CN104726347B - One plant of fox excrement mould fungal bacterial strain and the method for preparing left-handed 7 hydroxyl butylphenyl phthaleine using the bacterial strain - Google Patents
One plant of fox excrement mould fungal bacterial strain and the method for preparing left-handed 7 hydroxyl butylphenyl phthaleine using the bacterial strain Download PDFInfo
- Publication number
- CN104726347B CN104726347B CN201510113631.9A CN201510113631A CN104726347B CN 104726347 B CN104726347 B CN 104726347B CN 201510113631 A CN201510113631 A CN 201510113631A CN 104726347 B CN104726347 B CN 104726347B
- Authority
- CN
- China
- Prior art keywords
- grams
- bacterial strain
- handed
- hydroxyls
- ncc3421
- 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.)
- Active
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
- C12R2001/80—Penicillium
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Left-handed 7 hydroxyl butylphenyl phthaleine is prepared the invention discloses one plant of fox excrement mould fungal bacterial strain and using the bacterial strain（(S)‑(‑)‑3‑butyl‑7‑hydroxyphthalide）Method.The bacterial strain that the present invention is provided is one plant of fox excrement mould fungal bacterial strain NCC3421（Penicillium vulpinum）, it is preserved in China General Microbiological culture presevation administrative center（Abbreviation CGMCC）, deposit number is CGMCC No.9094.Under the condition of culture that the present invention is provided, the fermentation unit of left-handed 7 hydroxyl butylphenyl phthaleine has reached more than 1600mg/L, and fermentation product components are more single, it is easy to isolate and purify, and can be used for the industrialized production of left-handed 7 hydroxyl butylphenyl phthaleine.
Description
Technical field
The present invention relates to microbial technology field, specifically one plant fox excrement mould fungal bacterial strain and the bacterial strain is utilized
Prepare left-handed 7- hydroxyls butylphenyl phthaleine（(S)-(-)-3-butyl-7-hydroxyphthalide）Method.
Background technology
Left-handed 7- hydroxyls butylphenyl phthaleine is earliest isolated from fox excrement mould by Japanese scholars Makino M etc.
[Heterocycles, 1998, 48(9):1931-1933], other phthalide analog compounds should alone or in combination for the compound
With with prevention and treatment diabetes（US20090192218Al）, the illness relevant with impaired neurotransmission such as depressed and anxiety
（US20100184852A1）And the disease related to angiogenesis（US20060246157A1）.Report in the world at present
7- hydroxyl butylphenyl phthaleine access approaches have：Extraction, chemical synthesis, the separation from fox excrement mould nutrient solution from Ligusticum wallichii.
Disclosed in patent US20060246157A1, the left-handed 7- hydroxyls butylphenyl phthaleine of reactive compound for extracting from Ligusticum wallichii can be single
Solely or with vanillic aldehyde, coniferyl alcohol ferulic acid and falcarindiol it is combined, for treating and preventing the disease based on angiogenesis
Medicine and nutraceutical.It is by solvent extraction-silica gel column chromatography-HPLC liquid phases that the patent, which separates left-handed 7- hydroxyls butylphenyl phthaleine,
Preparation method is obtained, its complex steps.Left-handed 7- hydroxyls butylphenyl phthaleine content in Ligusticum wallichii is extremely low, and Ligusticum wallichii resource-constrained, is unsuitable for
Prepare left-handed 7- hydroxyls butylphenyl phthaleine on a large scale using Ligusticum wallichii as raw material.
Document Biosci Biotechnol Biochem, 2003,67 (10):2240-2244 has delivered 7- hydroxyl fourths
The synthetic method of two kinds of enantiomters of phthalide, is distinguished using Sharpless bishydroxy reagent A D-mix- α and AD-mix- β
Carry out asymmetric syntheses and obtain two kinds of optically pure enantiomers, and prove that Makino M in 1998 etc. are separated from fox excrement mould
The 7- hydroxyls butylphenyl phthaleine arrived is S configurations.
Document Tetrahedron Letters, 2014,55:1303-1305 reports, utilize lipase Novozym-
435 propargyl alcohols for splitting racemization obtain key intermediate (S)-propargyl alcohol acetate（Optical purity 95%）, then by Alder-
The left-handed 7- hydroxyls butylphenyl phthaleine of Rickert reaction synthesis.Key intermediate synthetic reaction includes 5 steps, yield 21.2%；From being closed
Key intermediate also needs 3 steps to react to synthesis finished product, yield 35.8%, therefore total recovery only has 7.6%, and whole process is related to
Pyroreaction simultaneously uses a variety of organic solvents, is not suitable for large-scale production.
Document Heterocycles, 1998,48 (9):1931-1934 reports are found that left-handed 7- hydroxyls butylphenyl phthaleine first,
Source is fox excrement mould nutrient solution.Left-handed 7- hydroxyls butylphthalide content is very low in nutrient solution, and contains multiple other components, separation
Purge process employs the absorption of HP-20 posts, silica gel column chromatography and HPLC and prepared, and complex process, product yield is low（10L zymotic fluids
Only obtain 16mg）, commercial application difficulty.
This patent is disclosed one plant of fox excrement mould fungal bacterial strain and the side of left-handed 7- hydroxyls butylphenyl phthaleine is prepared using the bacterial strain
Method, under the bacterial strain and condition of culture that the present invention is provided, the fermentation unit of left-handed 7- hydroxyls butylphenyl phthaleine has reached 1600mg/L
More than, and fermentation component is more single, separation purifying technique is simple, can be used for the industrial metaplasia of left-handed 7- hydroxyls butylphenyl phthaleine
Production.
The content of the invention
It is an object of the invention to provide the superior strain of one plant of left-handed 7- hydroxyls butylphenyl phthaleine, while providing one kind utilizes the bacterium
The method that strain prepares left-handed 7- hydroxyls butylphenyl phthaleine.
Left-handed 7- hydroxyls butylphenyl phthaleine producing strains provided by the present invention are fox excrement mould fungal bacterial strain, the entitled fox of its preservation
Excrement mould（Penicillium vulpinum）NCC3421, depositary institution is：China Committee for Culture Collection of Microorganisms is general
Logical microorganism center, preservation date on April 25th, 2014, deposit number is CGMCC No.9094, and preservation address is：Beijing
The institute 3 of Chaoyang District North Star West Road 1.
Fungal bacterial strain NCC3421 provided by the present invention, is accredited as through macroscopic view, microscopic morphology observation and Molecular Identification
Fox excrement mould（Penicillium vulpinum）.
Authentication method of the present invention is morphological feature and Molecular Identification.Bacterial strain NCC3421 spore liquid is inoculated in
PDA culture medium, 25 DEG C are cultivated 7 days in progress microscopic morphology observation under ESEM.By bacterial strain NCC3421 spore liquid dibbling in
4 kinds of general identification culture mediums：CYA（Cha Shi yeast agars）、MEA（Malt flour agar）、G25N（25% glycerophosphate agar）、
PDA（Potato dextrose agar）In, cultivated 7 days at 25 DEG C, carry out macroscopic form observation.ITS sequence survey is carried out to NCC3421
It is fixed, submit NCBI to be compared, phylogenetic tree construction is compared with type strain.By macroscopic view, microscopic morphology observation and molecule mirror
It is fixed, NCC3421 is accredited as fox excrement mould（Penicillium vulpinum）.
Fox excrement mould used in the present invention（Penicillium vulpinum）Fungal bacterial strain NCC3421 is from Sichuan Province
One plant of isolated wild strain in the mountain soil of Ya'an Baoxing County.
Collecting soil sample method of the present invention is with the sterile humic scalped close to plant root soil top layer
Soil, then takes and is placed in preservation in sterilized paper bag apart from earth's surface depth for 5 ~ 10cm about 30 grams of soil sample.
The separation method of pedotheque of the present invention fully shakes to take 1 gram of sample to be added in 10mL sterilized waters
Swing, then take 1mL suspensions to be added in another test tube equipped with 9mL sterilized waters and fully vibrate, the rest may be inferred carries out gradient
Dilution value is to 10-10Concentration.Each concentration takes 0.2mL to be coated with the double dish of PDA, then the quiescent culture at 26 DEG C, double dish cultivation cycles
For 3 ~ 10 days, during this period from double dish the different colony inoculation of picking form in PDA（Beijing bispin）Cultivated in inclined-plane,
The inclined-plane culture time is 14 days.One fungal strain is obtained by the above method, NCC3421 is named as.
Using preparation method of the present invention, the fermentation unit of its left-handed 7- hydroxyls butylphenyl phthaleine reached 1600mg/L with
On, and metabolite is single, therefore it can be applied in left-handed 7- hydroxyls butylphenyl phthaleine is prepared.
The method that utilization fox excrement mould fungal bacterial strain provided by the present invention prepares left-handed 7- hydroxyls butylphenyl phthaleine, including it is following
Step：
A, the seed liquor for preparing fox excrement mould fungal bacterial strain NCC3421：
Bacterial strain NCC3421 slant culture or spore liquid are inoculated in seed culture medium, 22 ~ 30 DEG C, 100 ~ 220
Rpm, 48 ~ 72 h of culture obtain seed liquor；
Wherein described seed culture medium is made by the following method：10.0 ~ 40.0 grams of cornstarch, glucose 10.0 ~
40.0 grams, 2.0 ~ 10.0 grams of hot moulding soybean cake powder, 1.0 ~ 6.0 grams of malt flour, 1.0 ~ 3.0 grams of dusty yeast, sodium chloride 0.5 ~ 2.0
Gram, 0.5 ~ 2.0 gram of epsom salt, add water and be settled to 1000mL, pH6.5 ~ 7.0,121 DEG C sterilizing 30min.
B, the zymotic fluid for preparing fox excrement mould fungal bacterial strain NCC3421：
Above-mentioned seed liquor is inoculated in fermentation medium with the inoculum concentration of percent by volume 3 ~ 10%, in shaking flask or fermentation tank
Middle fermentation, 22 ~ 30 DEG C of fermentation temperature, 100 ~ 220 rpm, the h of fermentation time 120 ~ 180 obtains zymotic fluid；
Wherein described fermentation medium is made by the following method：10.0 ~ 40.0 grams of glucose, soluble starch 10.0 ~
40.0 grams, 10.0 ~ 40.0 grams of glycerine, 4.0 ~ 20.0 grams of hot moulding soybean cake powder, 4.0 ~ 20.0 grams of cotton seed meal, corn steep liquor 4.0 ~ 15.0
Gram, 1.0 ~ 5.0 grams of potassium dihydrogen phosphate, 1.0 ~ 5.0 grams of ammonium sulfate, 0.5 ~ 2.0 gram of sodium chloride, 0.2 ~ 2.0 gram of epsom salt, plus
Water is settled to 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min.
C, above-mentioned zymotic fluid centrifuged, supernatant discarding, which is obtained, adds methanol, ethanol or acetone leaching in mycelium, mycelium
Carry, stir, filtering, filter residue repeats extraction once, merging extracts filtrate twice, and be concentrated under reduced pressure to obtain medicinal extract.
D, medicinal extract is added into ethyl acetate, chloroform or n-hexane extracted, stir, stand, separate to obtain supernatant, repeat to extract
Once, supernatant twice, plus anhydrous sodium sulfate dehydration are merged, filtering, filtrate decompression is dense dry to obtain oily crude extract.
E, by oily crude extract methanol or ethanol dissolving after, be added on HZ816, D312 or AB-8 chromatographic resin post capital,
Eluted using the polar organic solvent aqueous solution as eluant, eluent, HPLC detection effluxes are collected and contain left-handed 7- hydroxyls butylphenyl phthaleine
Part.It is concentrated under reduced pressure and is evaporated, obtains left-handed 7- hydroxyls butylphenyl phthaleine crude product.
F, by left-handed 7- hydroxyls butylphenyl phthaleine crude product be dissolved in the polar organic solvent aqueous solution crystallize, filtering, be dried in vacuo
Left-handed 7- hydroxyls butylphenyl phthaleine fine work.
Shake flask fermentation wherein described in step b, its rotating speed is 100 ~ 220rpm.
Ferment tank wherein described in step b, its tank pressure is that 0.05 ± 0.01Mpa, throughput are 10 ~ 30L/min,
Mixing speed is 400 ~ 500rpm.
Alcohol steep is added in wherein step c, the volume of addition is 1 ~ 3 times of mycelium volume, is preferably added to mycelium 2
The industrial alcohol of times volume.
Ethyl acetate extraction is added in wherein step d, the volume of addition is 3 ~ 5 times, preferably 5 times of medicinal extract volume.
Oily crude extract methanol dissolves in wherein step e, and consumption is 2 times of oily crude extract volume, and eluant, eluent is first
Alcohol solution, ethanol water or aqueous acetone solution, the preferred alcohol aqueous solution carry out three-level gradient elution, gradient be 30%, 50%,
70%.Post blade diameter length ratio is 1：3~1：10 preferably 1：5,5 ~ 10 times of bed volumes of every grade of elution, 2 ~ 4 times of bed volume/hours of flow velocity,
2 times of bed volume/hours of preferable flow rate.Collect left-handed 7- hydroxyls butylphthalide content 10mg/ml above section eluents.
The polar organic solvent aqueous solution is methanol aqueous solution, ethanol water or acetone water wherein used in step f crystallization
Solution, the concentration expressed in percentage by volume of polar organic solvent is 70 ~ 80%, is preferably crystallized with methanol aqueous solution.
Wherein step f crystallization process, every gram of dissolving crude product is crystallized in 20 ~ 30 milliliters of polar organic solvent aqueous solution,
Time is between 2 ~ 24h.
Above-mentioned optimum condition, it is higher that it obtains left-handed 7- hydroxyls butylphenyl phthaleine yield, better quality.
Fox excrement mould fungal bacterial strain NCC3421 used in the present invention is wild strain, its left-handed 7- hydroxyls butylphenyl phthaleine hair
Ferment unit has reached 1600 more than mg/L, and fermentation component is more single, it is easy to isolate and purify.Left-handed 7- hydroxyls in the present invention
Butylphenyl phthaleine preparation technology is easy, suitable for industrialized production, and product total recovery reaches 60 more than %.
Brief description of the drawings
Fig. 1 is the coremium of bacterial strain.
Fig. 2 is the conidiophore of bacterial strain.
Fig. 3 is the conidia chain of bacterial strain.
Fig. 4 is the colonial morphology that bacterial strain is cultivated 7 days on CYA.
Fig. 5 is the colonial morphology that bacterial strain is cultivated 7 days on MEA.
Fig. 6 is the colonial morphology that bacterial strain is cultivated 7 days on G25N.
Fig. 7 is the colonial morphology that bacterial strain is cultivated 7 days on PDA.
Fig. 8 is the phylogenetic tree built according to NCC3421 ITS sequence.
Fig. 9 is that NCC3421 zymotic fluid thallus extracts HPLC detects collection of illustrative plates.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
Experiment material used is industrial raw material unless otherwise specified in following embodiments；Analyze chromatographic column Inertsil ODS-
5 μm of mm of C18 4.6 × 250 of SP are purchased from Japanese Shimadzu, and organic solvent used in extraction process is purchased from Beijing Chemical Plant, and chromatogram is molten
Agent is purchased from Honeywell trade（Shanghai）Co., Ltd.
The separation and identification of embodiment 1, fox excrement mould NCC3421
（a）Bacterial strain NCC3421 separation
Fungal bacterial strain NCC3421 separation is PDA with culture medium（Beijing bispin）；
Take 1 gram of pedotheque（Pick up from Sichuan Province China province Ya'an Baoxing County mountain soil）, it is put into equipped with 9mL sterilized waters
In 50mL centrifuge tubes, fully vibration.Take 1mL suspensions to be put into the test tube equipped with 9mL sterilized waters, mix.Series is carried out successively
Gradient dilution is until 10-10.0.2mL is taken to apply flat board under each gradient.Applied flat board is placed in 26 DEG C of quiescent cultures, therefrom obtains
One fungal strain, is named as NCC3421.
（b）Bacterial strain NCC3421 identification --- morphological feature and Molecular Identification
Bacterial strain NCC3421 spore liquid is inoculated in PDA culture medium, 25 DEG C of cultures 7 days under ESEM after being shown
Titanium miniplate is observed.By bacterial strain NCC3421 spore liquid dibbling in 4 kinds of general identification culture mediums：CYA（Cha Shi yeast agars）、MEA
（Malt flour agar）、G25N（25% glycerophosphate agar）、PDA（Potato dextrose agar）In, 25 DEG C are cultivated 7 days, are carried out
Macroscopic form is observed.
ITS sequence measure is carried out to NCC3421, submits NCBI to be compared, phylogenetic tree construction, with type strain ratio
It is right.
1）Morphological feature
Bacterial strain NCC3421 microphoto, Fig. 1 is that the coremium of bacterial strain, Fig. 2 are that the conidiophore of bacterial strain, Fig. 3 are
The conidia chain of bacterial strain, as seen from the figure conidiophore combine closely the coremium in handle；Falx stem is thin and bends, surface
It is smooth；The irregular branch of penicillus, is entangled with into mutually hymenium；8-12 μm of metulae；There are 3-5 bottle stalk, 10- on each metulae
12 μm, bottle stalk neck is short and attenuates suddenly；Conidium is oval, smooth, general 3.5-4.0 μm, with long and narrow spore every, point
Raw sporogenesis long-chain, covering coremium top forms a column.
Morphological features of the bacterial strain NCC3421 in 4 kinds of general identification culture mediums is as follows：
NCC3421 is in CYA（Cha Shi yeast agars）Upper 7 day, colony diameter reaches 30-34mm；Bacterium colony superficial white is velvet-like；
There is the radial lines of relatively regular distribution；Centre produces large-scale coremium, and handle is close and irregular tufted growth, and top is covered
The abundanter celadon spore of lid；Annulus is presented in the coremium comparatively dense tufted growth of proximal edge position；There is liquid on the coremium of part
Drop；The radial lines in bacterium colony back is in clearly relatively regular distribution；Coremium back is in brown.NCC3421 is cultivated 7 days on CYA
The form of bacterium colony is shown in Fig. 4.
NCC3421 is in MEA（Malt flour agar）Upper 7 day, colony diameter reaches 24-26mm；Bacterium colony superficial white is velvet-like；Closely
Central part coremium tufted dense growth, is presented annulus, and coremium is more elongated, by base portion upwards in rhodo to white gradual change；
There is the extremely short small coremium of white at edge；Surface is without obvious radial lines；The radial lines in bacterium colony back is in clearly relatively regular point
Cloth；Coremium back is in brown.The form that NCC3421 cultivates 7 days bacterium colonies on MEA is shown in Fig. 5.
NCC3421 is in G25N（25% glycerophosphate agar）Upper 7 day, colony diameter reaches 16-18mm；Bacterium colony surface is white
Color is velvet-like；Central part slightly swells, comparatively dense tufted, is diametrically 4-6mm celadon subcirculars；There is the radiation of relatively regular distribution
Shape lines；Bacterium colony back is light yellow；Radial lines is clear, in brown color.NCC3421 cultivates the shape of 7 days bacterium colonies on G25N
State is shown in Fig. 6.
NCC3421 is in PDA（Potato dextrose agar）Upper 7 day, colony diameter reaches 24-26mm；Bacterium colony superficial white
Staple；There are 1-2 white short and small coremiums, top covering celadon spore in bacterium colony center once in a while；There is the extremely short small spore of white at edge
Obstruct beam；Bacterium colony back is white to light yellow；Coremium back is in brown.The form that NCC3421 cultivates 7 days bacterium colonies on PDA is shown in
Fig. 7.
2）Molecular Identification
Length is 582bp to NCC3421 ITS sequence after measured：
Tccgtaggtgaacctgcggaaggatcattaccgagctgacggcctctgggtccacctcccacccgtgtttatttacc
ttgttgcttcggcgggcccgccttaactggccgccggggggcttccgcccccgggcccgcgcccgccgaagacaccc
ccgaactctgtctgaagattgcagtctgagtgaaaatataaattatttaaaactttcaacaacggatctcttggttc
cggcatcgatgaagaacgcagcgaaatgcgatacgtaatgtgaattgcaaattcagtgaatcatcgagtctttgaac
gcacattgcgccccctggtattccggggggcatgcctgtccgagcgtcatttctgccctcaagcccggcttgtgtgt
tgggccccgtcccccgatcccgggggacgggcccgaaaggcagcggcggcaccgcgtccggtcctcgagcgtatggg
gctttgtcacccgctctgtaggcccggccggcgcctgccaccaacccaaatttttatccaggttgacctcggatcag
gtagggatacccgctgaacttaagcatatcaataagcggagga
Sequence submission NCBI is compared, and phylogenetic tree construction, it is according to what NCC3421 ITS sequence was built
System development tree（See Fig. 8）, it can be seen that the bacterium and the typical strain fox excrement mould in Penicillium mould subgenus（Penicillium vulpinumNRRL 1002）In a branch, illustrate that the affiliation between them is nearer.
According to macroscopic view, micro-morphology and Molecular Identification, NCC3421 is accredited as fox excrement mould（Penicillium vulpinum）.
The preparation of embodiment 2,5L tanks fermentation 7- hydroxyl butylphenyl phthaleines
(a) fox excrement penicillium bacterial strain NCC3421 seed liquors are prepared
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 26 DEG C, the rpm of rotating speed 200 cultivates 72 h and planted
Sub- liquid.The preparation method of seed culture medium is：20.0 grams of cornstarch, 10.0 grams of glucose, 2.0 grams of hot moulding soybean cake powder, wheat
6.0 grams of bud powder, 3.0 grams of dusty yeast, 2.0 grams of sodium chloride, 1.0 grams of epsom salt, plus running water dissolving are settled to 1000
Ml, 6.8,121 DEG C of 30 min of sterilizing of pH value.
(b) fox excrement penicillium bacterial strain NCC3421 zymotic fluid is prepared
Seed liquor is inoculated in the fermentation tank equipped with 5 L fermentation mediums, tank temperature 26 with the inoculum concentration of percent by volume 5%
DEG C, tank press 0.05 ± 0.01 Mpa, throughput 30 L/min, 220 rpm stirrings, ferment 168 h.The preparation side of fermentation medium
Method is：14.5 grams of glucose, 18.0 grams of soluble starch, 21.5 grams of glycerine, 15.5 grams of hot moulding soybean cake powder, 8.5 grams of cotton seed meal,
8.5 grams of corn steep liquor, 2.5 grams of potassium dihydrogen phosphate, 2.5 grams of ammonium sulfate, 1.2 grams of sodium chloride, 0.4 gram of epsom salt, plus running water
It is settled to 1000mL, pH6.5,121 DEG C of sterilizing 30min.Fermentation finish after through detecting that left-handed 7- hydroxyls butylphenyl phthaleine fermentation unit is
1650mg/L。
（c）Take the fox excrement penicillium bacterial strain NCC3421 L of zymotic fluid 2.0（Left-handed 7- hydroxyls butylphthalide content 1650mg/L）In
20min is centrifuged in 4000r/min centrifuge, mycelia about 500ml, plus the extraction of 1000ml industrial acetones is collected, stirs 1 hour,
Filtering, collects filtrate.Repeat extraction once.Merge filtrate about 2.2L twice.It is concentrated under reduced pressure and recovery acetone and removes big portion's moisture,
Obtain medicinal extract about 80g.
（d）80g medicinal extract adds 320ml chloroforms, stirs 30min, stands 1 hour, separates supernatant.It is repeated once.In merging
Clear liquid, evaporated under reduced pressure reclaims chloroform, obtains about 3.9ml oily crude extracts.
（e）Post, column internal diameter 3.2cm, post bed height about 16cm are filled with AB-8 resins.Post bed is rinsed with 500ml salt-free waters, it is standby
With.3.9ml crude extracts are dissolved in 8ml methanol, and filtering, filtrate is added on the AB-8 chromatographic columns capital handled well in two times, are added every time
Resin column all is rinsed with 200ml salt-free waters after sample, slightly stirring abacus knot is divided if necessary.Then respectively with 30%, 50%,
Each 1200ml of 70% ethanol water carries out gradient elution, flow velocity 8.5ml/min, HPLC detection eluent to resin column.In 70% ethanol
When water section is eluted, left-handed 7- hydroxyls butylphthalide content 10mg/ml above section eluents are collected, decompression boils off ethanol, filters
Drain to obtain left-handed 7- hydroxyls butylphenyl phthaleine crude product 2.5g.
（f）Left-handed 7- hydroxyls butylphenyl phthaleine crude product about 2.5g, is dissolved with 52ml methanol, and about 22ml distilled water is added dropwise, micro- aobvious muddy
It is turbid.Warm clarifies solution, and crystal is slowly separated out, and crystallizes 2 hours, filtering.Vacuum drying, obtains left-handed 7- hydroxyls butylphenyl phthaleine fine work
2.1g, content 99.5%, total recovery 63.3%.
（g）Left-handed 7- hydroxyls butylphenyl phthaleine, water white transparency sheet or column crystal；It is soluble in methanol, ethanol, chloroform, acetic acid
Ethyl ester, is dissolved in n-hexane, is slightly soluble in water；There is maximum absorption band at 234 nm, 298 nm；HRMS shows its molecular ion peak [M
+H]+For 207.1015；Survey [α]=- 48.5o(c, 0.8, CHCl3).Pure state is stablized at room temperature, stable under solution state, sun
Relatively stablize under light direct beam, less stable in the presence of soda acid.
The comparison such as table 1 that the hydrocarbon signals assignment of left-handed 7- hydroxyls butylphenyl phthaleine is reported with document *.Nuclear magnetic resonance tester
For the MHz of Varian Inova 500 (1H)、125MHz(13C)。
Table 1 is made by oneself and the left-handed 7- hydroxyls butylphenyl phthaleine of reported in literature13C-NMR and1H-NMR attribution data tables
Document * is reported as HETEROCYCLES, Vol.48, NO.9,1998, P1931-1934
（h）The liquid chromatographic detection condition of left-handed 7- hydroxyls butylphenyl phthaleine
Chromatographic column：Inertsil ODS-SP 5μm C18 4.6×250 mm
Mobile phase：A acetonitriles；B water, containing 0.05% phosphoric acid.Flow velocity 1ml/min.Gradient sets such as following table.
Detector：UV, 210nm.
NCC3421 zymotic fluid thallus extracts HPLC detects collection of illustrative plates（See Fig. 9）.From the UV 3D collection of illustrative plates of collection,
In NCC3421 zymotic fluid thalline ethanol extracts in addition to having some highly polar components, primary product is both left-handed 7- hydroxyls butylbenzene
Phthalein（Retention time is about 17.2min）.
（I）The liquid chromatogram quick detection condition of left-handed 7- hydroxyls butylphenyl phthaleine
Chromatographic column：Inertsil ODS-SP 5μm C18 4.6×250 mm
Mobile phase：Acetonitrile/water=60/40, containing 0.05 phosphoric acid；Flow velocity 1ml/min
Detector：UV, 210nm.
Under above-mentioned condition, the retention time of left-handed 7- hydroxyls butylphenyl phthaleine is about 5.9min.
The preparation of embodiment 3, shake flask fermentation 7- hydroxyl butylphenyl phthaleines
（a）Prepare fox excrement penicillium bacterial strain NCC3421 seed liquor
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 26 DEG C, the rpm of rotating speed 200 cultivates 72 h and planted
Sub- liquid.The preparation method of seed culture medium is：20.0 grams of cornstarch, 10.0 grams of glucose, 2.0 grams of hot moulding soybean cake powder, wheat
6.0 grams of bud powder, 3.0 grams of dusty yeast, 2.0 grams of sodium chloride, 1.0 grams of epsom salt, plus running water dissolving are settled to 1000
Ml, 6.5,121 DEG C of 30 min of sterilizing of pH value.
（b）Prepare fox excrement penicillium bacterial strain NCC3421 zymotic fluid
Seed liquor is inoculated in the 250mL shaking flasks equipped with 40mL fermentation mediums, 26 with the inoculum concentration of percent by volume 5%
DEG C, the rpm of rotating speed 200, ferment 144 h.The preparation method of fermentation medium is：20.5 grams of glucose, soluble starch 18.0
Gram, 21.5 grams of glycerine, 15.5 grams of hot moulding soybean cake powder, 15.5 grams of cotton seed meal, 8.5 grams of corn steep liquor, 2.5 grams of potassium dihydrogen phosphate, sulphur
Sour 2.5 grams of ammonium, 0.5 gram of sodium chloride, 0.2 gram of epsom salt, plus running water are settled to 1000mL, pH6.7,121 DEG C of sterilizings
30min.Ferment after finishing through detecting that left-handed 7- hydroxyls butylphenyl phthaleine fermentation unit is 1638mg/L.
（c）Take the fox excrement penicillium bacterial strain NCC3421 L of zymotic fluid 2.0（Left-handed 7- hydroxyls butylphthalide content 1638mg/L）In
20min is centrifuged in 4000r/min centrifuge, mycelia about 500ml, plus the extraction of 1000ml industrial alcohols is collected, stirs 1 hour,
Filtering, collects filtrate.Repeat extraction once.Merge filtrate about 2.2L twice.The recovery ethanol that is concentrated under reduced pressure simultaneously removes big portion's moisture,
Obtain medicinal extract about 77g.
（d）77g medicinal extract adds 250ml ethyl acetate, stirs 30min, stands 1 hour, separates supernatant.It is repeated once.Close
And supernatant, evaporated under reduced pressure recovery ethyl acetate, obtain about 3.7ml oily crude extracts.
（e）Post, column internal diameter 3.2cm, post bed height about 16cm are filled with HZ816 resins.Post bed is rinsed with 500ml salt-free waters,
It is standby.3.7ml crude extracts are dissolved in 8ml methanol, and filtering, filtrate is added on the HZ816 chromatographic columns capital handled well in two times, every time
Resin column all is rinsed with 200ml salt-free waters after sample-adding, slightly stirring abacus knot is divided if necessary.Then respectively with 30%, 50%,
Each 650ml of 70% ethanol water carries out gradient elution, flow velocity 4.5ml/min, HPLC detection eluent to resin column.When 70% ethanol water
When part is eluted, left-handed 7- hydroxyls butylphthalide content 10mg/ml above section eluents are collected, decompression boils off ethanol, and filtering is taken out
Do to obtain left-handed 7- hydroxyls butylphenyl phthaleine crude product 2.4g.
（f）Left-handed 7- hydroxyls butylphenyl phthaleine crude product about 2.4g, is dissolved with 56ml methanol, and about 14ml distilled water is added dropwise, micro- aobvious muddy
It is turbid.Warm clarifies solution, and crystal is slowly separated out, and crystallizes 10 hours, suction filtration, vacuum drying, obtains left-handed 7- hydroxyls butylphenyl phthaleine essence
Product 2.0g, content 99.4%, total recovery 60.7%.
The preparation of embodiment 4, shake flask fermentation 7- hydroxyl butylphenyl phthaleines
（a）Prepare fox excrement penicillium bacterial strain NCC3421 seed liquor
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 22 DEG C, the rpm of rotating speed 100 cultivates 66 h and planted
Sub- liquid.The preparation method of seed culture medium is：40.0 grams of cornstarch, 20.0 grams of glucose, 5.5 grams of hot moulding soybean cake powder, wheat
4.0 grams of bud powder, 3.0 grams of dusty yeast, 0.5 gram of sodium chloride, 0.5 gram of epsom salt, plus running water dissolving are settled to 1000
Ml, 6.5,121 DEG C of 30 min of sterilizing of pH value.
（b）Prepare fox excrement penicillium bacterial strain NCC3421 zymotic fluid
Seed liquor is inoculated in the 250mL shaking flasks equipped with 40mL fermentation mediums, 22 with the inoculum concentration of percent by volume 10%
DEG C, the rpm of rotating speed 100, ferment 180 h.The preparation method of fermentation medium is：10.0 grams of glucose, soluble starch 40.0
Gram, 10.0 grams of glycerine, 20.0 grams of hot moulding soybean cake powder, 4.0 grams of cotton seed meal, 4.0 grams of corn steep liquor, 1.0 grams of potassium dihydrogen phosphate, sulfuric acid
1.0 grams of ammonium, 2.0 grams of sodium chloride, 2.0 grams of epsom salt, plus running water are settled to 1000mL, pH6.5,121 DEG C of sterilizings
30min.Fermentation obtains about 2L zymotic fluids after finishing, and 7- hydroxyls butylphenyl phthaleine fermentation unit is 1696mg/L after testing.
（c）Take the fox excrement penicillium bacterial strain NCC3421 L of zymotic fluid 2.0（Left-handed 7- hydroxyls butylphthalide content 1696mg/L）In
20min is centrifuged in 4000r/min centrifuge, mycelia about 500ml, plus the extraction of 1000ml methanol is collected, stirs 1 hour, filtering,
Collect filtrate.Repeat extraction once.Merge filtrate about 2.2L twice.It is concentrated under reduced pressure and reclaims methanol and remove big portion's moisture, must soaks
Cream about 83g.
（d）83g medicinal extract adds 400ml n-hexanes, stirs 30min, stands 1 hour, separates supernatant.It is repeated once.Merge
Supernatant, evaporated under reduced pressure reclaims n-hexane, obtains about 3.5ml oily crude extracts.
（e）Post, column internal diameter 3.2cm, post bed height about 16cm are filled with D312 resins.Post bed is rinsed with 500ml salt-free waters, it is standby
With.3.5ml crude extracts are dissolved in 7ml methanol, and filtering, filtrate is added on the D312 chromatographic columns capital handled well in two times, are added every time
Resin column all is rinsed with 200ml salt-free waters after sample, slightly stirring abacus knot is divided if necessary.Then respectively with 30%, 50%,
Each 1000ml of 70% ethanol water carries out gradient elution, flow velocity 6ml/min, HPLC detection eluent to resin column.When 70% ethanol portion
When dividing elution, left-handed 7- hydroxyls butylphthalide content 10mg/ml above section eluents are collected, decompression boils off ethanol, and filtering is drained
Obtain left-handed 7- hydroxyls butylphenyl phthaleine crude product 2.5g.
（f）Left-handed 7- hydroxyls butylphenyl phthaleine crude product about 2.5g, is dissolved with 37ml methanol, and about 13ml distilled water is added dropwise, micro- aobvious muddy
It is turbid.Warm clarifies solution, and crystal is slowly separated out, and crystallizes 20 hours, suction filtration, filter cake vacuum drying, obtains left-handed 7- hydroxyls butylbenzene
Phthalein fine work 2.2g, content 99.5%, total recovery 64.5%.
The preparation of embodiment 5, shake flask fermentation 7- hydroxyl butylphenyl phthaleines
（a）Prepare fox excrement penicillium bacterial strain NCC3421 seed liquor
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 30 DEG C, the rpm of rotating speed 220 cultivates 48 h and planted
Sub- liquid.The preparation method of seed culture medium is：10.0 grams of cornstarch, 40.0 grams of glucose, 10.0 grams of hot moulding soybean cake powder, wheat
1.5 grams of bud powder, 1.0 grams of dusty yeast, 1.0 grams of sodium chloride, 2.0 grams of epsom salt, plus running water dissolving are settled to 1000
Ml, 7.0,121 DEG C of 30 min of sterilizing of pH value.
（b）Prepare fox excrement penicillium bacterial strain NCC3421 zymotic fluid
Seed liquor is inoculated in the 250mL shaking flasks equipped with 40mL fermentation mediums, 30 with the inoculum concentration of percent by volume 3%
DEG C, the rpm of rotating speed 220, ferment 120 h.The preparation method of fermentation medium is：40.0 grams of glucose, soluble starch 10.0
Gram, 40.0 grams of glycerine, 4.0 grams of hot moulding soybean cake powder, 20.0 grams of cotton seed meal, 15.0 grams of corn steep liquor, 5.0 grams of potassium dihydrogen phosphate, sulphur
Sour 5.0 grams of ammonium, 0.5 gram of sodium chloride, 1.0 grams of epsom salt, plus running water are settled to 1000mL, 7.0,121 DEG C of sterilizings of pH value
30min.Fermentation obtains zymotic fluid about 2L after finishing, and 7- hydroxyls butylphenyl phthaleine fermentation unit is 1739mg/L after testing.
（c）Take the fox excrement penicillium bacterial strain NCC3421 L of zymotic fluid 2.0（Left-handed 7- hydroxyls butylphthalide content 1739mg/L）In
20min is centrifuged in 4000r/min centrifuge, mycelia about 500ml, plus the extraction of 1000ml industrial acetones is collected, stirs 1 hour,
Filtering, collects filtrate.Repeat extraction once.Merge filtrate about 2.2L twice.It is concentrated under reduced pressure and recovery acetone and removes big portion's moisture,
Obtain medicinal extract about 85g.
（d）85g medicinal extract adds 300ml ethyl acetate, stirs 30min, stands 1 hour, separates supernatant.It is repeated once.Close
And supernatant, evaporated under reduced pressure recovery ethyl acetate, obtain about 4.1ml oily crude extracts.
（e）Post, column internal diameter 3.2cm, post bed height about 16cm are filled with D312 resins.Post bed is rinsed with 500ml salt-free waters, it is standby
With.4.1ml crude extracts are dissolved in 8ml methanol, and filtering, filtrate is added on the D312 chromatographic columns capital handled well in two times, are added every time
Resin column all is rinsed with 200ml salt-free waters after sample, slightly stirring abacus knot is divided if necessary.Then respectively with 30%, 50%,
Each 800ml of 70% ethanol water carries out gradient elution, flow velocity 5ml/min, HPLC detection eluent to resin column.In 70% ethanolic moiety
During elution, left-handed 7- hydroxyls butylphthalide content 10mg/ml above section eluents are collected, decompression boils off ethanol, and filtering is drained
Left-handed 7- hydroxyls butylphenyl phthaleine crude product 2.7g.
（f）Left-handed 7- hydroxyls butylphenyl phthaleine crude product about 2.7g, is dissolved with 60ml ethanol, and about 20ml distilled water is added dropwise, micro- aobvious muddy
It is turbid.Warm clarifies solution, and crystal is slowly separated out, and crystallizes 24 hours, suction filtration, vacuum drying, obtains left-handed 7- hydroxyls butylphenyl phthaleine essence
Product 2.3g, content 99.3%, total recovery 65.7%.
Claims (10)
1. one plant of fox excrement mould fungal bacterial strain NCC3421（Penicillium vulpinum）, its deposit number is CGMCC
No.9094。
2. the method that the bacterial strain described in a kind of utilization claim 1 prepares left-handed 7- hydroxyls butylphenyl phthaleine, comprises the following steps：
A, the seed liquor for preparing fox excrement mould fungal bacterial strain NCC3421：
Bacterial strain NCC3421 slant culture or spore liquid are inoculated in seed culture medium, 22 ~ 30 DEG C, 100 ~ 220 rpm, training
Support 48 ~ 72 h and obtain seed liquor；
Wherein described seed culture medium is made by the following method：10.0 ~ 40.0 grams of cornstarch, 10.0 ~ 40.0 grams of glucose,
2.0 ~ 10.0 grams of hot moulding soybean cake powder, 1.0 ~ 6.0 grams of malt flour, 1.0 ~ 3.0 grams of dusty yeast, 0.5 ~ 2.0 gram of sodium chloride, seven water sulphur
Sour 0.5 ~ 2.0 gram of magnesium, adds water and is settled to 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min；
B, the zymotic fluid for preparing fox excrement mould fungal bacterial strain NCC3421：
Above-mentioned seed liquor is inoculated in fermentation medium with the inoculum concentration of percent by volume 3 ~ 10%, sent out in shaking flask or fermentation tank
Ferment, 22 ~ 30 DEG C of fermentation temperature, 100 ~ 220 rpm, the h of fermentation time 120 ~ 180, obtains zymotic fluid；
Wherein described fermentation medium is made by the following method：10.0 ~ 40.0 grams of glucose, 10.0 ~ 40.0 grams of soluble starch,
10.0 ~ 40.0 grams of glycerine, 4.0 ~ 20.0 grams of hot moulding soybean cake powder, 4.0 ~ 20.0 grams of cotton seed meal, 4.0 ~ 15.0 grams of corn steep liquor, phosphoric acid
1.0 ~ 5.0 grams of potassium dihydrogen, 1.0 ~ 5.0 grams of ammonium sulfate, 0.5 ~ 2.0 gram of sodium chloride, 0.2 ~ 2.0 gram of epsom salt, add water constant volume
To 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min；
C, above-mentioned zymotic fluid centrifuged, supernatant discarding obtains mycelium, and methanol, ethanol or acetone leaching are added into mycelium
Carry, stir, filtering, filter residue repeats extraction once, merging extracts filtrate twice, and be concentrated under reduced pressure to obtain medicinal extract；
D, medicinal extract is added to ethyl acetate, chloroform or n-hexane extraction, stir, stand, separate to obtain supernatant, be repeated once, merge
Supernatant twice, plus anhydrous sodium sulfate dehydration, filtering, filtrate decompression is dense dry to obtain oily crude extract；
E, by oily crude extract methanol or ethanol dissolving after, HZ816, D312 or AB-8 chromatographic resin post capital are added on, with pole
Property aqueous solutions of organic solvent be that eluant, eluent is eluted, HPLC detection effluxes are collected containing left-handed 7- hydroxyls butylphenyl phthaleine part,
It is concentrated under reduced pressure and is evaporated, obtains left-handed 7- hydroxyls butylphenyl phthaleine crude product；
F, by above-mentioned left-handed 7- hydroxyls butylphenyl phthaleine crude product be dissolved in the polar organic solvent aqueous solution crystallize, filtering, be dried in vacuo
Left-handed 7- hydroxyls butylphenyl phthaleine fine work.
3. add alcohol steep in method according to claim 2, wherein step c.
4. ethyl acetate extraction is added in method according to claim 2, wherein step d.
5. oily crude extract methanol dissolves in method according to claim 2, wherein step e, chromatographic resin is
HZ816, eluant, eluent is methanol aqueous solution, ethanol water or aqueous acetone solution.
6. method according to claim 5, wherein step e eluant, eluents methanol aqueous solution, ethanol water or acetone are water-soluble
Liquid elutes for three-level gradient 30%, 50%, 70%, 5 ~ 10 times of bed volumes of every grade of elution, 2 ~ 4 times of bed volume/hours of flow velocity.
7. method according to claim 2, the polar organic solvent aqueous solution is that methanol is water-soluble wherein used in step f crystallization
Liquid, ethanol water or aqueous acetone solution, the concentration expressed in percentage by volume of polar organic solvent is 70 ~ 80%.
8. the crystallization process of method according to claim 7, wherein step f, every gram of dissolving crude product is in 20 ~ 30 milliliters of polarity
Crystallized in aqueous solutions of organic solvent, the time is between 2 ~ 24h.
9. method according to claim 2, the wherein shake flask fermentation described in step b, its rotating speed are 100 ~ 220rpm.
10. method according to claim 2, the wherein ferment tank described in step b, its tank pressure for 0.05 ±
0.01Mpa, throughput are 10 ~ 30L/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510113631.9A CN104726347B (en) | 2015-03-16 | 2015-03-16 | One plant of fox excrement mould fungal bacterial strain and the method for preparing left-handed 7 hydroxyl butylphenyl phthaleine using the bacterial strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510113631.9A CN104726347B (en) | 2015-03-16 | 2015-03-16 | One plant of fox excrement mould fungal bacterial strain and the method for preparing left-handed 7 hydroxyl butylphenyl phthaleine using the bacterial strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104726347A CN104726347A (en) | 2015-06-24 |
| CN104726347B true CN104726347B (en) | 2017-08-29 |
Family
ID=53450767
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510113631.9A Active CN104726347B (en) | 2015-03-16 | 2015-03-16 | One plant of fox excrement mould fungal bacterial strain and the method for preparing left-handed 7 hydroxyl butylphenyl phthaleine using the bacterial strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104726347B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226370B (en) * | 2019-07-15 | 2021-11-19 | 浙江泛亚生物医药股份有限公司 | New strain of penicillium fox dung and application thereof |
| CN112625016B (en) * | 2019-09-24 | 2024-06-07 | 华北制药集团新药研究开发有限责任公司 | 7-Hydroxy butylphthalide crystal form B and preparation method thereof |
| CN114762700B (en) * | 2021-01-13 | 2023-03-21 | 浙江泛亚生物医药股份有限公司 | Application of penicillium fox dung culture |
| CN114762699B (en) * | 2021-01-13 | 2023-06-20 | 浙江泛亚生物医药股份有限公司 | External preparation of penicillium bromhidrosis culture |
| CN115141861A (en) * | 2022-08-03 | 2022-10-04 | 华北制药集团新药研究开发有限责任公司 | Method for producing 7-hydroxy butylphthalide by fermenting penicillium fox dung |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7482027B2 (en) * | 2005-04-30 | 2009-01-27 | Angiolab, Inc. | Composition for the prevention or treatment of diseases associated with angiogenesis |
-
2015
- 2015-03-16 CN CN201510113631.9A patent/CN104726347B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| Antibacterial Polyketides from the Jellyfish-Derived Fungus Paecilomyces variotii;Juan Liu等;《Journal of Natural Products》;20111231;第74卷;1826-1829 * |
| Chemoenzymatic total synthesis of paecilocin A and 3-butyl-7-hydroxyphthalide;Ch. Sreelakshmi等;《Tetrahedron Letters》;20140103;第55卷;1303-1305 * |
| Studies on the metabolites of Penicillium Vulpinum;Mitsuko Makino等;《Heterocycles》;19981231;第48卷(第9期);1931-1934 * |
| Synthesis and Absolute Configuration of (-)-3-Butyl-7-hydroxyphthalide a Cytotoxic Metabolite of Penicillium vulpinum;Tomoya OHZEKI等;《Biosci.Biotechnol.Biochem》;20031231;第67卷(第10期);2240-2244 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104726347A (en) | 2015-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104726347B (en) | One plant of fox excrement mould fungal bacterial strain and the method for preparing left-handed 7 hydroxyl butylphenyl phthaleine using the bacterial strain | |
| CN102433364B (en) | Process for preparing rapamycin by using microbial fermentation method | |
| CN108753627B (en) | Marine aspergillus derived oxaanthraquinone compound, preparation method thereof and application thereof in preparation of antitumor agent | |
| CN108660082B (en) | Marine aspergillus derived oxaanthraquinone compound, preparation method thereof and application thereof in preparation of antibacterial agent | |
| CN102559828B (en) | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms | |
| CN106978350A (en) | One plant of aspergillus niger and its application in the preparation of Pu'er tea chlorins compound | |
| CN114606134A (en) | Sponge epiphyte and application thereof in preparation of oxaanthraquinone compounds | |
| CN103667072B (en) | A kind of Huperzia serrata endogenetic epiphyte and the application at preparation 8 α, 15 α-epoxidation selagine thereof | |
| CN106047713A (en) | Talaromyces pinophilum strain Li-93 and application thereof | |
| CN107189949A (en) | Rhizopus oryzae LJH3 and the application in bioconversion Sophoricoside prepares genistein | |
| CN104894173B (en) | A kind of preparation method of curcumin derivate | |
| CN103981104A (en) | Endophytic fungus and method for biologically converting glycyrrhizic acid into glycyrrhetinic glycoside by using same | |
| CN107099464A (en) | A kind of sclerotium aspergillus and its method for preparing penicillic acid | |
| CN105441504B (en) | A kind of preparation method of CYCLIC DIPEPTIDES compounds | |
| CN117106595A (en) | Industrial cannabis endophytic fungus HMY07 capable of producing flavonoid compounds and application thereof | |
| CN103589665B (en) | Celebrating sheng, a reed pipe wind instrument rhodococcus and the application in preparation (S)-4-chloro-3-hydroxyl ethyl butyrate thereof | |
| CN101100682B (en) | Technique for purifying spherosinin by fermenting green muscardine fungus | |
| CN113337432B (en) | Methylophilus for producing pyrroloquinoline quinone and application thereof | |
| CN109439705A (en) | A kind of microbe preparation method of subergorgic acid | |
| CN103146594B (en) | Sorangiumcellulosum strain and application thereof to synthesis of epothilone | |
| CN106282032B (en) | Penicillium citrinum LB and the application in phillygenol is prepared in bioconversion forsythin | |
| CN104059944B (en) | A kind of method utilizing post spore mould genus fungal bacterial strain to prepare ascochlorin | |
| CN102584615A (en) | Alkaloid compound as well as preparation method and application thereof | |
| CN104004065B (en) | A kind of Isolation and purification method of bleomycin race derivative | |
| CN112961783A (en) | Plant endophytic fungus and application thereof in preparation of spironolactone derivative |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |