CN109234175A - The Fusarium oxysporum DCLZJ-4 bacterial strain and application thereof of one plant of production chonglou saponin - Google Patents
The Fusarium oxysporum DCLZJ-4 bacterial strain and application thereof of one plant of production chonglou saponin Download PDFInfo
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Abstract
The present invention relates to a kind of Fusarium oxysporum DCLZJ-4 bacterial strain for producing chonglou saponin, it is Fusarium (Fusarium sp.) through microbial identification that this bacterial strain, which is to be separately cultured to obtain using the method that endogenetic fungus isolates and purifies from paris polyphylla,.The bacterial strain is preserved in China Microbiological bacterial strain preservation administration committee common micro-organisms center, address: China, Beijing, Institute of Microorganism, Academia Sinica；Postcode 100101, the deposit date is on October 30th, 2017, deposit numbers are as follows: CGMCC No.14791；The ITS rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.Above-mentioned bacterial strains can be used in fermentation and prepare chonglou saponin.The present invention prepares chonglou saponin for scale fermentation and provides simple one plant of character stabilization, culture medium, well-grown Fusarium oxysporum bacterial strain, the fermentation of practical simplicity and separation-extraction technology, and the medicinal extract after the extracted concentration of fermentation liquid can be used as the active constituent either pharmaceutical carrier of hemostasis class drug.
Description
Technical field
The present invention relates to a kind of Fusarium oxysporum bacterial strain, which can produce chonglou saponin, belong to microorganisms technical field.
Background technique
Endophyte of plant have powerful metabolic function, can generate with the same or similar active constituent of host, extensively
It is present in various plants, it can culture of isolated comes out from plant by suitable condition of culture.Endophyte of plant is ground at present
Study carefully and be gradually taken seriously, the various endophytes of different plants are found in succession.The diversity of endophyte type also gives its generation
The diversity for thanking to product, using the method for endophyte fermentation for excavating new resources with huge potentiality.As people are to medicine
It is novel, efficient, low to produce using the endophyte for generating corresponding active constituent can be metabolized with the gradually research of endophyte of plant
The active medicine of cost solves the status of rare Chinese medicinal plant resource scarcity, will become the emphasis of medicinal plant endophyte research.
Rhizoma Paridis has significant pharmacological activity, and clinical application is extensive, is a variety of Chinese patent drugs such as Yunnan Baiyao, Gong Xue Ning
One of primary raw material, but Paris polyphylla growth cycle is long, the speed of growth is slow, and achieving no breakthrough property of introduction and acclimatization progress, price occupies
It is high not under, genuine wild resource destroys serious, and Paris polyphylla resource faces exhausted danger.Research shows that in Paris polyphylla plant, there is also big
Endophyte is measured, active metabolite can be generated.Have some scholars at present and has filtered out some production saponin(es or its analog
Endophyte, but bacterial strain quantity and type are all few continues to filter out some new bacterial strains for producing saponin(es or sapogenin, to deeply opening
The research for the approach that exhibition microbial fermentation produces saponin(e or sapogenin is of great significance.It is filtered out from Paris polyphylla plant metabolizable
The endophyte of chonglou saponin is generated, provides a new approach to solve Paris polyphylla status in short supply.
Wang Q etc. acquires the endogenetic fungus in the paris polyphylla on the ground such as Songming, Golconda, Heqing, from root, stem tuber, Ye Zhongfen
From to 749 plants of endogenetic fungus, Wei Juan etc. respectively is isolated by 44 plants and 40 plants from wild paris polyphylla and the paris polyphylla of artificial culture
Endogenetic bacteria.Roc etc. has found the interior raw enterobacter cloacae that 1 plant can produce rhizoma paridis saponin I and 2 plants can produce polyphyllin Ⅵ
Interior raw Escherichia coli and bacillus amyloliquefaciens.Ren Zhi, Zhao Ming, Zhang Xiaojie, Cao Xiaodong etc. are also isolated to that steroidal can be produced
The Paris polyphylla endophyte of saponin(e.Illustrate the various in style of Paris polyphylla endophyte, there is very strong biological activity, saponins can be produced
Close object.
Paris polyphylla endophyte just starts to be concerned by people in recent years, but can especially be metabolized about Paris polyphylla endophyte
The research for generating corresponding steroid saponin ingredient is still relatively fewer, and is largely focused on magnificent Paris polyphylla aspect, and in paris polyphylla
In terms of the research of raw bacterium is also concentrated mainly on bio-diversity, the research for producing chonglou saponin metabolite to endophyte is also less.
Summary of the invention
The present invention solves the problems in background technique, provides a kind of Fusarium oxysporum for producing a variety of chonglou saponins
DCLZJ-4 bacterial strain.
The object of the present invention is to provide a kind of Fusarium oxysporum DCLZJ-4 bacterial strain for producing Paris polyphylla soap, preparation method, fermentation trainings
Application of the medicinal extract made of the method for supporting and its fermentation liquid in preparation treatment/prevention hemostasis class drug.
The object of the present invention is achieved like this,
A kind of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, deposit number are CGMCC No.14791.
The preparation method of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, it is characterised in that including cleaning and sterilizing, inoculation,
Separation, culture and preservation, specific steps are as follows:
A) cleaning and sterilizing: after the fresh Paris polyphylla stem tuber of acquisition is cleaned, the hard object part of surface necrosis is removed, with 75% second
Alcohol carries out surface sterilization 20min, then uses sterile water wash 3~5 times；
B) it is inoculated with: the Paris polyphylla stem tuber of step a) processing being put into the spare mortar that sterilizes in advance, 5mL sterile water is added to be ground into
White suspension；It takes 300 μ L suspensions in each PDA culture medium plate with pipettor, is smoothened with sterile painting stick；And in the 3rd,
5,7,9d is photographed to record respectively, is no longer separated if fungi covers with plate, and sterilization treatment；
C) be inoculated with: by step a) processing the crosscutting thin slice fritter at 1cm2 of Paris polyphylla stem tuber, first with 75% alcohol immersion 3~
5min, then with sterile water wash 2~3 times washes away remaining alcohol, is finally impregnated about 5 minutes with sterile water, with going out after taking-up
The filter paper of bacterium blots surface moisture；By treated, fritter is labelled on PDA solid plate, and 5 are put on each plate；And in the 3rd,
5,7,9d is photographed to record respectively, is no longer separated if fungi covers with plate, and sterilization treatment；
D) separate: the PDA culture medium plate that step b) or step c) are obtained cultivates 5~9d in 28 DEG C of constant incubators,
Observation in time, is inoculated with different fungies in PDA solid medium tablets with transfer needle, purifies repeatedly, until obtaining single
Bacterium colony；
E) it cultivates and saves: the fungi picking of completion will be purified on plate into the sloped tube that PDA is housed,
Then after 28 DEG C of constant temperature incubation 5d, after glycerol is added, silica gel plug, sealed membrane sealing are protected in -80 DEG C of ultra low temperature freezers beyond the Great Wall
It deposits.
The fermentation culture method of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, it is characterised in that be inoculated into strain
In 50ml PDA liquid medium, in 28 DEG C of constant-temperature table 150r/min activation cultures, the strain for taking 1ml to activate afterwards for 24 hours to dress
Have in the triangular flask of PDA liquid medium and ferments, liquid amount 100ml/ 250ml, 28 DEG C, 150r/min shaking table culture 7d；
After the above fermented and cultured, fermentation liquid is collected.
Every 1000ml PDA culture medium is made of potato 300g, sucrose 18g.
It is a kind of containing there are many medicinal extract of chonglou saponin, it is characterized in that with Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain
Fermentation liquid is raw material, three times with extracting n-butyl alcohol, is obtained after volatilizing n-butanol.
Containing the application there are many medicinal extract of chonglou saponin in preparation treatment/prevention hemostasis class drug.
The dosage form of drug is peroral dosage form or intravenous injection.
The dosage form of drug is tablet, hard capsule, soft capsule, powder, pill, granule.
A kind of pharmaceutical composition, containing there are many medicinal extract of chonglou saponin as active constituent either pharmaceutical carrier.
The present inventor screens one plant of novel strain, which is named as DCLZJ-4, belongs to a kind of Fusarium oxysporum
(Fusarium oxysporum), the bacterial strain are preserved in China Microbiological bacterial strain preservation administration committee common micro-organisms center,
Address: China, Beijing, Institute of Microorganism, Academia Sinica；Postcode 100101, the deposit date is on October 30th, 2017, protect
Hiding number are as follows: CGMCC No.14791；The ITS rDNA sequence of the bacterial strain is as shown in SEQ ID NO .1.
Above-mentioned bacterial strains can be used in fermentation and prepare chonglou saponin.Culture medium prescription used by microbial fermentation is PDA training
Support base: potato 300g, sucrose 18g.Optimal culture conditions are as follows: pH value 7.0,28 DEG C of cultivation temperature, shaking speed 150r/min.
Compared with prior art, the invention has the following advantages that Fusarium oxysporum DCLZJ-4 bacterial strain provided by the present invention
It can be metabolized and generate a variety of chonglou saponins.Bacterial strain provided by the invention can carry out large scale fermentation culture in a short time, and send out
Cost is relatively low for ferment, and is not limited by conditions such as time domain, seasons, therefore it is natural to solve Paris polyphylla by the approach of microbial fermentation
The increasingly deficient status of resource guarantees that the long-term sustainable of Paris polyphylla medicine resource utilizes.
Detailed description of the invention
Fig. 1 is the bacterium colony front of Fusarium oxysporum DCLZJ-4 bacterial strain.
Fig. 2 is the bacterium colony back side of Fusarium oxysporum DCLZJ-4 bacterial strain.
Fig. 3 is the thin-layer chromatogram of Fusarium oxysporum DCLZJ-4 Metabolite.
Fig. 4 is rhizoma paridis saponin I, II, VI, the VII total ion figure of standard items mixed liquor matter coupled HPLC.
Fig. 5 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC rhizoma paridis saponin I standard items level-one
Mass spectrogram.
Fig. 6 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC rhizoma paridis saponin I standard items second level
Mass spectrogram.
Fig. 7 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC chonglou saponin Ⅱ standard items level-one
Mass spectrogram.
Fig. 8 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC chonglou saponin Ⅱ standard items second level
Mass spectrogram.
Fig. 9 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC polyphyllin Ⅵ standard items level-one
Mass spectrogram.
Figure 10 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC polyphyllin Ⅵ standard items two
Grade mass spectrogram.
Figure 11 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC VII standard items of chonglou saponin one
Grade mass spectrogram.
Figure 12 be rhizoma paridis saponin I, II, VI, VII standard items mixed liquor matter coupled HPLC VII standard items of chonglou saponin two
Grade mass spectrogram.
Figure 13 is rhizoma paridis saponin I mass spectrogram in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 14 is rhizoma paridis saponin I ion figure in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 15 is chonglou saponin Ⅱ mass spectrogram in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 16 is chonglou saponin Ⅱ ion figure in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 17 is polyphyllin Ⅵ mass spectrogram in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 18 is polyphyllin Ⅵ ion figure in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 19 is VII mass spectrogram of chonglou saponin in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
Figure 20 is VII ion figure of chonglou saponin in Fusarium oxysporum DCLZJ-4 Metabolite LC-MS chromatography.
The explanation of preservation biomaterial
The preservation of bacterial strain: the bacterial strain is preserved in China Microbiological bacterial strain preservation administration committee common micro-organisms center, address: in
State, Beijing, Institute of Microorganism, Academia Sinica；Postcode 100101, the deposit date is on October 30th, 2017, deposit numbers
Are as follows: CGMCC No.14791；The ITS rDNA sequence of the bacterial strain is as shown in SEQ ID NO .1.
Specific embodiment
Detailed specific description done to the present invention combined with specific embodiments below, but protection scope of the present invention not office
It is limited to following embodiment.
A kind of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, deposit number are CGMCC No.14791.
The preparation method of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, it is characterised in that including cleaning and sterilizing, inoculation,
Separation, culture and preservation, specific steps are as follows:
A) cleaning and sterilizing: after the fresh Paris polyphylla stem tuber of acquisition is cleaned, the hard object part of surface necrosis is removed, with 75% second
Alcohol carries out surface sterilization 20min, then uses sterile water wash 3~5 times；
B) it is inoculated with: the Paris polyphylla stem tuber of step a) processing being put into the spare mortar that sterilizes in advance, 5mL sterile water is added to be ground into
White suspension；It takes 300 μ L suspensions in each PDA culture medium plate with pipettor, is smoothened with sterile painting stick；And in the 3rd,
5,7,9d is photographed to record respectively, is no longer separated if fungi covers with plate, and sterilization treatment；
C) be inoculated with: by step a) processing the crosscutting thin slice fritter at 1cm2 of Paris polyphylla stem tuber, first with 75% alcohol immersion 3~
5min, then with sterile water wash 2~3 times washes away remaining alcohol, is finally impregnated about 5 minutes with sterile water, with going out after taking-up
The filter paper of bacterium blots surface moisture；By treated, fritter is labelled on PDA solid plate, and 5 are put on each plate；And in the 3rd,
5,7,9d is photographed to record respectively, is no longer separated if fungi covers with plate, and sterilization treatment；
D) separate: the PDA solid plate culture that step b) or step c) are obtained be based on culture 5 in 28 DEG C of constant incubators~
9d is observed in time, is inoculated with different fungies in PDA solid medium tablets with transfer needle, purifies repeatedly, until obtaining single
Bacterium colony；
E) it cultivates and saves: by the fungi picking for purifying completion on plate into the inclined-plane solid medium test tube that PDA is housed,
Then after 28 DEG C of constant temperature incubation 5d, after glycerol is added, silica gel plug, sealed membrane sealing are protected in -80 DEG C of ultra low temperature freezers beyond the Great Wall
It deposits.
The fermentation culture method of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, it is characterised in that be inoculated into strain
In 50ml PDA liquid medium, in 28 DEG C of constant-temperature table 150r/min activation cultures, the strain for taking 1ml to activate afterwards for 24 hours to dress
Have in the triangular flask of PDA liquid medium and ferments, liquid amount 100ml/ 250ml, 28 DEG C, 150r/min shaking table culture 7d；
After the above fermented and cultured, fermentation liquid is collected.
PDA culture medium is made of potato 300g, sucrose 18g.
It is a kind of containing there are many medicinal extract of chonglou saponin, it is characterized in that with Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain
Fermentation liquid is raw material, three times with extracting n-butyl alcohol, is obtained after volatilizing n-butanol.
Containing the application there are many medicinal extract of chonglou saponin in preparation treatment/prevention hemostasis class drug.
The dosage form of drug is peroral dosage form or intravenous injection.
The dosage form of drug is tablet, hard capsule, soft capsule, powder, pill, granule.
A kind of pharmaceutical composition, containing there are many medicinal extract of chonglou saponin as active constituent either pharmaceutical carrier.
In order to make the objectives, technical solutions, and advantages of the present invention clearer, by the following examples and test number
According to the present invention is described in further detail.It should be appreciated that specific embodiment described herein is only used to explain this hair
It is bright, it is not limited to the technical solution.
Embodiment 1
Separation, the culture of bacterial strain:
After the fresh Paris polyphylla stem tuber of acquisition is cleaned, the hard object part of surface necrosis is removed, surface is carried out with 75% ethyl alcohol and disappears
Then malicious 20min is used sterile water wash 3~5 times, is put into the spare mortar that sterilizes in advance, 5mL sterile water is added to be ground into white
Suspension.It takes 300 μ L suspensions in each PDA culture medium plate with pipettor, is smoothened with sterile painting stick, trained in 28 DEG C of constant temperature
It supports and cultivates 5~9d in case, observe in time, be inoculated with different fungies in PDA solid medium tablets with transfer needle, it is repeatedly pure
Change, until obtaining single bacterium colony.And photographed to record respectively in the 3rd, 5,7,9d, no longer divided if fungi covers with plate
From, and sterilization treatment.
The stem tuber of surface sterilization is cleaned simultaneously, then the crosscutting thin slice fritter at 1cm2, first impregnates 3 with 75% alcohol
~5min, then with sterile water wash 2~3 times, remaining alcohol is washed away, it is finally impregnated about 5 minutes with sterile water, is used after taking-up
The filter paper of sterilizing blots surface moisture.By treated, fritter is labelled on PDA solid plate, 5 is put on each plate, in 28 DEG C
5~9d is cultivated in constant incubator, is observed in time, is inoculated with different fungies in PDA solid medium tablets, instead with transfer needle
Multiple purifying, until obtaining single bacterium colony.And photographed to record respectively in the 3rd, 5,7,9d, it is no longer carried out if fungi covers with plate
Separation, and sterilization treatment.
By the fungi picking for purifying completion on plate into the inclined-plane solid medium test tube that PDA is housed, then in 28 DEG C
After constant temperature incubation 5d, after glycerol is added, sealed membrane sealing saves, each bacterial strain at least saves 3 in -80 DEG C of ultra low temperature freezers
Part.
Fig. 1 is the bacterium colony front of Fusarium oxysporum DCLZJ-4 bacterial strain.
Fig. 2 is the bacterium colony back side of Fusarium oxysporum DCLZJ-4 bacterial strain.
Embodiment 2
The fermented and cultured of bacterial strain:
The strain of preservation is inoculated into 50ml PDA liquid medium, in 28 DEG C of constant-temperature table 150r/min activation cultures, for 24 hours
The strain for taking 1ml to activate afterwards ferments into the triangular flask for be equipped with PDA liquid medium, liquid amount 100ml/ 250ml, and 28
DEG C, 150r/min shaking table culture 7d.After the above fermented and cultured, fermentation liquid is collected.
Embodiment 3
The screening of bacterial strain:
Three times with extracting n-butyl alcohol, each 100ml is volatilized to be dissolved with methanol after n-butanol and is metabolized each bacterial strain fermentation liquor sample
Product utilizes chonglou saponin I, II, VI, VII in silica gel thin-layer chromatography detection metabolite.The solvent used is (chloroform-first
Alcohol-water) system, color developing agent is phosphomolybdic acid, and screening obtains to produce the fungi of steroid saponin.
Fig. 3 is the thin-layer chromatogram of Fusarium oxysporum DCLZJ-4 Metabolite.It can be seen in figure 3 that fermentation liquid
(the second from left) and with the displacement that the compound (right side one) that methanol extracts chonglou saponin in Paris polyphylla standard medicinal material has many blue spots it is
It is consistent, thus it is speculated that there are many chonglou saponins in this Metabolite, to verify whether that strictly chonglou saponin need to carry out liquid matter
The identification of coupled HPLC method.
Embodiment 4
The fungus metabolite that can produce steroid saponin that embodiment 3 is obtained, is detected with LC-MS chromatography, by it
Spectrogram, data are compared with standard items data, substantially determine in sample whether contain corresponding chonglou saponin.
Rhizoma paridis saponin I, II, VI, VII standard items are mixed and carry out LC-MS chromatography.Fig. 4 is LC-MS chromatography
Total ion figure, Fig. 5 and Fig. 6 are respectively the first mass spectrometric figure and second order ms figure of rhizoma paridis saponin I standard items, Fig. 7 and Fig. 8 difference
For the first mass spectrometric figure and second order ms figure of chonglou saponin Ⅱ standard items, Fig. 9 and Figure 10 are respectively polyphyllin Ⅵ standard items
First mass spectrometric figure and second order ms figure, Figure 11 and Figure 12 are respectively the first mass spectrometric figure and second order ms of VII standard items of chonglou saponin
Figure.
The Fusarium oxysporum DCLZJ-4 Metabolite that embodiment 3 is obtained carries out LC-MS chromatography, Figure 13
It is respectively rhizoma paridis saponin I mass spectrogram and ion figure with Figure 14, Figure 15 and Figure 16 are respectively chonglou saponin Ⅱ mass spectrogram and ion figure,
Figure 17 and Figure 18 is respectively polyphyllin Ⅵ mass spectrogram and ion figure, Figure 19 and Figure 20 be respectively VII mass spectrogram of chonglou saponin and from
Subgraph.
From the point of view of Fig. 3-Figure 20, rhizoma paridis saponin I, II, VI, VII are contained in Fusarium oxysporum DCLZJ-4 Metabolite.
By the Fusarium oxysporum DCLZJ-4 bacterial strain of preservation according to the method for embodiment 2 and embodiment 3 carry out fermented and cultured and
Post-processing, obtains other 7 parts of Fusarium oxysporums DCLZJ-4 Metabolite sample, carries out LC-MS chromatography respectively,
Contain rhizoma paridis saponin I, II, VI, VII in sample as the result is shown.
Embodiment 5
Molecular Identification is carried out to the bacterial strain.Upstream primer: ITS1 (5 '-TCCGTAGGTGAACCTGCGG -3 '), downstream primer:
ITS4(5'-TCCTCCGCTTATTGATATGC -3') .It is similar that sequencing result carries out Blast in GenBank nucleic acid database
Property analysis, through Blast sequence alignment and phylogenetic analysis, the homology of DCLZJ-4 bacterial strain and Fusarium oxysporum is 99.0%,
Determine that it is Fusarium oxysporum (Fusarium oxysporum).
Embodiment 6
The optimization of fermentation condition: the fermentation medium component of microorganism allows for the needs for meeting the breeding of bacterial strain fast-growth,
Fermentation culture conditions most suitable simultaneously can guarantee a large amount of accumulation of its secondary metabolites again.The present invention passes through single factor test and orthogonal
The fermentation medium components of DCLZJ-4 bacterial strain are optimized in test experiment, the optimum medium formula after optimization are as follows: determine
Nutrient media components and corresponding concentration and then the most suitable initial pH value for generating chonglou saponin, training further are metabolized to the bacterial strain
It supports temperature and shaking speed is optimized, the final optimum medium for determining DCLZJ-4 bacterial strain is: potato 300g, sucrose
18g.Optimal culture conditions are as follows: pH value 7.0,28 DEG C of cultivation temperature, shaking speed 150r/min.
SEQUENCE LISTING
<110>Yunnan University of Traditional Chinese Medicine
The Fusarium oxysporum DCLZJ-4 bacterial strain and application thereof of<120>one plants of production chonglou saponins
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 548
<212> DNA
<213>the ITS rDNA sequence of bacterial strain
<400> 1
ttttcctccg cttattgata tgcttaagtt cagcgggtat tcctacctga tccgaggtca 60
acattcagaa gttggggttt aacggcgtgg ccgcgacgat taccagtaac gagggtttta 120
ctactacgct atggaagctc gacgtgaccg ccaatcaatt tgaggaacgc gaattaacgc 180
gagtcccaac accaagctgt gcttgagggt tgaaatgacg ctcgaacagg catgcccgcc 240
agaatactgg cgggcgcaat gtgcgttcaa agattcgatg attcactgaa ttctgcaatt 300
cacattactt atcgcatttt gctgcgttct tcatcgatgc cagaaccaag agatccgttg 360
ttgaaagttt tgatttattt atggttttac tcagaagtta catatagaaa cagagtttag 420
gggtcctctg gcgggccgtc ccgttttacc gggagcgggc tgatccgccg aggcaacaag 480
tggtatgttc acaggggttt gggagttgta aactcggtaa tgatccctcc gcaggttcac 540
cctacgga 548
<210> 2
<211> 19
<212> DNA
<213> ITS1
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 20
<212> DNA
<213> ITS4
<400> 3
tcctccgctt attgatatgc 20
Claims (9)
1. a kind of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain, deposit number is CGMCC No.14791.
2. a kind of preparation method of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain as described in claim 1, feature exist
In including cleaning and sterilizing, inoculation, separation, culture and preservation, specific steps are as follows:
A) cleaning and sterilizing: after the fresh Paris polyphylla stem tuber of acquisition is cleaned, the hard object part of surface necrosis is removed, with 75% second
Alcohol carries out surface sterilization 20min, then uses sterile water wash 3~5 times；
B) it is inoculated with: the Paris polyphylla stem tuber of step a) processing being put into the spare mortar that sterilizes in advance, adds 5mL sterile water to be ground into white
Color suspension；It takes 300 μ L suspensions in each PDA solid medium tablets with pipettor, is smoothened with sterile painting stick；And in
3,5,7,9d is photographed to record respectively, is no longer separated if fungi covers with plate, and sterilization treatment；
C) it is inoculated with: the Paris polyphylla stem tuber of step a) processing is crosscutting at 1cm2Thin slice fritter, first with 75% alcohol impregnate 3~
5min, then with sterile water wash 2~3 times washes away remaining alcohol, is finally impregnated about 5 minutes with sterile water, with going out after taking-up
The filter paper of bacterium blots surface moisture；By treated, fritter is labelled in PDA solid medium tablets, and 5 are put on each plate；And
It photographs to record, is no longer separated if fungi covers with plate, and sterilization treatment respectively in the 3rd, 5,7,9d；
D) separate: the PDA culture medium plate that step b) or step c) are obtained cultivates 5~9d in 28 DEG C of constant incubators, and
When observe, be inoculated with different fungies in PDA solid medium tablets with transfer needle, purify repeatedly, until obtain single bacterium
It falls；
E) it cultivates and saves: the fungi picking of completion will be purified on plate into the sloped tube that PDA is housed, so
Afterwards after 28 DEG C of constant temperature incubation 5d, after glycerol is added, silica gel plug, sealed membrane sealing are protected in -80 DEG C of ultra low temperature freezers beyond the Great Wall
It deposits.
3. a kind of fermentation culture method of Fusarium Fusarium oxysporum DCLZJ-4 bacterial strain as described in claim 1, feature
It is for strain to be inoculated into 50ml PDA liquid medium, in 28 DEG C of constant-temperature table 150r/min activation cultures, takes afterwards for 24 hours
The strain of 1ml activation ferments into the triangular flask for be equipped with PDA liquid medium, liquid amount 100ml/ 250ml, 28 DEG C,
150r/min shaking table culture 7d；After the above fermented and cultured, fermentation liquid is collected.
4. according to the method in claim 2 or 3, it is characterised in that the every 1000ml culture medium of the PDA culture medium is by soil
Beans 300g, sucrose 18g add water to form.
5. it is a kind of containing there are many medicinal extract of chonglou saponin, it is characterized in that with Fusarium Fusarium oxysporum described in claim 1
The fermentation liquid of DCLZJ-4 bacterial strain is that raw material three times with extracting n-butyl alcohol obtains after volatilizing n-butanol.
6. a kind of application of medicinal extract as claimed in claim 5 in preparation treatment/prevention hemostasis class drug.
7. application according to claim 6, it is characterised in that the dosage form of the drug is peroral dosage form or intravenous injection
Agent.
8. application according to claim 6, it is characterised in that the dosage form of the drug be tablet, hard capsule, soft capsule,
Powder, pill, granule.
9. a kind of pharmaceutical composition, it is characterized in that comprising any medicinal extract of claim 5 ~ 8 as active constituent either
Pharmaceutical carrier.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811347659.9A CN109234175B (en) | 2018-11-13 | 2018-11-13 | Fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin and application thereof |
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| CN201811347659.9A CN109234175B (en) | 2018-11-13 | 2018-11-13 | Fusarium oxysporum DCLZJ-4 strain for producing paris polyphylla saponin and application thereof |
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| CN109234175B CN109234175B (en) | 2021-08-13 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016519A (en) * | 2019-04-30 | 2019-07-16 | 中国热带农业科学院环境与植物保护研究所 | A kind of banana is withered germina number-four biological strain DCL deletion mutant body and its tiny RNA |
| CN113832039A (en) * | 2021-09-27 | 2021-12-24 | 黑龙江省农业科学院经济作物研究所 | Industrial hemp endophytic fungus with antifungal activity and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107723245A (en) * | 2017-10-18 | 2018-02-23 | 湖北文理学院 | It is a kind of to assist tuber of dwarf lilyturf endogenous fusanin category fungi and the application in steroid saponin is prepared |
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2018
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107723245A (en) * | 2017-10-18 | 2018-02-23 | 湖北文理学院 | It is a kind of to assist tuber of dwarf lilyturf endogenous fusanin category fungi and the application in steroid saponin is prepared |
Non-Patent Citations (2)
| Title |
|---|
| TAO LIU等: "Levels of rhizome endophytic fungi fluctuate in Paris polyphylla var.yunnanensis as plants age", 《PLANT DIVERSITY》 * |
| 魏超: "产皂苷的重楼内生真菌的分离、鉴定及退化研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110016519A (en) * | 2019-04-30 | 2019-07-16 | 中国热带农业科学院环境与植物保护研究所 | A kind of banana is withered germina number-four biological strain DCL deletion mutant body and its tiny RNA |
| CN113832039A (en) * | 2021-09-27 | 2021-12-24 | 黑龙江省农业科学院经济作物研究所 | Industrial hemp endophytic fungus with antifungal activity and application thereof |
| CN113832039B (en) * | 2021-09-27 | 2023-08-01 | 黑龙江省农业科学院经济作物研究所 | Industrial cannabis endophytic fungus with antifungal activity and application thereof |
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| Publication number | Publication date |
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| CN109234175B (en) | 2021-08-13 |
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