CN101875905B - Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof - Google Patents
Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof Download PDFInfo
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- CN101875905B CN101875905B CN2009102374094A CN200910237409A CN101875905B CN 101875905 B CN101875905 B CN 101875905B CN 2009102374094 A CN2009102374094 A CN 2009102374094A CN 200910237409 A CN200910237409 A CN 200910237409A CN 101875905 B CN101875905 B CN 101875905B
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Abstract
The invention discloses a mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof. The mao bamboo seed endophytic fungi strain (strain number is ZZZ-817) capable of efficiently producing the hypocrellin is obtained by solid culture, shaking flask fermentation and extract measurement of separated mao bamboo seed endophytic fungi strains from Guangxi, and the microbial preservation number of the strain is CGMCC No: 3135. When the mao bamboo seed endophytic fungi strain is used for producing the hypocrellin by fermentation, 1,760.9 milligrams of hypocrellin can be obtained in each liter of fermentation liquor, and the extraction rate of the hypocrellin is as high as 152.4 percent. Compared with the conventional related research on the content of hypocrellin active substances produced by microbes, the hypocrellin obtained by the strain of the invention has high content, and the strain can realize scale production of the hypocrellin and has higher application value.
Description
Technical field
The present invention relates to a fungus strains, relate in particular to the mao bamboon seed endogenetic fungal bacterial strain that a strain can the High-efficient Production hypocrellin, the invention still further relates to the application of this mao bamboon seed endogenetic fungal bacterial strain in the preparation hypocrellin, belong to the preparation field of hypocrellin.
Background technology
Hypocrellin (hypocrellin) is the main effective constituent of little bamboo fless and tabasheer sporophore; belong to perylene quinone compound; It is present known photosensitizers good in visible region; Have significant Phototherapy, be used for treating tetter clinically, like leukoplakia vulvae and softening keloid.Recent study finds that it has good photosensitive killing tumor cell and the effect that suppresses hiv virus HIV-1, and as novel photo-activation pesticide and potential photoelectric conversion material, has obtained the extensive concern of biological educational circles and medical circle.
At present both at home and abroad the pharmacological action and the using value of hypocrellin have been launched research extensively and profoundly, and obtained a lot of substances and breakthrough progress, market is also increasing to the demand of hypocrellin.Up to now, hypocrellin is directly from little bamboo fless and bamboo parasitic fungus mushroom entity, to separate, and little bamboo fless only is distributed in partial area, Yunnan, yields poorly; Though it is wider that bamboo parasitic fungus is taken place; But spontaneous generation often receives the influence of the multiple factors such as weather, host; And " harvesting " can cause resource exhaustion every year; Therefore directly utilize little bamboo fless and tabasheer sporophore to extract activeconstituents and often receive multiple restriction, be difficult to extensive industrialization and be used, utilize microorganism strains fermentative prodn hypocrellin can get rid of above-mentioned drawback; Be easy to suitability for industrialized production, but its prerequisite is to need to separate the microorganism strains that obtains the efficient fermentative prodn hypocrellin of ability.
Summary of the invention
The present invention's technical problem at first to be solved is the deficiency that overcomes prior art, and the efficiently bacterial strain of fermentative prodn hypocrellin of a strain is provided;
The present invention's technical problem at first to be solved realizes through following technical scheme:
The present invention carries out solid culture, shake flask fermentation and extraction and determination through the mao bamboon seed endogenetic fungal bacterial strain that a strain is separated from Guangxi; Obtained the efficiently mao bamboon seed endogenetic fungal bacterial strain of fermentative prodn hypocrellin (bacterial strain number for ZZZ-817) of a strain, its microbial preservation number is CGMCC NO:3135; Preservation date is: on June 26th, 2009; Its classification name is: (Shiraia bambusicola); Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The present invention mainly obtains above-mentioned bamboo parasitic fungus bacterial strain through following method from the mao bamboon seed endogenetic fungus separation that comes from Lingchuan town, Lingchuan County, Guangxi Zhuang Autonomous Region:
Get the mao bamboon seed and carry out surface sterilization with 75% ethanol earlier, remove surface structure, cut into 1cm under the aseptic condition
3The tissue block of size is soaked 30-60s then in 10% chlorine bleach liquor, use aseptic water washing again three times, transfers to contain on the PDA solid medium plate of (containing 0.4% paraxin), cultivates for 20 ℃, separates, and promptly gets.
Through Molecular Identification, the present invention separates the classification position that obtains mao bamboon seed endogenetic fungal bacterial strain ZZZ-817 and belongs to: Ascomycota (Ascomycota), Pseudosphaeriales (Pleosporales), tabasheer Pseudomonas (Shiraia), tabasheer (Shiraia bambusicola).
The biological characteristics of mao bamboon seed endogenetic fungal bacterial strain ZZZ-817 bacterial strain of the present invention is following:
1, host
Endogenetic fungus of the present invention separates from mao bamboon (Phyllostachys edulis (Carr.) H.De Lehaie) seed.About bamboo parasitic fungus host's scope, bibliographical information is inconsistent, and people such as vast stretch of wooded country duckweed think that host is more widely arranged; Relying people's such as radiance observation to think that then the bamboo parasitic fungus host range is very narrow, mainly is short fringe Sinobambusa kind.Hou Chenglin etc. find that in some forest farm investigation of bamboo kind garden, Guangde County, Anhui Province and Jingde County bamboo parasitic fungus is generally taken place in recent years on short fringe bamboo, but other various bamboo kinds that are adjacent comprise that the member that Phyllostachys and fishscale bamboo belong to but has no symptom.But the report that does not also have bamboo parasitic fungus on the relevant mao bamboon up to now.
2, cultivate proterties
Mao bamboon endophyte bacterial strain ZZZ-817 is incubation growth on 26 ℃, PDA solid medium.Cover with the Pi Shi plate about about 10d, be the concentric annular growth, the speed of growth is through measuring about 0.336mm/d.
According to formula:
The average daily speed of growth (the mmd of mycelium
-1)=colony radius increases length (mm)/cultivation fate (d)
Another technical problem to be solved by this invention is that above-mentioned mao bamboon seed endogenetic fungus ZZZ-817 bacterial strain is applied to produce hypocrellin;
Another technical problem to be solved by this invention realizes through following technical scheme:
The application of mao bamboon seed endogenetic fungus ZZZ-817 bacterial strain in producing hypocrellin comprises:
(1) mao bamboon seed endogenetic fungus ZZZ-817 inoculation being carried out flat board to solid medium cultivates;
(2) picking bacterium piece inserts in the liquid seed culture medium from flat board, cultivates;
(3) liquid seeds of enlarged culturing is linked into carries out fermentation culture in the liquid shaking bottle fermention medium;
(4) mycelium is separated with fermented liquid, obtain mycelium; From mycelium, extract again and obtain hypocrellin.
Wherein, the solid medium described in the step (1) is preferably the PDA substratum;
The composition of the liquid seed culture medium described in the step (2) is preferably the potato glucose substratum, and its compound method comprises: yam (peeling) 200g, and stripping and slicing digestion 20min gets filtrating and adds glucose 20g, adds water to 1000mL, the pH nature; Sterilization promptly gets.
Cultivation described in the step (2) is preferably cultivated under following condition: rotating speed is 150r/min, 26 ℃ of culture temperature, incubation time 60h;
The composition of the liquid shaking bottle fermention medium described in the step (3) is preferably: peeling yam 200g, stripping and slicing digestion 20min gets filtrating, adds glucose 20g, KH
2PO
41g, MgSO
40.5g and CuSO
40.03g, add water to 1000mL, adjust pH to 6; Sterilization promptly gets.
Fermentation culture described in the step (3) is preferably carried out fermentation culture under following condition: rotating speed 150r/min, 26 ℃ of culture temperature, incubation time 144h;
The method that obtains hypocrellin of from mycelium, extracting described in the step (3) is preferably: mycelium is used purified rinse water, suction filtration; Collect mycelium, freeze overnight is dried to constant weight through vacuum freeze drying again; Exsiccant mycelium powder pure (with acetone through the apparatus,Soxhlet's) about 4～5h of refluxing extraction is to colourless, concentrated, promptly get.
Test-results shows that use mao bamboon seed endogenetic fungal bacterial strain fermentative prodn hypocrellin of the present invention, every liter of fermented liquid can obtain hypocrellin 1760.9mg, and the extraction yield of hypocrellin is up to 152.4%.With some scholars were to the correlative study comparison of bamboo parasitic fungus product active substance hypocrellin content in the last few years, bacterial strain gained hypocrellin content of the present invention is higher, can accomplish scale production, and has higher using value.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Embodiment
1, the moity of used substratum and preparation:
1) solid medium (potato dextrose agar, PDA substratum): yam (peeling) 200g, stripping and slicing digestion 20min gets filtrating, adds glucose 20g and agar 18g, adds water to 1000mL, pH nature, 1 * 10
5The Pa 30min that sterilizes promptly gets.
2) liquid seed culture medium (potato glucose substratum): yam (peeling) 200g, stripping and slicing digestion 20min gets filtrating and adds glucose 20g, adds water to 1000mL, pH nature, 1 * 10
5The Pa 30min that sterilizes promptly gets.
3) liquid shaking bottle fermention medium: peeling yam 200g, stripping and slicing digestion 20min gets filtrating, adds glucose 20g, KH
2PO
41g, MgSO
40.5g and CuSO
40.03g, add water to 1000mL, pH6,1 * 10
5The Pa 30min that sterilizes promptly gets.
2, cultivation and detection method
1) the dull and stereotyped cultivation: solid medium, places the petridish central position to carry out flat board with inoculating needle picking mao bamboon seed of the same size endogenetic fungus ZZZ-817 bacterial strain bacterium piece and cultivates 26 ℃ of constant temperature after the conventional sterilization of 30min through 121 ℃.
2) liquid seeds enlarged culturing: the suitable mao bamboon seed endogenetic fungus ZZZ-817 bacterial strain bacterium piece of picking 5 piece sizes inserts in the seed culture medium the plate that covers with mycelia after activation, and liquid amount is 80mL/250mL, and rotating speed is 150r/min, 26 ℃ of cultivations, 60h.
3) shake flask fermentation is cultivated: the liquid seeds of enlarged culturing is inserted in the fermentation culture triangular flask by 10% inoculum size, and liquid amount 100mL/250mL, rotating speed 150r/min cultivates 144h for 26 ℃.
4) test sample is treated in preparation: with fermenting culture through the B suction filtration separate mycelium and fermented liquid, discard fermented liquid, mycelium is with purified rinse water three times; Suction filtration; Collect mycelium and put into petridish, freeze overnight, again through the dry 1250min of vacuum freeze drying to constant weight.Dried mycelium is placed on vacuum drier and preserves, and is subsequent use.
5) hypocrellin extracts: exsiccant mycelium powder to be measured is pure, and the filter paper parcel.Extremely colourless with acetone through the about 4～5h of apparatus,Soxhlet's refluxing extraction, concentrate through Rotary Evaporators again, acetone is heavily molten, and constant volume 4 ℃, keeps in Dark Place to 20mL.
6) detection of hypocrellin extraction yield: make solvent with acetone, adopt spectrophotometry to measure the light absorption value that extracts liquid to be measured in the 465nm wavelength.The OD value is between 0.2-0.8.Try to achieve concentration according to typical curve, calculate output (explain: the hypocrellin standard substance are provided by Yunnan Province Institute of Micro-biology, and purity is more than 98%).
Hypocrellin extraction yield (%)=(extractive content mg/ mycelium dry weight g) x100
Test-results shows, adopts mao bamboon seed endogenetic fungus ZZZ-817 bacterial strain of the present invention to ferment, and can obtain hypocrellin 1760.9mg in every liter of fermented liquid, and the extraction yield of hypocrellin is up to 152.4%.
Claims (9)
1. a plant height produces the mao bamboon seed endogenetic fungus bamboo parasitic fungus bacterial strain (Shiraia bambusicola) of hypocrellin, and its microbial preservation number is CGMCC NO:3135.
2. the purposes of the described mao bamboon seed of claim 1 endogenetic fungus bamboo parasitic fungus bacterial strain in the fermentative prodn hypocrellin.
3. according to the described purposes of claim 2, it is characterized in that, may further comprise the steps:
(1) said mao bamboon seed endogenetic fungus bamboo parasitic fungus inoculation is carried out the flat board cultivation to solid medium;
(2) picking bacterium piece inserts in the liquid seed culture medium from flat board, cultivates, and obtains liquid seeds;
(3) liquid seeds is linked into carries out fermentation culture in the liquid shaking bottle fermention medium;
(4) mycelium is separated with fermented liquid, from mycelium, extract again and obtain hypocrellin.
4. 3 described purposes as requested, it is characterized in that: the solid medium described in the step (1) is the PDA substratum.
5. 3 described purposes as requested, it is characterized in that: the compound method of the liquid seed culture medium described in the step (2) is: peeling yam 200g, stripping and slicing digestion 20min gets filtrating, adds glucose 20g, adds water to 1000mL, the pH nature.
6. 3 described purposes as requested, it is characterized in that the cultivation described in the step (2) is under following condition, to cultivate: rotating speed is 150r/min, 26 ℃ of culture temperature, incubation time 60h.
7. 3 described purposes as requested, it is characterized in that the compound method of the liquid shaking bottle fermention medium described in the step (3) is: peeling yam 200g, stripping and slicing digestion 20min gets filtrating, adds glucose 20g, KH
2PO
41g, MgSO
40.5g and CuSO
40.03g, adding water to 1000mL, adjust pH is 6.
8. 3 described purposes as requested is characterized in that the fermentation culture described in the step (3) is carried out fermentation culture under following condition: rotating speed 150r/min, 26 ℃ of culture temperature, incubation time 144h.
9. 3 described purposes as requested is characterized in that, the method that obtains hypocrellin of from mycelium, extracting described in the step (4) comprises: mycelium is used purified rinse water, suction filtration; Collect mycelium, freeze overnight is dried to constant weight through vacuum freeze drying again; The exsiccant mycelium powder is pure extremely colourless with acetone refluxing extraction 4～5h, concentrate, promptly get.
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| CN2009102374094A CN101875905B (en) | 2009-11-06 | 2009-11-06 | Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof |
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| CN2009102374094A CN101875905B (en) | 2009-11-06 | 2009-11-06 | Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof |
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| CN101875905B true CN101875905B (en) | 2012-07-25 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102465154B (en) * | 2010-11-04 | 2013-12-04 | 苏州大学 | Method for improving hypocrellin yield in shiraia bambusicola |
| CN103898188B (en) * | 2014-03-28 | 2016-04-13 | 浙江大学 | LC-MS studies sample preparation and the detection method of Pyricularia oryzae metabolism group |
| CN110172409B (en) * | 2019-04-26 | 2020-04-28 | 华南理工大学 | Shiraia bambusicola strain capable of highly producing hypocrellin A and application thereof |
| CN111218407B (en) * | 2020-01-15 | 2020-12-08 | 山东国力生物科技有限公司 | Shiraia bambusicola with high yield of hypocrellin and culture method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186932A (en) * | 2007-11-11 | 2008-05-28 | 杨海龙 | Method for synchronously producing hypocrellin and shiraia bambusicola polysaccharides |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186932A (en) * | 2007-11-11 | 2008-05-28 | 杨海龙 | Method for synchronously producing hypocrellin and shiraia bambusicola polysaccharides |
Non-Patent Citations (2)
| Title |
|---|
| 刘永翔等.一种简单的竹黄菌（Shiraia bambusicola）单孢分离方法.《菌物研究》.2008,第6卷(第1期),第49-56页. * |
| 梁晓辉等.药用真菌竹黄的研究进展.《食品与生物技术学报》.2008,第27卷(第5期),第21-26页. * |
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