CN108865895A - Paecilomyces hepiali chen ZJB18001 and its application - Google Patents
Paecilomyces hepiali chen ZJB18001 and its application Download PDFInfo
- Publication number
- CN108865895A CN108865895A CN201810618920.8A CN201810618920A CN108865895A CN 108865895 A CN108865895 A CN 108865895A CN 201810618920 A CN201810618920 A CN 201810618920A CN 108865895 A CN108865895 A CN 108865895A
- Authority
- CN
- China
- Prior art keywords
- zjb18001
- paecilomyces hepiali
- cordycepin
- application
- hepiali chen
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/79—Paecilomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of peacilomyce hepiahi bacterium strains and its application for producing cordycepin, described peacilomyce hepiahi bacterium strain (Paecilomyces hepial) ZJB2018001 be it is separating obtained from wild type cordyceps sinensis, separated, purified by the cordyceps sinensis to wild type, identified and obtain peacilomyce hepiahi bacterium strain.And cordycepin content is made to improve about 3 times by carrying out physics and chemistry behavior to this plant of bacterium.
Description
(1) technical field
The screening that the present invention relates to one plant from cordyceps sinensis fungal component Paecilomyces hepiali chen cordycepin superior strain and its
Using.
(2) background technique
Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is that section ergot fungus cordyceps sinensis bacterium is parasitic
Stroma on Lepidoptera (Lepidoptera) Hepialidae insect (Hepialus armoricanus Oberthur) larva
And the complex (including stroma and polypide) in larva corpse.Cordyceps sinensis is a kind of civil usual rare tonic in China
Material, nutritional ingredient is higher than ginseng, can be used as medicine, also edible, is superior delicacies, has very high nutritive value, and has and exempts from
The extensive pharmacological actions such as epidemic disease adjusting, antibacterial, antitumor, anti-oxidant, anti-aging, reducing blood sugar and blood lipid.It is clinically long to the deficiency syndrome of the lung
Cough is panted, hemoptysis of pulmonary tuberculosis, night sweat, kidney deficiency soreness of waist and knee joint, impotence and seminal emission, under the red blood cell after neurasthenia and chemotherapy, radiotherapy
It drops all effective in cure.
Aweto is a kind of sac fungus, in bat moth larvae body formed sclerotium to grow cordyceps sinensis stroma with
Ascus, this process belong to the cordyceps sinensis epigamous stage, and cordyceps sinensis at this time claims epigamous.From the ascospore of cordyceps sinensis
It germinates into mycelia (or generation conidium --- germination is at mycelia) and infects bat moth larvae, to Chinese caterpillar fungus bacterial filament in larva body
Interior proliferation, the process category aweto strain asexual reproduction stage for digesting and assimilating the whole nutrition of larva body.And in artificial culture, liquid
It is the aweto in imperfect stage used in the actual productions such as body fermentation, thus the identification of Anamorph of Cordyceps Sinensis is very heavy
It wants.Paecilomyces hepiali chen (Paecilomyces hepial) has proved to be one kind of the fungal component of cordyceps sinensis, has and day
The right identical active constituent of cordyceps sinensis and drug effect.
Cordycepin, also known as cordycepin, i.e. cordycepin, its molecular formula are C10H10N5O3, it is nitrogenous glycocide
Nucleic acid derivative belongs to purine alkaloid.It is usually used in treating the diseases such as tumour as a kind of active constituent of cordyceps sinensis.It is early
A kind of adenosine class activity is separated to from the virgin pulp liquid of Cordyceps militaris (Cordyceps militaris) in 1950, Cuningham etc.
Substance is named as cordycepin, a kind of nucleotide that it is formed by adenosine and by the deoxypentose of carbon Zhi Jian, referred to as
3'-Deoxyadenosine (3 '-deoxyadenosine, 3 '-dA), later by further identification, which is proved to be China
The effective component of traditional Chinese medicine cordyceps sinensis (C.sinensis), Chinese name are known as cordycepin, also known as cordycepin, cordyceps sinensis
Rhzomorph.The nucleoside antibiotic that cordycepin is separated from fungi as first, have antibacterial, it is anti-oxidant, antitumor and
Enhance the remarkable effects such as immune function of human body, is increasingly valued by people.
(3) summary of the invention
Object of the present invention is to separate identification Paecilomyces hepiali chen, and improve the content of cordycepin in Paecilomyces hepiali chen.It is logical
It crosses and screening separation is carried out to wild cordyceps sinensis, utilize morphology, molecular biology and Biolog Metabolic fingerprinting phase
In conjunction with method obtained Paecilomyces hepiali chen (Paecilomyces hepial) original strain is identified.Then to obtaining
The original strain obtained carries out physics and chemistry behavior, obtains cordycepin content and stablizes the bacterial strain ZJB18001 improved, makes it artificial
It can be more widely used in fermentation.
The technical solution adopted by the present invention is that：
The present invention provides a kind of Paecilomyces hepiali chen (Paecilomyces hepiali) from cordyceps sinensis
ZJB18001, the Paecilomyces hepiali chen ZJB18001 are preserved in China typical culture collection center, and preservation date 2018 2
The moon 6, deposit number CCTCC NO:M 2018091, preservation address are Wuhan, China, Wuhan University, postcode 430072.
The present invention also provides a kind of Paecilomyces hepiali chen ZJB18001 to improve the application in cordycepin output, described
Using being that Paecilomyces hepiali chen ZJB18001 is seeded to fermentation medium, fermented and cultured under the conditions of 18-25 DEG C, 150rpm,
Fermentation liquid is centrifuged, the thallus containing cordycepin is obtained, thallus is dry, extract cordycepin；The fermentation medium final concentration group
Become：Glucose 18-25g/L, peptone 4-8g/L, KH2PO41-3g/L, MgSO40.4-0.8g/L, solvent are tap water, pH
5.5-6.5。
Further, the fermented and cultured carries out in the fermenter, and fermentation condition is 18-25 DEG C, and ferment pressure tank
0.05MPa, speed of agitator 100-300rpm (preferably 150rpm), ventilation ratio 0.08-1.5vvm (preferably 0.5vvm).
Further, the Paecilomyces hepiali chen ZJB18001 first carries out activation culture and seed culture before fermented and cultured,
Seed liquor is seeded to fermentation medium with the inoculum concentration of volumetric concentration 2-10% (preferably 5%) again, the activation culture is：It will
Paecilomyces hepiali chen ZJB18001 is seeded to PDA plate, and 25 DEG C are cultivated 5 days, obtains activation thallus；The seed culture is：It chooses
Activation thallus is taken to be inoculated in seed culture medium, 25 DEG C, 150rpm is cultivated 3 days, obtains seed liquor；Seed culture medium final concentration group
At：Peptone 5g/L, yeast powder 2g/L, glucose 20g/L, KH2PO41g/L, MgSO40.5g/L, solvent are tap water, pH
It is natural.
Further, the fermentation medium final concentration group becomes：Glucose 20g/L, peptone 5g/L, KH2PO41g/L,
MgSO40.5g/L, solvent are tap water, pH 5.5-6.5.
The wild cordyceps acquired from Yushu district, Qinghai clean up and in carrying out after 1% mercuric chloride strict sterilization
The sterile cutting operation of sclerotium, and interior sclerotium is placed on a kind of slant medium and is sprouted, object is sprouted for subsequent pure
Culture.Preliminary Identification is carried out to the single colonie form after pure culture with morphologic method first, then utilizes molecular biology
Method Molecular Identification is carried out to isolate, 18S rDNA sequence finally further takes as shown in SEQ ID No.1
The method of Biolog Metabolic fingerprinting is identified as the fungal component Paecilomyces hepiali chen (Paecilomyces of aweto
hepial).A variety of mutagenesis methods such as later use physical chemistry carry out mutagenesis to its spore suspension, obtain cordycepin output raising
About 3 times of bacterial strain ZJB18001.
Due to the otherness of cordyceps sinensis locality, leading to the species being separated to also has certain otherness, as long as its
There is 95% or more homology with the 18S rDNA sequence, belong to the column of the scope of the present invention.The change may include core
Missing, insertion or the replacement of nucleotide sequence nucleotide.
The beneficial effects are mainly reflected as follows：The present invention identifies a kind of separation of Paecilomyces hepiali chen novel strain
It is studied in detail, provides the morphological observations, 18S rDNA sequence and Biolog of this plant of Paecilomyces hepiali chen
Metabolic fingerprinting.And the present invention produces the metabolism of peacilomyce hepiahi bacterium plant mutant using mutagenesis methods such as physics, chemistry
About 3 times of new strains ZJB18001 of the output increased of object cordycepin, wild type Paecilomyces hepiali chen cordycepin without mutagenesis
Yield is 0.187-0.27mg/g, cordycepin output 0.562-0.811mg/g, reduces the production cost of cordycepin, favorably
In the wide popularization and application of Paecilomyces hepiali chen.The induced-mutation technique that the present invention utilizes has the mutagenesis of aweto important
Directive significance.
(4) Detailed description of the invention
Single colonie form on Fig. 1 solid medium.
Mycelium morphology under the observation of Fig. 2 Electronic Speculum.
Fig. 3 18S rDNA sequence PCR amplification argrose electrophoretogram.
Fig. 4 18s rDNA phylogenetic tree.
Glucose content change curve in Fig. 5 fermentation liquid.
PH change curve in Fig. 6 fermentation liquid.
Dry cell weight (g/L) change curve in Fig. 7 course of fermentation.
The liquid phase spectrogram of Fig. 8 cordycepin mark product (A), original strain (B) and mutant strain ZJB18001 (C).
The standard curve of Fig. 9 cordycepin.
The liquid mass spectrogram of Figure 10 cordycepin mark product (A, B) and sample (C, D).
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This：
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is conventional biochemical reagent unless otherwise specified.
Embodiment 1：The screening and culturing and identification of wild cordyceps fungal component Paecilomyces hepiali chen
1, screening and culturing
45 plants of fructifications sturdy winter full, polypide is coarse is excavated from 4000-4300 meters of Yushu district, Qinghai height above sea level of hillside
The complete bacterium worm complex of worm summer grass, host insect are bat moth larvae.It is put into 25 DEG C of incubators and conserves.Son is removed with cleaning brush
Solid object surface impurity is rinsed, then carried out disinfection with 0.1% mercuric chloride with axenic purification water, sterile scalpel is used in super-clean bench
The interior sclerotium part of polypide is splitted, the tissue at three positions of picking upper, middle and lower is placed on sterilized and contains gentamicin (50mg/
L it on potato culture), is placed in 25 DEG C of constant incubators and cultivates, observe and record daily.Chorista is cultivated in potato
Start to sprout after cultivating 3 days on base, be transferred in potato fluid nutrient medium at this time, 25 DEG C, 150r/min shaken cultivation, is observed
Record.After shaken cultivation 3 days, mycelium is covered in potato fluid nutrient medium.
Potato (PDA) culture medium：Potato 200g/L, glucose 20g/L, agar 20g/L, solvent are tap water, pH
It is worth nature.First by peeling potatoes, be cut into block and boiling added to boil half an hour, then use three layers of filtered through gauze, then plus glucose with
Agar dissolves Hou Jiashui and complements to 1000mL, 115 DEG C of sterilizing 30min.
Potato fluid nutrient medium：Potato 200g/L, glucose 20g/L, solvent are tap water, and pH value is natural.
2, the Morphological Identification of cordyceps sinensis isolate
It draws a little step 1 mycelium to be coated on potato slant medium, media surface covers with white single after 5 days
Bacterium colony, as shown in Figure 1.It is observed in the growing state and single colonie form of potato culture, and utilizes scanning electron microscope
Microorganism on fixed glass slide is observed, the form of microorganism is understood.Identified, mycelial growth is slow, and inclined-plane is big
Culture 5d is about needed, mycelium is fine and close, is not easy picking, swells in snowy white, with the extension of incubation time, the mycelium back side is slowly
Brown color is presented, surface is more rounded, and mycelia is fluffy outward, as shown in Figure 1.Electronic Speculum observes mycelium morphology and shows that hyphal development is good
It is good, it is elongated, there is a tabula, 1.5~2.0 μm of diameter, it can be observed that forming the spore device of spore, as a result as shown in Figure 2.Through form
Identification is learned, tentatively judges that the bacterium colony that this isolate is formed is Paecilomyces hepiali chen.
3, the molecular biology identification of cordyceps sinensis isolate
(1) 18S rDNA design of primers
A pair of 18S rDNA primer p1 is designed according to fungi conservative region:5 '-TCCGTAGGTGAACCTGCCG-3 ' and p2:
5'p2:CCTCCGCTTATTGATATGC-3'.Primer is synthesized by Beijing Qing Ke company.
(2) genome extracts
Microbial gene is extracted using Rapid nucleic acid extraction apparatus and microbial genome extracts kit (MP company of the U.S.)
Group DNA.
(3) 18S rDNA sequence amplification
Using genomic DNA as template, PCR amplification (BioRad company of the U.S., PTC200 are carried out using universal primer p1 and p2
Amplification instrument), reaction condition is：95 DEG C of initial denaturation 5min, loop parameter are 95 DEG C of denaturation 30s, and 56 DEG C of renaturation 30s, 72 DEG C extend
60s, after repeating 35 circulations, 72 DEG C are continued to extend 10min, final to identify PCR product with 0.9% agarose gel electrophoresis.
(4) connection of target gene and conversion (kit：PMD18-T Vector, TaKaRa code D101A)
1) linked system：PMD18-T 1 μ L, Slution1 4 μ L, target gene 5 μ L.
2) connection, conversion condition：
Connection：16 DEG C, 16h；Inactivation：65 DEG C, 15min；10 μ L reaction systems are gone in competent cell JM109, ice
Bathe 30min；Thermal shock：42 DEG C, 90s；Ice bath：2~3min；It is added 800 μ L LB liquid mediums, 37 DEG C, 250rpm, 1h；Coating
Resistance containing Amp (100mg/L) LB plate；37 DEG C of incubator overnight incubations.
LB liquid medium composition：Yeast powder 5g/L, peptone 10g/L, sodium chloride 10g/L, solvent are deionized water, pH
It is worth nature.
LB plate composition：Yeast powder 5g/L, peptone 10g/L, sodium chloride 10g/L, agar powder 20g/L, solvent be go from
Sub- water, pH value are natural.
(5) recombinant bacterium screens：
1) plate picking single colonie, label；Each single colonie half is for being inoculated with Tube propagation；The other half is used for bacterium colony
PCR；
2) Tube propagation：5mL LB liquid medium/test tube, 3 μ L Amp (100mg/L)/5mLLB；37 DEG C, 250rpm training
It supports overnight, centrifugation obtains recombinant bacterial strain；
3) bacterium colony PCR：Single colonie is chosen into 50 μ L sterile waters, boiling water bath 30min；
PCR system is：2X Phanta Max Buffer 25 μ L, dNTP Mix (10mM) 1 μ L, upstream and downstream primer p1, p2
(50mM) each 1 μ L, 1 μ L, Phanta Max Super-Fidelity DNA Polymerase of bacterium solution, the 1 μ L after boiling, adds water
20 μ L complement to the total system of 50 μ L.
Primer：
p1:5 '-TCCGTAGGTGAACCTGCCG-3 ' and
p2:5'–TCCTCCGCTTATTGATATGC-3'.Primer is synthesized by Shanghai Sangon Biotech Company.
4) electrophoresis detection.
(6) plasmid extracts (kit：AxyPrep Plasmid DNA small volume of reagent box)
1) take 2 in 2mL step (5)) culture solution of overnight incubation, 12000g is centrifuged 1min, abandons supernatant；
2) plus 250 μ L Buffer S1 (4 DEG C of freezer storages), suspension are uniform；
3) add 250 μ L Buffer S2, spin upside down mildly and fully 4~6 times, crack thallus sufficiently, until shape
At bright solution, the time is no more than 5min；
4) add 350 μ L Buffer S3, mildly and fully overturn mixing 6~8 times, 12000g is centrifuged 10min；
5) supernatant is transferred to preparation pipe (being placed in 2mL centrifuge tube (kit offer)), 12000g is centrifuged 1min, abandons filter
Liquid；
6) plus 500 μ L Bufffer W1,12000g are centrifuged 1min, abandon filtrate；
7) plus 700 μ L Bufffer W2,12000g are centrifuged 1min, abandon filtrate, then wash one with 700 μ L Bufffer W2
It is secondary, abandon filtrate；
8) pipe will be prepared and put back into centrifuge tube, 12000g is centrifuged 1min；
9) pipe will be prepared, new 1.5mL centrifuge tube (kit offer) is provided, add 60~80 μ L super preparing periosteum center
Pure water (65 DEG C of preheatings), is stored at room temperature 1min, 12000g is centrifuged 1min；
10) -20 DEG C of preservations.
7,18S rDNA Sequence Detection
It is sequenced, 18S rDNA sequencing result is carried out using software Blast same with automatic sequence instrument after extracting plasmid
The analysis of source property.Using the cell total DNA extracted as template, the 18S rDNA sequence of bacterial strain is expanded using primer p1, p2 of design,
PCR product is carried out to 0.9% agarose gel electrophoresis, a length, which has successfully been obtained, through PCR amplification as can be seen from Figure 3 is about
The segment of 0.55kb, meets expected results.
Primer：
p1:5 '-TCCGTAGGTGAACCTGCCG-3 ' and
p2:5'–TCCTCCGCTTATTGATATGC-3'.Primer is synthesized by Shanghai Sangon Biotech Company.
8, the measurement and analysis of 18S rDNA sequence
By the 18S rDNA piece obtained containing this experiment after the segment of PCR amplification is cloned into carrier T, after extracting plasmid
The recombinant plasmid of section confirms segment physical length through sequencing.The physical length of sample is 561bp, following (the SEQ ID of sequence
NO.1)：
SEQ ZJB18001:561bp；
Composition 129A；175C；141G；116T；0OTHER
PercentaGe:23.0%A；31.2%C；25.1%G；20.7%T；0.0%OTHER
MolecuLar WeiGht(kDa):ssDNA:172.61dsDNA:345.89
ORIGIN
The data saved in the sequence and genBank of acquisition carry out similarity analysis discovery, the identified microorganism of this experiment
With Paecilomyces hepiali homology highest (homology, 100%/560bp, based on 18S rDNA), according to
Microbial molecules science of heredity identity principle, the homology based on 18S rDNA sequence are higher than 95%, and identification bacterium substantially belongs to compare
Bacterium.18S rDNA sequence is analyzed by phylogenetic tree, and affiliation and Paecilomyces hepiali are nearest, such as Fig. 4
It is shown.Therefore, the microorganism of this experimental identification is that Paecilomyces belongs to hepiali kind.
Embodiment 2：The Biolog Metabolic fingerprinting of cordyceps sinensis isolate is identified
Utilization of carbon source situation is measured using Biolog (FF) automatic identifying system.Due to the morphological feature and training of test strain
It supports feature and is similar to fungi, so this test is measured the utilization of carbon source situation of test strain using FF identification microplate.
1, prepared by bacteria suspension：
(1) aseptic cotton carrier is taken to dip in the inoculation liquid (FF-IF) of Biolog strain idenfication system wet；
(2) cotton swab is glued in 1 bacterium colony surface scrolls of embodiment and takes spore, be careful not to take culture medium out of；
(3) cotton swab is rotated along inner wall on inoculation liquid pipe liquid level, makes Spore adhesion on inner wall, while spore uniformly being beaten
It dissipates；
(4) inclination inoculation liquid pipe, is dispersed spore in inoculation liquid with cotton swab.If any small cenobium, should be allowed to sink to pipe
Bottom；
(5) turbidity is adjusted：
(a) power supply, adjustment pointer to " 0 " scale are closed；
(b) blank inoculation liquid pipe outer wall is dried, is placed in nephelometer, powers on, adjustment pointer to 100%T；
(c) accuracy of transmissometer is checked using turbidity standards, and is adjusted to standard turbidity value；
(d) bacteria suspension pipe outer wall is dried, nephelometer is inserted into, reads bacteria suspension turbidity value；
(e) turbidity value is adjusted by addition spore, makes it within the scope of 75% plus spore %.
2, FF microplate is inoculated with：
(1) bacteria suspension prepared is poured into loading slot, using eight electric pipettors, is inoculated in microplate 96
Kong Zhong；
(2) microplate is identified using FF, inoculum concentration is 100 holes μ L/.
3, FF microplate culture：
Be inoculated with filamentous fungi FF identification microplate at 26 DEG C, in air culture to for 24 hours, 48h, 72h, 96h, 168h and
240h, culture environment are unsuitable overly moist.
4, FF microplate reading and result save：
(1) " MicroLog " application program is opened, username and password is inputted, " OK " is clicked and enters main interface；
(2) it is introduced into the interface " Setup ", is clicked " initialize reader ", Initialize installation is carried out, waits to interface
When " ComNot Open " key of upper red becomes " Ready " of green, click " Read "；
(3) after entering the interface " Read ", reader mode-Reader is selected, such as using artificial reading, into manual mould
Formula (Manual)；At " Data File Name ", input data saves title and saves address afterwards；Click " Read New
Plate " selects microplate type and incubation time, and filamentous fungi type is selected in " Strain type " drop-down menu；
(4) microplate is put on people's readout instrument bracket, closes readout instrument lid, prepare reading；
(5) " Read Next " key is pressed to start to read.
(6) result is saved with PDF format.
5, the interpretation of result of Biolog identification
Bacterial strain is investigated to the metabolic condition of 95 kinds of different carbon sources using Biolog automatic microbe identification systems：By original bacteria
Strain is inoculated in fungi PDA culture medium, 25 DEG C constant temperature incubation 5 days, the thallus on plate is washed down with aseptic cotton carrier, with inoculation liquid
(FF-IF) it mixes, bacteria suspension is made, is adjusted with nephelometer to 75%T/FF.Bacteria suspension is added in respectively with 8 hole electric plus liquid devices
In each hole of Biolog FF micropore identification plate, every hole 100uL.Micropore identification plate is placed in 14 micropore incubators, is being trained respectively
It supports to place it in for 24 hours, after 48h, 72h, 96h, 168h and 240h on Biolog readout instrument and reads result.Through Biolog readout instrument point
Metabolic Fingerprinting is analysed, bacterial strain can utilize more by force 26 kinds of carbon sources, cannot utilize to other 69 kinds of carbon sources or Utilization ability is weaker and standard
After database comparison, discovery and the index of similarity of aweto phorozoon are greater than 0.5.Biolog system provides 168h identification
As a result, as shown in table 1.
Utilization ability of the 1. bacterial strain ZJB18001 of table to 95 kinds of carbon sources on Biolog FF plate
Embodiment 3：The shake flask fermentation of Paecilomyces hepiali chen
1, the preparation of seed liquor：The original strain that embodiment 1 obtains is inoculated on PDA solid medium, 25 DEG C of cultures 3
After it, there is single colonie, picking single colonie is inoculated in seed culture medium, and 25 DEG C, 150rpm is cultivated 3 days.
Seed culture based formulas：Peptone 5g, yeast powder 2g, glucose 20g, KH2PO41g, MgSO40.5g adds water fixed
Hold to 1L, pH is naturally, 115 DEG C of sterilizing 30min.
2, fermented and cultured
The shaking flask of 250mL specification fills 50mL fermentation medium, is inoculated with seed according to the inoculum concentration of volumetric concentration 2% when fermentation
Liquid, 25 DEG C, 150rpm fermented and cultured 5 days.In fermentation process, glucose content changes such as Fig. 5, pH in fermentation liquid in fermentation liquid
Variation such as Fig. 6, dry cell weight variation such as Fig. 7.
Fermentative medium formula：Peptone 5g, glucose 20g, KH2PO41g, MgSO40.5g adds water to be settled to 1L, pH
It is natural.
Embodiment 4：The screening of high yield cordycepin mutant strain
1, the preparation of spore suspension：It is taken at the original strain cultivated in 25 DEG C of PDA culture mediums five days, scrapes bacterium with inoculation shovel
Body, and be placed in the triangle reaction flask for the Tween 80 aqueous solution that joined volumetric concentration 0.05%, it is previously added in reaction flask
Magnetic rotor, after mycelium is added, wrapping is better than high-speed stirred one hour on magnetic stirring apparatus.Then with three layers of filtered through gauze,
Obtained filtrate is required spore suspension.All utensils used above have sterilized.
2, it is counted using blood counting chamber：Take the spore suspension prepared on blood counting chamber, put under the microscope into
Row counts, and takes upper left, lower-left, upper right, and bottom right and intermediate five points are counted, and then obtains total spore in spore suspension
Number, dilution control spore count is about 106/mL。
3、Co60Method of mutagenesis：Take the spore suspension in step 2, with 0GY, 200GY, 400GY, 600GY, 800GY,
The Co of seven various doses of 1000GY and 1200GY60Mutagenesis is carried out to spore.It will be through Co60The spore physiological saline of mutagenesis is dilute
It releases to 105Coated plate is cultivated 3 days in PDA culture medium, 25 DEG C afterwards, and the preliminary biggish single colonie of picking colony is carried out according to embodiment 3
Thallus detection cordycepin output (method is with embodiment 6) is collected in fermentation, until obtaining high productive mutant.It is mutated number, mutation rate
It is as shown in table 2 with lethality.
Table 2Co60Method of mutagenesis mutagenic processes
4, EMS-UV method of mutagenesis：The enhanced variant that step 3 is screened is prepared into spore suspension according to step 1 method
5mL is placed in diameter 9cm sterilized petri dishes, in the UV lamp oscillation irradiation 90s at 25cm, and an amount is taken to turn to exist side by side in sterile test tube
It immerses in ice water after 2h, sampling is coated in PDA plate, and 25 DEG C are protected from light culture 3 days, thallus after mutagenesis is collected, at EMS
Reason, processing mode：Every 1mL thallus suspension uses the PBS buffer solution of 50mM, the pH7.0 of ethylmethane sulfonate containing 5mg/mL (EMS)
0.5h is stirred, 8000rpm is centrifuged 5min, collects thallus and uses sterile water wash 3 times, PDA solid medium is coated on after resuspension
In, 25 DEG C are protected from light culture, the partially yellow bacterial strain of preliminary screening color, and picking single colonie is fermented according to embodiment 3, collect bacterium
Body is by HPLC method detection cordycepin output (method is with embodiment 6), until obtaining cordycepin output promotes apparent mutation
Strain.It is as shown in table 3 to be mutated number, mutation rate and lethality.
Table 3 is ultraviolet-nitrosoguanidine complex mutation process
Step 3 and 4 is a wheel mutation, and the superior strain mutagenic obtained for every wheel is re-used as initial strains according to upper
It states method and carries out complex mutation.It is screened from 500 mutant through 3 wheels, it is 0.562- that screening, which obtains cordycepin output,
0.811mg/g, it is final to obtain the highest mutant strain ZJB18001 of yield, i.e. Paecilomyces hepiali chen (Paecilomyces
Hepial) ZJB18001 is preserved in China typical culture collection center (CCTCC), and preservation date 2 months 2018 6, preservation
Number CCTCC NO:M 2018091, preservation address are Wuhan, China, Wuhan University, postcode 430072.
The present invention includes but is not limited only to above two mutagenesis method.
Embodiment 5：Paecilomyces hepiali chen CCTCC NO:The submerged fermentation of M 2018091
(1) inclined-plane culture：Paecilomyces hepiali chen CCTCC NO:M 2018091 is inoculated on PDA solid medium, and 25 DEG C
After culture 3 days, there is single colonie, obtains activation thallus.
(2) seed culture：Picking activation thallus single colonie is inoculated in seed culture medium, and 25 DEG C, 150rpm is cultivated 3 days, obtains
Obtain seed liquor.Seed culture based formulas：Peptone 5g, yeast powder 2g, glucose 20g, KH2PO41g, MgSO40.5g adds certainly
Water is settled to 1L, and pH is naturally, 115 DEG C of sterilizing 30min.
(3) fermented and cultured：Step (2) seed liquor is seeded to fermentation medium with the inoculum concentration of volumetric concentration 5%, in gas
In lift-type fermentor, tank presses 0.05Mpa, speed of agitator 150rpm, and fermentor ventilatory capacity is 0.5V/Vmin；Cultivation temperature 25
DEG C, culture 40 days put tank, obtain fermentation liquid, fermentation liquid is centrifuged, obtain mycelium to fermentation termination.
Embodiment 6：The rapidly extracting and detection of cordycepin
1, the Paecilomyces hepiali chen CCTCC NO of the acquisition of embodiment 5 is accurately weighed:2018091 mycelium 0.2g of M is added
Into the pure methanol of 10mL, 65Hz ultrasonic extraction 2 times, each 30min, 4 DEG C of refrigerators are set with pure methanol constant volume to 10mL again after extraction
It is spare.1mL extracting solution is drawn, is used for high performance liquid chromatography detection after filtering using 0.22um organic phase filter membrane.
2, liquid-phase condition：Mobile phase is methanol-water, gradient elution, 0-4min methanol：Water=15:85,4-8min methanol：Water
=20:80,8-12min methanol：Water=25:75,12-16min methanol：Water=20:80,16-20min methanol：Water=15:85.
Pillar is that C18 (4.6mm*250mm) Detection wavelength is 260nm.The above method detects cordycepin, obtains cordycepin mark product
(A), the chromatograms of original strain (B) and ZJB18001 (C) are as shown in Figure 8.
3, the drafting of the standard curve of cordycepin：Cordycepin standard items 0.1g is taken, 10mL methanol is dissolved in, obtaining concentration is
The standard solution of 1g/L then takes out the standard solution of 200uL, with 5 times of methanol dilution, is taking 200uL from diluted solution,
With 5 times of pure methanol dilution, being repeated four times to mark bent concentration is 1g/L, 0.2g/L, 0.04g/L, 0.008g/L, 0.0016g/L.With
Liquid phase detection standard curve be：Y=42967x+37.423 (x is that cordycepin concentration mg/L, y are peak area), R2=
0.9972, as shown in Figure 9.
4, the liquid quality detection of cordycepin：Utilizing liquid phase -- mass spectrum, which is used in conjunction, detects the cordycepin in sample, liquid phase --
Mass Spectrometry Conditions are：Mobile phase is methanol-water, flow velocity 0.4ml/min, gradient elution, 0-8min methanol：Water=15:85,8-
16min methanol：Water=20:80,16-24min methanol：Water=25:75,24-32min methanol：Water=20:80.Pillar is C18
(4.6mm*250mm) Detection wavelength is 260nm.Liquid chromatography mass figure obtained is as shown in Figure 10.Without the wild of mutagenesis
Type Paecilomyces hepiali chen cordycepin output is 0.187mg/g, cordycepin output 0.562mg/g.
Sequence table
<110>Zhejiang Polytechnical University
<120>Paecilomyces hepiali chen ZJB18001 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 561
<212> DNA
<213>Paecilomyces hepiali chen (Paecilomyces hepial)
<400> 1
tgcggaggga tcattaccga gttttcaact cccaaaccct tttgtgaaca tacctatcgt 60
tgcttcggcg gactcgcccc agcgtccggc cggccccgcg ccggccgcgg cctggatcca 120
ggcggccgcc ggagaccccc aaactctgta ttctcagtat cttctgaatc cgccgcaagg 180
caaaacaaat gaatcaaaac tttcaacaac ggatctcttg gttctggcat cgatgaagaa 240
cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc gaatctttga 300
acgcacattg cgcccgccag cattctggcg ggcatgcctg ttcgagcgtc atttcaaccc 360
tcgacttccc tttggggaaa tcggcgttgg ggaccggccg tataccgccg gccccgaaat 420
gaagtggcgg cccgtccgcg gcgacctctg cgtagtaatc caactcgcac cggaaccccg 480
acgtggccac gccgtaaaac ccccgacttc tgaacgttga cctcggatca ggtaggaata 540
cccgctgaac ttaagcatat c 561
Claims (6)
1. a kind of Paecilomyces hepiali chen (Paecilomyces hepiali) ZJB18001 from cordyceps sinensis, is preserved in China
Type Tissue Collection, preservation date 2 months 2018 6, deposit number CCTCC NO:M 2018091, preservation address are
Wuhan, China, Wuhan University, postcode 430072.
2. Paecilomyces hepiali chen ZJB18001 described in a kind of claim 1 is improving the application in cordycepin output.
3. application as claimed in claim 2, it is characterised in that the application is to be seeded to Paecilomyces hepiali chen ZJB18001
Fermentation medium, fermented and cultured under the conditions of 18-25 DEG C, 150rpm, fermentation liquid is centrifuged, and obtains the thallus containing cordycepin, will
Thallus is dry, extracts cordycepin；The fermentation medium final concentration group becomes：Glucose 18-25g/L, peptone 4-8g/L,
KH2PO41-3g/L, MgSO40.4-0.8g/L, solvent are tap water, pH 5.5-6.5.
4. application as claimed in claim 3, it is characterised in that the fermented and cultured carries out in the fermenter, and fermentation condition is
18-25 DEG C, ferment pressure tank 0.05MPa, speed of agitator 100-300rpm, ventilation ratio 0.08-1.5vvm.
5. application as claimed in claim 3, it is characterised in that the Paecilomyces hepiali chen ZJB18001 is first before fermented and cultured
Activation culture and seed culture are carried out, then seed liquor is seeded to fermentation medium with the inoculum concentration of volumetric concentration 2-10%, institute
Stating activation culture is：Paecilomyces hepiali chen ZJB18001 is seeded to PDA plate, 25 DEG C are cultivated 5 days, and activation thallus is obtained；Institute
Stating seed culture is：Picking activation thallus is inoculated in seed culture medium, and 25 DEG C, 150rpm is cultivated 3 days, obtains seed liquor；Seed
Culture medium final concentration composition：Peptone 5g/L, yeast powder 2g/L, glucose 20g/L, KH2PO41g/L, MgSO40.5g/L, it is molten
Agent is tap water, and pH is natural.
6. application as claimed in claim 3, it is characterised in that the fermentation medium final concentration group becomes：Glucose 20g/L,
Peptone 5g/L, KH2PO41g/L, MgSO40.5g/L, solvent are tap water, pH 5.5-6.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810618920.8A CN108865895B (en) | 2018-06-15 | 2018-06-15 | Paecilomyces hepiali ZJB18001 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810618920.8A CN108865895B (en) | 2018-06-15 | 2018-06-15 | Paecilomyces hepiali ZJB18001 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108865895A true CN108865895A (en) | 2018-11-23 |
| CN108865895B CN108865895B (en) | 2020-12-25 |
Family
ID=64339034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810618920.8A Active CN108865895B (en) | 2018-06-15 | 2018-06-15 | Paecilomyces hepiali ZJB18001 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108865895B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355208A (en) * | 2018-12-10 | 2019-02-19 | 华南农业大学 | A kind of highly pathogenicity biocontrol microorganisms Java cordyceps sinensis and its application |
| CN109699683A (en) * | 2018-12-10 | 2019-05-03 | 华南农业大学 | A kind of talcum matrix Java cordyceps sinensis spore preparation |
| CN110470754A (en) * | 2019-07-31 | 2019-11-19 | 武汉大学 | A kind of method of cordycepin content in high effective liquid chromatography for measuring cordyceps sinensis |
| TWI723368B (en) * | 2019-03-29 | 2021-04-01 | 葡萄王生技股份有限公司 | Method for manufacturing and the use of paecilomyces hepiali mycelia active substance for preventing and/or ameliorating acute lung injury |
| CN114149925A (en) * | 2021-11-08 | 2022-03-08 | 锬酃藏虫草生物科技(深圳)有限公司 | Application method of paecilomyces hepiali S2 in tea planting |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101045069A (en) * | 2007-04-10 | 2007-10-03 | 深圳市中科海外科技有限公司 | Method for extracting caterpillar fungus oil by using supercritical fluid of carbon dioxide |
| CN101942394A (en) * | 2010-09-28 | 2011-01-12 | 贵州大学 | Verticillium Paecilomyces varioti strain and application thereof |
| CN103627770A (en) * | 2012-08-23 | 2014-03-12 | 云南云百草实验室有限公司 | Method used for biotransformation of pseudo-ginseng using two paecilomyces funguses |
| KR20150057936A (en) * | 2013-11-18 | 2015-05-28 | (주)케비젠 | Paecilomyces hepiali CBG-CS-1 strain isolated form Cordyceps sinensis and method for culturing thereof |
| CN104911109A (en) * | 2015-05-28 | 2015-09-16 | 吉林大学 | High-yield paecilomyces hepiali mutant strain and cultivation method |
-
2018
- 2018-06-15 CN CN201810618920.8A patent/CN108865895B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101045069A (en) * | 2007-04-10 | 2007-10-03 | 深圳市中科海外科技有限公司 | Method for extracting caterpillar fungus oil by using supercritical fluid of carbon dioxide |
| CN101942394A (en) * | 2010-09-28 | 2011-01-12 | 贵州大学 | Verticillium Paecilomyces varioti strain and application thereof |
| CN103627770A (en) * | 2012-08-23 | 2014-03-12 | 云南云百草实验室有限公司 | Method used for biotransformation of pseudo-ginseng using two paecilomyces funguses |
| KR20150057936A (en) * | 2013-11-18 | 2015-05-28 | (주)케비젠 | Paecilomyces hepiali CBG-CS-1 strain isolated form Cordyceps sinensis and method for culturing thereof |
| CN104911109A (en) * | 2015-05-28 | 2015-09-16 | 吉林大学 | High-yield paecilomyces hepiali mutant strain and cultivation method |
Non-Patent Citations (5)
| Title |
|---|
| PANG, FANG等: ""Transcriptome analysis of Paecilomyces hepiali at different growth stages and culture additives to reveal putative genes in cordycepin biosynthesis"", 《GENOMICS》 * |
| 李瑞雪等: ""蝉拟青霉高产虫草素菌株液体培养工艺的研究"", 《徐州工程学院学报》 * |
| 武阳阳等: ""3种拟青霉发酵三七及其产物检测"", 《食品与发酵工业》 * |
| 洪露露: ""蝙蝠蛾拟青霉虫草素高产菌株的选育及其培养条件优化"", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》 * |
| 王蕾等: ""虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究"", 《菌物学报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355208A (en) * | 2018-12-10 | 2019-02-19 | 华南农业大学 | A kind of highly pathogenicity biocontrol microorganisms Java cordyceps sinensis and its application |
| CN109699683A (en) * | 2018-12-10 | 2019-05-03 | 华南农业大学 | A kind of talcum matrix Java cordyceps sinensis spore preparation |
| CN109699683B (en) * | 2018-12-10 | 2021-03-12 | 华南农业大学 | Talcum matrix Java cordyceps militaris spore preparation |
| TWI723368B (en) * | 2019-03-29 | 2021-04-01 | 葡萄王生技股份有限公司 | Method for manufacturing and the use of paecilomyces hepiali mycelia active substance for preventing and/or ameliorating acute lung injury |
| CN110470754A (en) * | 2019-07-31 | 2019-11-19 | 武汉大学 | A kind of method of cordycepin content in high effective liquid chromatography for measuring cordyceps sinensis |
| CN110470754B (en) * | 2019-07-31 | 2021-05-04 | 武汉大学 | Method for determining cordycepin content in cordyceps sinensis by high performance liquid chromatography |
| CN114149925A (en) * | 2021-11-08 | 2022-03-08 | 锬酃藏虫草生物科技(深圳)有限公司 | Application method of paecilomyces hepiali S2 in tea planting |
| CN114149925B (en) * | 2021-11-08 | 2023-11-14 | 锬酃藏虫草生物科技(深圳)有限公司 | Application method of paecilomyces hepiali S2 in tea planting |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108865895B (en) | 2020-12-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108865895A (en) | Paecilomyces hepiali chen ZJB18001 and its application | |
| CN109082382B (en) | Cordyceps sinensis hirsutella sinensis ZJB18002 and application thereof | |
| CN106222118B (en) | One streptomycete category actinomyces and application thereof | |
| US20220369648A1 (en) | Endophytic falciphora oryzae fo-r20 and its application | |
| US12010999B2 (en) | Application of endophytic Falciphora oryzae FO-R20 in controlling panicle blast | |
| CN116790410B (en) | Bacillus cereus YZ-228 fermentation preparation and preparation method and application thereof | |
| CN115197853B (en) | Endophyte Epicoccum thailandicum LF-28 strain and application thereof | |
| CN114766285B (en) | Ganoderma lucidum strain L4495 and cultivation method and application thereof | |
| CN115287194B (en) | Medicinal wild rice endophytic fungi YYA21 and application thereof | |
| CN106635822B (en) | A kind of elm mushroom bacterial strain and its cross breeding method | |
| CN115806913A (en) | Streptomyces nojiri (Streptomyces nojiriensis) strain 9-13 and application thereof | |
| CN111004727B (en) | Endophytic fungus Z1 for increasing biomass of casuarina equisetifolia in high-salt environment | |
| CN113046249A (en) | Verticillium lecanii LL-01 and biocontrol application thereof | |
| Long et al. | Tiankengomelania guangxiense, gen. et sp. nov., a dark septate endophytic fungus, promotes the growth of the medicinal orchid Dendrobium officinale | |
| CN110172411A (en) | A kind of scab shape Xylaria strain ZJ1811 and its cultural method and application | |
| CN113444646B (en) | Mould for producing beta-caryophyllene and application thereof | |
| CN112126592B (en) | Hirsutella sinensis mutant strain and application thereof in nucleoside production | |
| CN116064242B (en) | Lanlight collar mould and its separation method and application | |
| CN114134052B (en) | Mao Cumu mould YW411, culture method, microbial inoculum and application thereof | |
| CN114456951B (en) | Horizontalium fungus strain for promoting growth of ginseng, and method and application thereof | |
| CN112300948B (en) | Phellinus igniarius laser pulse intense light combined mutagenesis strain and ultrasonic enhanced fermentation method thereof | |
| CN113736669B (en) | Cordyceps sinensis fungus new species and separation and identification method thereof | |
| CN112980694B (en) | Penicillium chrysogenum SF-85 strain and application thereof | |
| CN105039176B (en) | Tree peony endogenetic fungus and its application | |
| CN105039174B (en) | Produce tree peony endogenetic fungus and its application of Paeonol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |