not appear in the newspapers at present.
Summary of the invention
The invention provides a strain improves Schisandra chinensis main active ingredient content through fermentation method endogenetic fungus.
The present invention improves the endogenetic fungus of Schisandra chinensis main active ingredient content through fermentation method; It is broom shape aspergillus (Aspergillus penicillioides) WT4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M2012043, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012; It after cultivating 7 days on the PDA solid medium, mycelia quality velvet shape, the conidium structure that tool is a large amount of; Sap green, edge white, no transudate; Light musty is arranged, and the bacterium colony reverse side is in various degree little Huang to sap green, and it is loose cylindric that the bacterial strain conidial head is; Conidiophore is elongated, top capsule flask shape, and capsule nearly clavate in minority top is spherical or subsphaeroidal when conidium is ripe.
Pass through the endogenetic fungus that fermentation method improves Schisandra chinensis main active ingredient content among the present invention; It is broom shape aspergillus (Aspergillus penicillioides) WT4; According to " classification of fungi ", " fungi identification handbook " and " Dendrochium diagram ", the colony characteristics and the spore shape of visible WT4 bacterial strain are very close with the aspergillus tubigensis characteristic.
Pass through the endogenetic fungus that fermentation method improves Schisandra chinensis main active ingredient content among the present invention; It is broom shape aspergillus (Aspergillus penicillioides) WT4; Its ITS sequence is committed to the NCBI webpage; Through Blast search and the high sequence of resulting sequence similarity, and through MEGA5.03 software building systematic evolution tree, the ITS sequence of WJ1 bacterial strain and the similarity of Aspergillus bacterial strain have reached more than 98%; Combining form is learned observations, confirms that the WT4 bacterial strain is broom shape aspergillus (Aspergillus penicillioides).
The present invention improves the endogenetic fungus of Schisandra chinensis main active ingredient content through fermentation method; It is broom shape aspergillus (Aspergillus penicillioides) WT4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M2012043, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
It is substrate that the present invention adopts Schisandra chinensis; With Schisandra chinensis endogenetic fungus WT4 fermentation Schisandra chinensis; Be intended to improve 4 kinds of main lignanoid component contents in the Schisandra chinensis; For efficiently utilizing Schisandra chinensis, save the medicine source, for the research and development of natural drug and utilization provide a favourable approach, also for protecting medicinal plant in imminent danger that practical terms of settlement is provided.
Compare article with schisandrin, Wuweizi ester A, deoxyschizandrin and 4 kinds of Schisandra chinensis main active ingredient of Wuweizisu B; Utilization HPLC method is measured the dynamic change of Schisandra chinensis its effective constituent before and after endogenetic fungus WT4 fermentation, identifies its kind through morphological observation and 18S rDNA sequential analysis at last.
Broom shape aspergillus (Aspergillus penicillioides) WT4 among the present invention; It can make main active ingredient such as schisandrin in the Schisandra chinensis, Wuweizi ester A, deoxyschizandrin and Wuweizisu B significantly improve; Therefore in the raw material production that with acquisition schisandrin, Wuweizi ester A, deoxyschizandrin and Wuweizisu B is purpose, can select Schisandra chinensis endogenetic fungus WT4 fermentation Schisandra chinensis to improve yield, save the medicine source.
Description of drawings
Fig. 1 is the colonial morphology figure of broom shape aspergillus (Aspergillus penicillioides) WT4 in the embodiment one;
Fig. 2 is the electrophorogram of the pcr amplification of broom shape aspergillus (Aspergillus penicillioides) WT4 in the embodiment one, and wherein the M swimming lane is represented Marker, and the WJ1 swimming lane is represented WT4;
Fig. 3 is the phyletic evolution tree graph of broom shape aspergillus (Aspergillus penicillioides) WT4 in the embodiment one;
Fig. 4 is the HPLC color atlas that mixes reference substance in the embodiment one, and wherein 1 representes schisandrin, 2 expression Wuweizi ester As, 3 expression deoxyschizandrins and 4 expression Wuweizisu Bs;
Fig. 5 is the HPLC color atlas of Schisandra chinensis in the embodiment one, and wherein 1 representes schisandrin, 2 expression Wuweizi ester As, 3 expression deoxyschizandrins and 4 expression Wuweizisu Bs;
Fig. 6 is the HPLC color atlas of Schisandra chinensis after the WT4 fermentation in the embodiment one, wherein 1 expression schisandrin, 2 expression Wuweizi ester As, 3 expression deoxyschizandrins and 4 expression Wuweizisu Bs.
Embodiment
Embodiment one: this embodiment improves the endogenetic fungus of Schisandra chinensis main active ingredient content through fermentation method; It is broom shape aspergillus (Aspergillus penicillioides) WT4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M2012043, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
Broom shape aspergillus (Aspergillus penicillioides) WT4 is after cultivating 7 days in this embodiment on the PDA solid medium, mycelia quality velvet shape, the conidium structure that tool is a large amount of; Sap green, edge white, no transudate; Light musty is arranged, and the bacterium colony reverse side is in various degree little Huang to sap green, and it is loose cylindric that the bacterial strain conidial head is; Conidiophore is elongated, top capsule flask shape, and capsule nearly clavate in minority top is when conidium is ripe spherical or subsphaeroidal (as shown in Figure 1).
Broom shape aspergillus (Aspergillus penicillioides) WT4 in this embodiment, its growth temperature is 28 ℃, growth pH value nature.
Broom shape aspergillus (Aspergillus penicillioides) WT4 in this embodiment; This strains separation is from healthy medicinal plant Schisandra chinensis plant ratan; Pick up from area, cap mountain, Heilongjiang Province; And being accredited as the plant that magnoliaceae schisandra belongs to Schisandra Chinensis (Turcz.) Baill. through the Fu Kezhi researcher of Heilongjiang Province Academy of Traditional Chinese Medicine Pharmaceutical Manufacturing Plant, sample is existing in life science institute of Heilongjiang University bio-pharmaceuticals laboratory; It carries out separation and Culture according to the following steps:
Get Schisandra chinensis healthy plant ratan, water is rinsed well, gets phloem; Alcohol-pickled 3-5min with 75%, aseptic water washing 4 times is cut into the fritter of 0.5cm with aseptic hilt rattan; Be placed on yam liquid separation culture medium surface then, placed 28 ℃ of constant temperature culture 3-7 days, treat that its notching edge grows bacterium colony after; The most advanced and sophisticated mycelia of picking fungi forwards purifying in the new substratum to, and bacterial colony carries out the doubling dilution purifying and cultivates; Be coated on the sterilized water of last flushing Schisandra chinensis on the yam solid separation culture medium surface with aseptic spreading rod in addition, in 28 ℃ of constant temperature culture, with this as control group.
The yam liquid separation culture medium:
Potato culture (PDA): every L is made up of the yam of 200g, the sucrose of 20g and the water of surplus, pH nature, 121 ℃ of autoclaving 30min; Wherein peeling potatoes is cut into small pieces and boils 30min, with 6 layers of filtered through gauze.
The yam solid separation culture medium:
Potato culture (PDA): every L is by the yam of 200g, the sucrose of 20g, and the agar powder of 16g and the water of surplus are formed, pH nature, 121 ℃ of autoclaving 30min; Wherein peeling potatoes is cut into small pieces and boils 30min, with 6 layers of filtered through gauze.
Inclined-plane solid medium: get the 5mL solid potato culture medium and be loaded in the test tube, behind 121 ℃ of autoclaving 30min, be paved into the inclined-plane cooling, in order to preserving bacterial classification.
Test is seen table 1 with key instrument equipment and reagent;
Table 1
| Instrument | Model | Production unit |
| The airbath vibrator | HZQ-C | Dongming, Harbin City Medical Instruments factory |
| The precise electronic balance | PL303 | Plum Teller-Tuo benefit Instr Ltd. |
| Electro-heating standing-temperature cultivator | DNP9082 | Shanghai accurate experimental installation ltd |
| The electric heating constant temperature air dry oven | DHG-9140 | Shanghai accurate experimental installation ltd |
| Portable pressuresteam sterilization pot | YXQ-SG46 | Zhenghai prosperous medicine equipment of gold ltd |
| Bechtop | SZX | Nantong scientific instrument factory |
| Electrothermal oven | 800W | Yongfeng, Harbin City electrical apparatus factory |
| Agar powder | Analytical pure | Haiyang Chemical Plant, Qingdao |
| NaCl | Analytical pure | Tianjin recovery fine chemistry industry institute |
| Sucrose | Analytical pure | Tianjin recovery fine |
| 95% ethanol | Analytical pure | Tianjin Jin Dong days positive fine chemistry chemical reagent works |
The result: from northern schisandra stems, isolate endogenetic fungus WT4, and in the negative control contrast dull and stereotyped with the control liquid substratum in all do not have any bacterium and grow, repeatedly repeat all so, prove that the bacterium of being assigned to is a plant endogenesis epiphyte, rather than surperficial epiphyte.
Molecular Identification: WT4 fermented liquid suction filtration gets mycelium with 25% ethanol rinsing 2 times, the deionized water rinsing of sterilization 2 times, the centrifugal supernatant that goes; Carry out the segmental pcr amplification of purpose after extracting total DNA, the PCR product that will contain target stripe all carries out point sample again, the agarose gel electrophoresis with 2%; Under uv lamp, the purpose band is downcut with scalper; Reclaim test kit with DNA glue and reclaim, preparation competence Bacillus coli cells, with the PCR product with after pMD 18-T carrier is connected; Connect product and join in the competent cell, carry out bacterium colony PCR checking.The PCR that picking list bacterium colony carries out the intestinal bacteria transformant detects, prove the purpose fragment successfully transforms get in the competent cell after, will clone positive strain and serve extra large living worker's biotechnology Services Co., Ltd and check order.The nucleotides sequence of being measured is listed in the ncbi database Application of B LAST and analyzes and carry out homology relatively; And select the 18S rDNA sequence application software MEGA5.03 constructing system of corresponding kind representative strain to grow according to the ultimate principle of molecular systematics research and set, confirm the classification position of aimed strain.
The WT4 genome is after the extraction of worker UNIQ-10 pillar fungal gene group extraction agent box is given birth in Shanghai; The gel imaging result is as shown in Figure 2 after adopting pcr amplification; Near 1300bp (base sequence is seen SEQ ID NO:1), obtain an amplified fragments, prove successfully from the WT4 bacterial strain, to extract its genomic dna.
ITS sequence according to the WT4 bacterial strain is committed to the NCBI webpage; Through Blast search and the high sequence of resulting sequence similarity; And through MAGA5.03 software component systematic evolution tree (as shown in Figure 3); The ITS sequence of WT4 bacterial strain and the similarity of Aspergillus bacterial strain have all reached more than 98%, and combining form is learned observations, confirms that the WT4 bacterial strain is broom shape aspergillus (Aspergillus penicillioides).
The HPLC method is measured the dynamic change of endogenetic fungus WT4 to its effective constituent before and after the Schisandra chinensis fermentation:
1. the preparation of trial-product and reference substance solution
(1) chromatographic condition chromatographic column: Venusil XBP-C
18Post (post 4.6mm * 250mm, 5 μ m, USA); Moving phase: methyl alcohol-acetonitrile-water (33: 33: 34); Flow velocity: 1mLmin
-1Column temperature: 25 ℃; Detect wavelength: 250nm; Sample size: 10 μ L;
(2) preparation of reference substance solution
The preparation of reference substance solution: precision takes by weighing schisandrin, Wuweizi ester A, deoxyschizandrin and Wuweizisu B standard substance, and each is an amount of, is that solvent is processed the reference substance storing solution that mass concentration is 1.0mg/mL respectively with methyl alcohol.Accurately measure above-mentioned each 1mL of reference substance storing solution, processing mass concentration is the mixing reference substance solution that is 0.25mg/mL;
(3) need testing solution preparation
The preparation of fermented sample: get the exsiccant Schisandra chinensis and pulverize, cross 60 mesh sieves.Accurately take by weighing the Schisandra chinensis 15g after the pulverizing, place Erlenmeyer flask, add water, after the sealing,, take out, as substrate in 121 ℃ of autoclaving 30min according to 1: 5 solid-liquid ratio;
With endogenetic fungus WT4 activation in PDA substratum separately, in the endogenetic fungus WT4 that activation is good, add sterilized water, process 1 * 10
7The bacteria suspension of CFU/mL.Each bacteria suspension of 5mL is joined in the substrate after the sterilization; Other gets substrate and adds the 5mL sterilized water as the blank sample, and each 10 parts of parallel sample are put into shaking table in 37 ℃ with all samples, cultivate 15 days, take out, and freeze-drying, subsequent use.
The preparation of positive reference substance solution: get exsiccant Schisandra chinensis fruit, pulverize, cross 40 mesh sieves.Precision takes by weighing 3 parts of each 1.0g of Schisandra chinensis powder, accurately adds 10mL methyl alcohol, weighs, and supersound extraction 60min takes out, and puts coldly, supplies the solvent that subtracts mistake with methyl alcohol, with behind the 0.45 μ m filtering with microporous membrane as positive reference substance solution.
The preparation of fermented sample need testing solution: precision takes by weighing the Schisandra chinensis sample 1.0g after endogenetic fungus WT4 fermentation; The accurate 10mL methyl alcohol that adds, supersound extraction 60min takes out; Put cold; Supply the solvent that subtracts mistake with methyl alcohol, with 0.45 μ m filtering with microporous membrane, i.e. Schisandra chinensis fermented sample need testing solution.
The preparation of blank article solution: get blank sample 1.0g, the accurate 10mL methyl alcohol that adds, supersound extraction 60min takes out, and puts coldly, supplies the solvent that subtracts mistake with methyl alcohol, with 0.45 μ m filtering with microporous membrane, i.e. blank article solution.
The preparation of fermented liquid reference substance solution: precision is measured each 100mL of Schisandra chinensis endogenetic fungus WT4 fermented liquid, and rotary evaporation is concentrated into 10mL, with 0.45 μ m filtering with microporous membrane, is the fermented liquid reference substance solution.
(4) sample determination is under " (1) " chromatographic condition, and precision is drawn 10 μ L (three times are parallel) reference substance solution and each need testing solution respectively, sample introduction, and the record peak area is by external standard method calculating content.
2. detected result
1. the separation of shizandra berry effective constituent under the chromatographic condition
Under " (1) " chromatographic condition; Schisandrin, Wuweizi ester A, deoxyschizandrin and Wuweizisu B are all separated preferably in the trial-product; Not only the RT of its chromatographic peak is consistent with reference substance, and each chromatographic peak has also all obtained confirming that preferably the result sees Fig. 4 through standard addition method; Fig. 5, Fig. 6.
2. endogenetic fungus WT4 is to the dynamic change of its effective constituent before and after the Schisandra chinensis fermentation
The content analytical results of Schisandra chinensis main 4 kinds of lignanoid's activeconstituentss before and after endogenetic fungus WT4 fermentation is seen table 2.
Table 2
Annotate: compare with positive (crude drug) control group
*P＜0.05,
*P＜0.01;
Compare △ P＜0.05 with the blank group, △ △ P＜0.01.
Conclusion: adopt Schisandra chinensis endogenetic fungus WT4 that Schisandra chinensis is fermented; The HPLC method is measured the content of the 4 kinds of main active ingredient in Schisandra chinensis fermentation front and back; The result shows that Schisandra chinensis endogenetic fungus WT4 can make the Schisandra chinensis main active ingredient be able to a certain extent significantly improve.
Claims (1)
1. a strain improves the endogenetic fungus of Schisandra chinensis main active ingredient content through fermentation method; It is characterized in that it is broom shape aspergillus (Aspergillus penicillioides) WT4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M2012043, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210126169 CN102660466B (en) | 2012-04-26 | 2012-04-26 | Endophytic fungi for improving content of main active ingredients of schisandra chinensis through fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210126169 CN102660466B (en) | 2012-04-26 | 2012-04-26 | Endophytic fungi for improving content of main active ingredients of schisandra chinensis through fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102660466A true CN102660466A (en) | 2012-09-12 |
| CN102660466B CN102660466B (en) | 2013-06-12 |
Family
ID=46770048
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210126169 Expired - Fee Related CN102660466B (en) | 2012-04-26 | 2012-04-26 | Endophytic fungi for improving content of main active ingredients of schisandra chinensis through fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102660466B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966109A (en) * | 2014-05-23 | 2014-08-06 | 黑龙江大学 | Schisandra fruit endophytic fungus strain capable of producing protocatechuic aldehyde |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280279A (en) * | 2008-04-09 | 2008-10-08 | 东北林业大学 | Phomopsis capable of producing gallic acid |
| CN102061265A (en) * | 2010-11-19 | 2011-05-18 | 天津工业大学 | Method for separating and screening anti-tumor active endophytic fungi of schisandra |
-
2012
- 2012-04-26 CN CN 201210126169 patent/CN102660466B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280279A (en) * | 2008-04-09 | 2008-10-08 | 东北林业大学 | Phomopsis capable of producing gallic acid |
| CN102061265A (en) * | 2010-11-19 | 2011-05-18 | 天津工业大学 | Method for separating and screening anti-tumor active endophytic fungi of schisandra |
Non-Patent Citations (2)
| Title |
|---|
| 崔宇等: "药用植物内生菌研究概况", 《黑龙江医药》 * |
| 郑春英等: "药用植物五味子内生真菌的分离及其抑菌活性研究", 《中国药学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966109A (en) * | 2014-05-23 | 2014-08-06 | 黑龙江大学 | Schisandra fruit endophytic fungus strain capable of producing protocatechuic aldehyde |
| CN103966109B (en) * | 2014-05-23 | 2016-04-13 | 黑龙江大学 | The shizandra berry endogenetic fungus of rancinamycin IV is produced in one strain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102660466B (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102994401B (en) | A kind of method preparing apple rot pathogen transformant and GFP labeled strain | |
| CN102643756B (en) | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice | |
| CN105219655A (en) | Flavus biocontrol strain and the application thereof of aflatoxin do not produced to sterilant resistance | |
| CN102660467B (en) | Liquorice endophytic fungus for producing glycyrrhetinic acids | |
| CN102220247B (en) | Glycyrrhiza endophytic fungi for producing glycyrrhetic acid | |
| CN104472221B (en) | A kind of method by natural cordyceps fructification fermented hypha | |
| CN102676404B (en) | Endophytic fungi capable of improving content of isofraxidin in Radix Acanthopanacis Semticosi through fermenting Radix Acanthopanacis Semticosi | |
| CN102660466B (en) | Endophytic fungi for improving content of main active ingredients of schisandra chinensis through fermentation method | |
| Hao et al. | Isolation and identification of swainsonine-producing fungi found in locoweeds and their rhizosphere soil | |
| CN102676405B (en) | Liquorice endophytic fungus for generating liquiritin | |
| CN102628018B (en) | Strain of endophytic fungus of fermenting schisandra chinensis to improve main active components of schisandra chinensis | |
| CN103146614B (en) | Camptotheca endophytic bacterium LY214 for producing camptothecin and application thereof | |
| CN1702176A (en) | One-step dual PCR method for detecting fire blight of pear | |
| CN105695335A (en) | New aspergillus flavus and use thereof | |
| CN105316240A (en) | Rhizopycnis sp. NYN8G01 and application thereof | |
| CN104278070A (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| CN106032523A (en) | Simple method for separating and purifying apple ring spot bacteria | |
| CN102643755B (en) | Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice | |
| CN105238697A (en) | Peony endophytic fungi and process for producing paeonol with peony endophytic fungi | |
| CN103849572A (en) | Endophytic fungus strain YCEF005 and application thereof | |
| CN103074236A (en) | Camptotheca endophytic fungi for producing camptothecin and application thereof | |
| CN102943103B (en) | Penicillium fungus M1 and application thereof to increase of saponins yield in fermentation process of ginseng or Americginseng | |
| CN103966109B (en) | The shizandra berry endogenetic fungus of rancinamycin IV is produced in one strain | |
| CN103849573A (en) | Strain YCEF053 of endophytic fungus and use thereof | |
| CN113736669B (en) | Cordyceps sinensis fungus new species and separation and identification method thereof |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130612 Termination date: 20180426 |