CN101942394A - Verticillium Paecilomyces varioti strain and application thereof - Google Patents
Verticillium Paecilomyces varioti strain and application thereof Download PDFInfo
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- CN101942394A CN101942394A CN 201010187300 CN201010187300A CN101942394A CN 101942394 A CN101942394 A CN 101942394A CN 201010187300 CN201010187300 CN 201010187300 CN 201010187300 A CN201010187300 A CN 201010187300A CN 101942394 A CN101942394 A CN 101942394A
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Abstract
The invention discloses a Verticillium Paecilomyces varioti strain. When the strain is treated on a czapek's medium at 25 DEG C for 14 days, the bacterial colony has a diameter of 35-55 mm, is raised, is of white villus, is in an irregular shape and has a white back side. Hyphae have a width of 1.2-1.8 mu m and can form conidiophores with a length of 9.6-19.8 mu m, majority of the phialides are cylindrical or tapered and are often whorled or alternate and grow on aerial hyphae in opposite, the hyphae gradually or suddenly tapers upwards and have a thin neck of 7.8-14.4*1.2-2.4 mu m, and the length is almost 1.2 times longer than that of the base of the Verticillium Paecilomyces varioti. The conidiophores are transparent and smooth and can be distinctly divided into two spores with different sizes, wherein the macro spores are in the shape of fusiformis or ellipse and have a size of 1.8-3.0*1.8-2.4 mu m, and the micro spores are subglobose or elliptic, have a size of 1.2-1.8*0.6-1.2 mu m and can form a linear chain or cluster. The invention also discloses an application of the Verticillium Paecilomyces varioti strain. The strain can be used for producing Cordycepin by fermentation and has the advantages of easy culture, storage and industrial production.
Description
Technical field
The present invention relates to biological technical field, relate in particular to wheel branch Paecilomyces varioti bacterial strain, also relate to its purposes simultaneously.
Background technology
Cordycepin (cordycepin) is the ucleosides antibiont that a class is separated from Cordyceps militaris (L.) Link. first, has multiple biological activity value, and Cordyceps militaris (L.) Link. can be used as the substitute of Cordyceps sinensis, the authentication of passing through new food in 2009.Along with market is more and more to the demand of cordycepin, and cordycepin content is not high in the wild Cordyceps militaris (L.) Link., generally 0.1% or lower, is difficult to satisfy commercial cordycepin content and will reaches 2% requirement.Along with the price of wild Cordyceps sinensis rises on the way, risen at present to about 200,000 yuan of per kilograms, make wild cordyceps militaris be subjected to mad the harvesting.So consider from the conservation of resources aspect, the Cordyceps militaris (L.) Link. that searching can be produced cordycepin substitutes bacterial strain, enlarges the medicine source of cordycepin, is the problem that the utmost point need solve.
Summary of the invention
A kind of bacterial strain that the objective of the invention is to overcome above-mentioned shortcoming and provide is cultivated easily and is preserved and suitability for industrialized production, can produce the wheel branch Paecilomyces varioti bacterial strain of cordycepin.
Another object of the present invention is to provide this to take turns the purposes of a Paecilomyces varioti bacterial strain aspect the product cordycepin.
This bacterial classification is deposited in Chinese typical culture collection center (address: Chinese Wuhan Wuhan University) on December 14th, 2009, and name is called: a wheel branch Paecilomyces varioti (Paecilomyces verticillatusGZ), preserving number: CCTCC NO:M 209302.
The Paecilomyces varioti (Paecilomyces verticillatus GZ) of taking turns of the present invention has following Microbiological Characteristics:
1, morphological characteristic: on the wheel branch Paecilomyces varioti GZ Cha Shi substratum, 25 ℃, 14 days, colony diameter 35-55mm, protuberance, white fine hair shape is irregular shape; Back side white.The wide about 1.2-1.8 μ m of mycelia, can form the conidiophore that is about 9.6-19.8 μ m, most bottle stalk columns or taper, normal verticillate or alternate and to being born on the aerial hyphae, upwards be tapered or attenuate suddenly, 7.8-14.4 * 1.2-2.4 μ m, neck is very thin, to wheel branch Paecilomyces varioti, be 1.2 double-lengths of base portion sometimes approximately.Conidium is transparent, and is smooth, and two kinds of spores that vary in size are obviously arranged, the megaspore fusiformis, and ellipse, 1.8-3.0 * 1.8-2.4 μ m, sporule is subsphaeroidal, oval, and 1.2-1.8 * 0.6-1.2 μ m becomes straight chain or poly-agglomerating.
2, cultivate characteristic: take turns branch Paecilomyces varioti GZ on sabouraud culture medium, 25 ℃, 14 days, colony diameter 35-40mm, open and flat, median rise, white, felted has the irregular zanjon line of 3-5 bar, and the edge is irregular; It is light yellow that the back side is.The wide 0.5-1.5 μ of mycelia m separates, and is transparent, smooth.Short or the nothing of conidiophore; Whorl is made up of 3-5 bottle stalk.Bottle metulae portion's column or fusiformis are expanded, and upwards attenuate gradually or suddenly; Neck is wide less than 0.5 μ m.Conidium is transparent, and is smooth, subsphaeroidal to oval, and 1.5-2.5 * 1.2-2.5 μ m is arranged in long-chain.
Wheel branch Paecilomyces varioti GZ is on the PDA solid medium, and is open and flat about the about 55mm of colony diameter, fine and close blanket shape, canescence; The back side is light yellow.
3, physiology characteristic: in pH 5～7,20 to 30 ℃ down can normal growth.
4, bacteria characteristic: according to Brown ﹠amp; The criteria for classification of Smith (1957) and paecilomyces colony morphology characteristic: hyphal development is good, the conidium bunchiness, and does not expand on the conidiophore top, sporophore top wheel shaft shape branch, vertical wheel shaft shape branch differs in size, and spore is bordering on sphere.Conidium and penicillus do not have colloid outward and cover.The conidium wall is thin, and base portion does not have the ring-type thickening, and doleiform stigma irregular arrangement push up elongatedly and crooked, and the spore clump is not green.Conidiophore and branch are than the dispersion more of Penicillium.Antagonistic strain is accredited as Paecilomyces varioti (Paecilomyces spp).
The cultural method that wheel branch Paecilomyces varioti (Paecilomyces verticillatus GZ) is produced cordycepin is as follows:
Take turns branch Paecilomyces varioti (Paecilomyces verticillatus GZ) bacterial strain on the PDA inclined-plane, 27 ℃, 7d, activate 3 times, be linked in the PDA liquid seed culture medium 140rpm, 26 ℃, shake-flask culture 3d gets the female kind of liquid, be linked in the fermention medium of white sugar 2%, maltose 1%, extractum carnis 2%, yeast extract paste 3% with 0.04 (v/v) inoculum size, 140rpm, 27 ℃, shake-flask culture 7d, obtain liquid to be measured, carry out HPLC and measure cordycepin content.
The wheel branch Paecilomyces varioti that the present invention mainly obtains based on screening (Paecilomyces verticillatus GZ), carry out complete fermenting process, the result proves that the fermentation of wheel branch Paecilomyces varioti can produce cordycepin, this to a certain extent ease people to the demand of cordycepin.
Embodiment
The screening method of wheel branch Paecilomyces varioti bacterial strain comprises the following steps:
(1) separation method of bacterial strain:
A) preparation of pedotheque bacterium liquid
In the laboratory, pick up except that foreign material such as dead twigs and withered leaves, finger stones gathering the pedotheque of drying under the natural condition, every part of soil soil sample takes by weighing 10g, be dissolved in respectively in the 250mL triangular flask that the 90mL sterilized water is housed and has granulated glass sphere, 20-30min fully vibrates, leave standstill 20min after soil sample is fully dissolved, make soil sample become soil bacteria liquid
B) preparation of soil diluent: from soil bacteria liquid, draw 1mL soil bacteria liquid with an aseptic transfer pipet, add abundant mixing in the test tube that fills the 9mL sterilized water, from then on drawing 1mL in the test tube with aseptic transfer pipet then adds in another test tube that fills the 9mL sterilized water, mix, by 10 times of method dilutions, be diluted in 10 with this type of
-1-10
-4
C) mix the inoculation of bacterium method: adopt and mix the inoculation of bacterium method, draw the 1ml suspension in diameter is 9 centimetres sterilization culture dish, pour into then and melted and be cooled to 45 ℃ two anti-Ma Dingshi substratum, fully mixing waits to solidify back inverted insulation cultivation.Cultivated 3 to 4 days for 25 ℃.
(2) separation and purification of bacterial strain
A) bacterium colony mark: treat to grow the macroscopic bacterium colony that varies in size in two anti-Ma Dingshi substratum, promptly carry out primary dcreening operation and observe, to having the bacterium colony mark of different conidial fructifications at microscopically.
B) plate streak inoculation: select for use smooth and slick and sly transfering loop picking under aseptic condition to carry out a small amount of bacterium colony spore of bacterium colony mark, and 3 to 4 successive parallel lines of A zoning on two anti-Ma Dingshi culture medium flat plates immediately, drawn the residual bacterium of burning immediately behind the A district on the transfering loop.Transfering loop behind the residual bacterium of burning-off is cooled off, and make the B district forward the top to, transfering loop arrives the B district by A district (bacterium source region) with the cingula, draw several fine and close parallel lines immediately, drawn the residual bacterium of burning immediately behind the B district on the transfering loop, and performed the mark in A district and B district, left standstill 20min with being about to flat board, streak plate is inverted in the constant incubator, and constant temperature was cultivated 3 to 4 days for 25 ℃.
C) purifying of single bacterium colony: select typically independently single bacterium colony in the B district, move and be connected on two anti-Ma Dingshi culture medium flat plates, treat to grow in the substratum the macroscopic bacterium colony that varies in size, promptly carrying out primary dcreening operation at microscopically observes, to have paecilomyces and produce the bacterium colony mark of embracing structure, and move on the Ma Dingshi flat board constant temperature culture immediately. behind purifying, be transferred to slant tube and preserve.
D) preservation of fungi: with single bacterium colony, move and be connected on the PDA slant medium, be inverted in the constant incubator, cultivated 3 to 4 days for 25 ℃, wait to observe pollute after, move in 4 ℃ refrigerator, preserve standby.
(3) evaluation of bacterial strain
A) cultivate: solid medium is made flat board,, promptly pick a small amount of spore point of antibacterial fungi and be connected on dull and stereotyped central position, observe in 25 ℃ of cultivation certain hours then with the inoculation needle point with a connection inoculation, can be with the naked eye during observation or by microscope.
B) making of sample: on clean glass slide, drip the cotton blue staining fluid of lactic acid phenylic acid, get a little mycelia that has spore with dissecting needle in dye liquor from the edge of mold colony, carefully mycelia is chosen into state of nature again, cover with cover glass then, note not producing bubble.In warm kiln, put a few days, allow a moisture evaporation part, cover glass and slide glass are close to, get final product mounting.During mounting, clean around the cover glass earlier, and around cover glass, be coated with a circle balsam or a synthetic gum with the gauze or the absorbent cotton of cleaning, air-dry back the sealing sample, in oily mirror observation conidial fructification feature down.
C) photomicrograph: under the Motic digit microscope, observe and choose the visual field of tool typical case properties and characteristics and take pictures, directly measure related data and be described record.
D) identification of morphology of bacterial classification: with reference to Raper (1965) and Brown ﹠amp; Categorizing system and the criteria for classification of Smith (1957) adopt the intersection selected characteristic, mainly cultivate proterties (colony characteristics) according to macroscopic, and individual morphology feature and some biological characteristicses of microcosmic carry out classical sort research.The research pertinent literature, the similarities and differences of analysis-by-synthesis contrast correlated characteristic are carried out identification of morphology to the bacterial strain that screens.
The cultural method of wheel branch Paecilomyces varioti bacterial strain may further comprise the steps:
(1) actication of culture
To take turns branch Paecilomyces varioti (Paecilomyces verticillatus GZ) bacterial classification and be connected on the PDA inclined-plane, 26 ℃ leave standstill and cultivated 7 days, transfer continuously 2 times, and it is standby to be stored in 4 ℃ of refrigerators.
(2) preparation of spore suspension
The bacterial strain that activation is good washes spore with aseptic tween physiological saline, changes in the aseptic triangular flask that has granulated glass sphere, and spore is fully broken up in vibration, removes by filter mycelia with 4 layers of aseptic lens paper, makes the homogeneous spore suspension.
(3) preparation of seed liquor
With spore suspension, be inoculated in the seed culture fluid with 5% (v/v) inoculum size, 26 ℃, 140rpm, shake-flask culture three days.
(4) fermentation culture
Getting seed liquor 5% (v/v) is inoculated in the fermention medium: white sugar 2%, maltose 1%, extractum carnis 2%, yeast extract paste 3%, 140rpm, 26 ℃, shake-flask culture 7 days.
Wheel branch Paecilomyces varioti bacterial strain produces the cordycepin experiment:
(1) preparation of liquid A liquid to be measured
Wheel branch Paecilomyces varioti (Paecilomyces verticillatus GZ) fermentation is after 7 days, in fermented liquid and mycelium transposition centrifuge tube together, the centrifugal 20min of 4000r/min carries out bacterium liquid and separates, get its supernatant liquor and after 0.45um water micro-filtrate membrane filtration is removed impurity, refrigerate, to be measured.
(2) preparation of liquid B liquid to be measured
A wheel branch Paecilomyces varioti (Paecilomyces verticillatus GZ) fermentation is after 7 days, in fermented liquid and mycelium transposition centrifuge tube together, and the centrifugal 20min of 4000r/min, carry out bacterium liquid and separate, remove its supernatant liquor, wash mycelia with distilled water, centrifugal again, so repeat secondary.Substantially the mycelium that is not had substratum is placed in the reduced vacuum loft drier, dries to constant weight for 55 ℃, grinds, and crosses 60 mesh sieves, gets hypha powder.Precision takes by weighing dried hypha powder 0.10g and places the sand pipe, adds 20% ethanol 5ml, and supersound process 30min is through 0.45um water micro-filtrate membrane filtration, to be measured.
(3) preparation of cordycepin reference liquid
Precision weighs the 1.00mg cordycepin, places the 10mL volumetric flask, is settled to scale with the dissolving of first solution, promptly gets the cordycepin standardized solution, places in the refrigerator standby.
(4) HPLC measures
HPLC measures the 1100 HPLC analyzing and testing with Agilent, by Ultraviolet Detector, chromatographic column adopting Kromasil c18 (4.6mmx250mm, 5ul praticle size, Scientific systems Inc., AP, USA); Moving phase is 0.272gKH
2PO
4Be dissolved in methyl alcohol/distilled water (15: 85), 40 ℃ of column temperatures, flow velocity are 0.8mL/min, and the detection wavelength is 259nm, and sample size is 10ul; Appearance time is about 4.60min.
The sample of measuring with HPLC goes out the content of peak area and known cordycepin standard specimen, calculates the concentration of testing sample, the relative content of calculating cordycepin according to the concentration and the extension rate of lixiviate again according to following formula.
The C sample is the cordycepin concentration of testing sample
The A sample is the peak area of testing sample
A is designated as the peak area of cordycepin standard specimen
0.10mg/ml be the concentration of cordycepin standard specimen
Through HPLC bioassay standard product (Fig. 1) and wheel branch Paecilomyces varioti (Paecilomyces verticillatus GZ) (Fig. 2) and Cordyceps militaris (L.) Link. Paecilomyces varioti (Peacilomyces militaris 08-01) HPLC figure (Fig. 3).
Claims (2)
1. branch Paecilomyces varioti (Paecilomyces verticillatus GZ) CCTCC NO:M209302 is taken turns in a strain.
2. the purposes of a Paecilomyces varioti aspect the product cordycepin of taking turns as claimed in claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865895A (en) * | 2018-06-15 | 2018-11-23 | 浙江工业大学 | Paecilomyces hepiali chen ZJB18001 and its application |
| CN109799221A (en) * | 2019-01-07 | 2019-05-24 | 北京青木子科技发展有限公司 | A kind of removable teaching Raman spectroscopy system and its control method |
| CN111157312A (en) * | 2020-01-08 | 2020-05-15 | 中华人民共和国京唐港海关 | Method for manufacturing permanent fungus slide specimen |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472330A (en) * | 2003-06-23 | 2004-02-04 | 贵州大学 | Method for increasing content of cordyceps sinensis |
| CN101531968A (en) * | 2008-05-04 | 2009-09-16 | 贵州大学 | Method for improving output of cordyceps militars fruiting body and cordycepin by adopting red yeast rice synergistic fermentation |
| CN101962665A (en) * | 2010-08-03 | 2011-02-02 | 贵州大学 | Method for improving yield of cordycepin in Verticillium Paecilomyces GZ |
-
2010
- 2010-09-28 CN CN 201010187300 patent/CN101942394A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472330A (en) * | 2003-06-23 | 2004-02-04 | 贵州大学 | Method for increasing content of cordyceps sinensis |
| CN101531968A (en) * | 2008-05-04 | 2009-09-16 | 贵州大学 | Method for improving output of cordyceps militars fruiting body and cordycepin by adopting red yeast rice synergistic fermentation |
| CN101962665A (en) * | 2010-08-03 | 2011-02-02 | 贵州大学 | Method for improving yield of cordycepin in Verticillium Paecilomyces GZ |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865895A (en) * | 2018-06-15 | 2018-11-23 | 浙江工业大学 | Paecilomyces hepiali chen ZJB18001 and its application |
| CN109799221A (en) * | 2019-01-07 | 2019-05-24 | 北京青木子科技发展有限公司 | A kind of removable teaching Raman spectroscopy system and its control method |
| CN111157312A (en) * | 2020-01-08 | 2020-05-15 | 中华人民共和国京唐港海关 | Method for manufacturing permanent fungus slide specimen |
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Open date: 20110112 |