CN107201316A - A kind of Aspergillus and its production lung read rhzomorph B0Method - Google Patents
A kind of Aspergillus and its production lung read rhzomorph B0Method Download PDFInfo
- Publication number
- CN107201316A CN107201316A CN201710575306.3A CN201710575306A CN107201316A CN 107201316 A CN107201316 A CN 107201316A CN 201710575306 A CN201710575306 A CN 201710575306A CN 107201316 A CN107201316 A CN 107201316A
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- Prior art keywords
- rhzomorph
- lung
- medium
- seed culture
- culture medium
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- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 description 1
- LRFVTYWOQMYALW-UHFFFAOYSA-N Xanthine Natural products O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 1
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
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- 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
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- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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- 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
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Abstract
The invention discloses a kind of new Aspergillus strain and its application.The Strain Designation is Glarea lozoyensis HS 2158, and deposit number is CGMCC NO.13367.The bacterial strain HS 2158 of the present invention reads rhzomorph B for lung0Producing Strain, and the lung produced reads rhzomorph C0、E0、B5、B0The accessory substances such as serine analogs account for lung and read rhzomorph B0Degree it is all relatively low, reduce lung and read rhzomorph B0Extraction and separating difficulty, therefore bacterial strain HS 2158 of the present invention has good application value.
Description
Technical field
The present invention relates to microbial engineering field, more particularly to a kind of Aspergillus and its read rhzomorph B preparing lung0
(Pneumocandin B0) in application.
Background technology
Caspofungin (Caspofungin) is developed by Merck ＆ Co., Inc., trade name Cancidas (Cancidas), and structural formula is as follows
Shown in formula 1, list within 2001, be widely used in more than 70 countries in the U.S. first.At present, the product has turned into whole body and used
The trump medicine in antifungal market.
Caspofungin is that (enzyme is not found glucan synthase inhibitors in the intracellular of mammal, is many cause a disease
Enzyme required for property fungi synthetic cell wall major part).The enzyme can be catalyzed the glucan base generation in transhipment uridine 5'-diphosphate
β -1,3-D- glucans.It is required that β -1,3-D- glucan synthases grow for fungi, and suppressing the enzyme can make cell wall structure different
Often, cytoclasis, cellular content seepage are caused.Caspofungin has broad-spectrum antifungal activity, can treat resistance to Fluconazole
Mycotoruloides, Eurotium and some region nosomycosises of medicine, to cassette pneumocystosis etc. effectively, pass through Noncompetition inhibition
Fungal cell wall in β -1,3-D- glucan synthases activity, and then cause the cracking of fungal cell wall and intracellular
The change of exosmosis pressure is so that fungal cell thoroughly be killed.
Lung reads rhzomorph B0(Pneumocandin B0) be the synthesis of caspofungin raw material, be a class by Glarea
The natural antifungal lipopeptide antibiotic that lozoyensis is produced, Glarea lozoyensis decapacitation produces A0And B0Two kinds main
Outside product, moreover it is possible to produce C0、D0、E0、B1And B2Deng 16 kinds of analogs, the modification of different aminoacids side chain or the amino acid order of connection
Difference result in the structure diversity that lung reads bacteriums compound.The lung being currently known reads bacteriums compound and the like
Structure as shown in following formula 2 and table 1 below：
The lung of table 1 reads the substituent of bacteriums compound
| Pneumocandin | R1 | R2 | R3 | R4 | R5 | R6 | R7 |
| A0 | CH3 | OH | OH | OH | CH3 | OH | OH |
| B0 | H | OH | OH | OH | CH3 | OH | OH |
| C0 | OH | H | OH | OH | CH3 | OH | OH |
| D0 | OH | OH | OH | OH | CH3 | OH | OH |
| E0 | H | H | OH | OH | CH3 | OH | OH |
| A1 | CH3 | OH | OH | OH | CH3 | H | OH |
| A2 | CH3 | OH | H | H | CH3 | OH | OH |
| A3 | CH3 | OH | OH | H | CH3 | H | H |
| A4 | CH3 | OH | H | H | CH3 | H | H |
| B1 | H | OH | OH | OH | CH3 | H | OH |
| B2 | H | oH | H | H | CH3 | OH | OH |
| B5 | H | OH | OH | H | CH3 | OH | OH |
| B6 | H | oH | H | oH | CH3 | OH | OH |
| D2 | OH | OH | H | H | CH3 | OH | OH |
| B0Serine analogue | H | OH | OH | OH | H | OH | OH |
| B5Serine analogue | H | OH | OH | H | H | OH | OH |
(Protoplast Mutation screening high yield knob is not in Chinese antibiotic magazine the 3rd phase of volume 41 in March, 2016 by Qin Tingting etc.
Kangding B0Bacterial strain) report Merck ＆ Co., Inc. and select lung by nitrosoguanidine mutagenesis and read rhzomorph B0Producing strains ATCC74030's comes
Source, fermentation titer is 241 μ g/ml, and by carbon source optimizing using mannitol as under conditions of carbon source, lung reads rhzomorph B0Potency reaches
800μg/ml.Pass through the preparation and regeneration of protoplast again, going out lung using atmospheric pressure at room plasma (ARTP) mutagenesis screening reads
Rhzomorph B0Superior strain, potency is 1130 μ g/ml.
WO00/08197, which discloses strains A TCC74030 fermentations and prepares lung, reads rhzomorph B0When, using 125g/L fructose as carbon source,
The method for reducing impurity by adding amino acid and trace element, wherein addition 15g/L proline can read lung rhzomorph C0
4.4% is reduced to, but lung reads rhzomorph E0Increase to 2.7%, addition trace element zinc makes lung read rhzomorph B0Serine analogs and B5
Content reduces 1% respectively, but lung reads rhzomorph E0Content adds one times and lung reads rhzomorph B0Potency reduces 50%, addition
Trace element cobalt can make lung read rhzomorph E0Content is reduced to 1.8% from 2.2%, but lung reads rhzomorph B0Serine analogs and
B5Content is doubled and lung reads rhzomorph B0Potency reduces 25%, and addition amino acid and trace element during the fermentation, increases
Production cost is added.
Lung, which can be efficiently separated, even by the silicagel column of large volume reads rhzomorph B0Analogue, but in species level
Improve lung and read rhzomorph B0Potency and reduce the content of accessory substance simultaneously so that the cost for reducing downstream separation is necessary, because
This, find it is a kind of it is new, efficient, accessory substance is low, the lung that is easy to extract reads rhzomorph B0(Pneumocandin B0) production
The work of strain is still in proceeding.
The content of the invention
An object of the present invention is to provide a kind of new bacterial strain Glarea lozoyensis HS-2158 its preservation volumes
Number be CGMCC NO.13367.
Rhzomorph B is read preparing lung the present invention also aims to the bacterial strain HS-2158 of offer0Or answering in its analog
With the analog is that lung reads rhzomorph A0, C0, D0, E0, A1, A2, A3, A4, B1, B2, B5, B6, D2, B0Serine analogs, B5Silk
Propylhomoserin analog, preferably lung read rhzomorph C0、B5、E0、B0Serine analogs.
Lung is prepared using bacterial strain HS-2158 present invention also offers one kind and reads rhzomorph B0Method.This method is included bacterium
Strain HS-2158 carries out bacterium colony culture in solid medium, then carries out seed culture in seed culture medium, is inoculated in fermentation training
Support in base and carry out fermented and cultured.
In preferred embodiments, the time of the bacterium colony culture is 5~15 days, preferably 7~12 days；The seed training
The foster time is 24~120 hours, preferably 48~100 hours；The temperature of the seed culture is 20~30 DEG C, preferably 22~28
℃.The time of the fermented and cultured is 96~360 hours, preferably 216-340 hours；The temperature of the fermented and cultured is 20~30
DEG C, preferably 22~28 DEG C.
In preferred embodiments, the concentration of described seed culture medium each component in the medium is：Carbon source 5.0~
70.0g/L, 5.0~60.0g/L of nitrogen source, 0~5.5g/L of inorganic salts, the pH of described seed culture medium is 5.0~7.0；And/or
The concentration of fermentation medium each component in the medium is：5.0~170.0g/L of carbon source, 5.0~65.0g/ of nitrogen source
L, 0~21.0g/L of inorganic salts, 5.0~30.0g/L of amino acid, the pH of described fermentation medium is 5.0~7.5；
In preferred embodiments, the carbon source of described seed culture medium is selected from glucose, cornstarch, lactose, wheat
One or more in bud dextrin, the nitrogen source of described seed culture medium is selected from soybean cake powder, cottonseed meal, peptone, peanut
One or more in cake powder, Dried Corn Steep Liquor Powder, the inorganic salts of described seed culture medium are selected from sodium nitrate, potassium dihydrogen phosphate,
One or more in dipotassium hydrogen phosphate, potassium nitrate, ammonium sulfate, calcium carbonate, zinc sulfate；And/or
The carbon source of described fermentation medium is selected from glucose, lactose, fructose, mannitol, sorbierite, maltodextrin, jade
One or more in rice starch, soya-bean oil, methyl oleate, the nitrogen source of described fermentation medium is selected from extraction from yeast powder, yeast
Powder, beef extract, soy peptone, peptone, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, bran
One or more in skin, the inorganic salts of described fermentation medium are selected from ammonium chloride, ammonium nitrate, potassium dihydrogen phosphate, phosphoric acid hydrogen
Dipotassium, ammonium sulfate, calcium carbonate, ferrous sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, iron chloride, manganese sulfate, zinc sulfate,
One or more in copper sulphate, the amino acid of described fermentation medium be selected from TYR, L-PROLINE, L-threonine,
One or more in L-ornithine hydrochloride, Valine, L-arginine.
It is preferred that, described seed culture medium contain 5.0~20.0g/L of cornstarch, 10.0~50.0g/L of glucose,
10.0~30.0g/L of soybean cake powder, 5.0~15.0g/L of peptone, 5.0~15.0g/L of groundnut meal, potassium dihydrogen phosphate 0.5~
2.0g/L, 0.5~1.5g/L of ammonium sulfate, 0.5~2.0g/L of calcium carbonate, the pH of described seed culture medium is 6.0~7.0；With/
Or
Described fermentation medium contains 90.0~150.0g/L of sorbierite, 5.0~50.0g/L of glucose, soybean cake powder
5.0~20.0g/L, 5.0~15.0g/L of peptone, 10.0~30.0g/L of cottonseed meal, 1.0~7.0g/L of potassium dihydrogen phosphate,
0.5~4.0g/L of ammonium sulfate, 0.5~5.0g/L of calcium chloride, 0.8~5.0g/L of zinc sulfate, 5.0~30.0g/L of L-PROLINE, institute
The pH for the fermentation medium stated is 5.0~7.0.
It is furthermore preferred that described seed culture medium contains cornstarch 10.0g/L, glucose 30.0g/L, soybean cake powder
20.0g/L, peptone 8.0g/L, groundnut meal 10.0g/L, potassium dihydrogen phosphate 1.0g/L, ammonium sulfate 0.8g/L, calcium carbonate
1.0g/L, the pH of described seed culture medium is 6.5；And/or
Described fermentation medium contains sorbierite 100.0g/L, glucose 5.0g/L, soybean cake powder 20.0g/L, albumen
Peptone 5.0g/L, cottonseed meal 30.0g/L, potassium dihydrogen phosphate 5.0g/L, ammonium sulfate 3.0g/L, calcium chloride 4.0g/L, zinc sulfate
1.0g/L, L-PROLINE 15.0g/L, the pH of described fermentation medium is 7.0.
Lung reads rhzomorph B0And the like can pass through following condition carry out liquid phase (HPLC) detection：
A, lung read rhzomorph B0、B5、E0、B0The high efficient liquid phase analysis method of serine analogs：Chromatographic column：ODS posts 4.6mm
× 250mm, column temperature：40℃；Mobile phase: acetonitrile: water=60: 40 (volume ratios), flow velocity：1.0ml/min, Detection wavelength：
210nm, sample size：10μl；
B, lung read rhzomorph B0With C0High efficient liquid phase analysis method：Chromatographic column：Silicon column 4.6mm × 250mm；Mobile phase: just oneself
Alkane: methanol: water=70: 30: 2 (volume ratios), flow velocity：1.0ml/min, Detection wavelength：276nm, sample size：10μl.
Remarks：When carrying out HPLC detections, detect that lung reads rhzomorph B by detection method a first0、B5、E0、B0Serine is similar
The potency of thing, then obtains lung by detection method b and reads rhzomorph B0With C0Ratio, then rhzomorph B is read with lung in detection method a0's
Potency calculates lung and reads rhzomorph C0Potency.
Viscosity of the present invention can be detected under the following conditions, instrument：BROOKFIELD, rotor：16s, rotating speed：
30rpm。
Bacterial strain HS-2158 of the present invention reads rhzomorph B with lung in the prior art0Producing strains compare have advantages below：
1. bacterial strain HS-2158 of the present invention produces lung and reads rhzomorph B0Fermentation unit it is high, and can steady production lung read rhzomorph B0。
2. bacterial strain HS-2158 of the present invention reduces lung and reads rhzomorph C0、E0、B5、B0The accessory substances such as serine analogs account for lung thought
Rhzomorph B0Degree, reduce lung and read rhzomorph B0Extraction and separating difficulty, be conducive to the lung for obtaining high-purity to read rhzomorph
B0。
3. lung is disclosed in the prior art reads rhzomorph B0The fermenting carbon source of producing strains is fructose or mannitol, and bacterium of the present invention
Strain HS-2158 can utilize sorbierite well, can be fermented in using sorbierite as the fermentation medium of primary carbon source, and lung is read
Rhzomorph B0Potency can reach 7625 μ g/ml, other significantly larger than known producing strains, the zymotic fluid obtained after culture glues
Degree is low, reduces the purification difficulty of tunning.
4. disclose in the prior art some to reduce respectively by adding proline, threonine, trace element zinc, nickel etc.
Impurity, and bacterial strain HS-2158 of the present invention can reduce lung simultaneously and read rhzomorph C0、E0、B5、B0The accessory substances such as serine analogs are accounted for
Lung reads rhzomorph B0Degree.
5. bacterial strain HS-2158 of the present invention genetic stability is good, with good prospects for commercial application.
Lung of the present invention reads rhzomorph B0Producing strains are Glarea lozoyensis HS-2158 (CGMCC
NO.13367)。
The microorganism fungus kind of bacterial strain HS-2158 (CGMCC NO.13367) of the present invention is on 01 09th, 2017 is deposited in
State's Microbiological Culture Collection administration committee common micro-organisms center (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state), deposit number is CGMCC NO.13367, and Classification And Nomenclature is Glarea lozoyensis, and
Register on the books, it was demonstrated that survival.
Embodiment
The reagent that regulation culture medium material liquid pH value is used in following embodiments is NaOH commonly used in the art molten
Liquid or hydrochloric acid.
Solid medium uses deionized water constant volume in following embodiments, and seed and fermentation medium are carried out using running water
Constant volume, each culture medium weighs raw material by the formula, and each raw material is sufficiently mixed, adds water to required volume, finally leads to
Cross NaOH solution or hydrochloric acid is adjusted to required pH value.
The bacterium source of embodiment 1
Bacterial strain HS-2158 systems of the present invention original strain isolated from the Xinanjiang River of Zhejiang, then obtained by mutagenesis
Mutant strain.
Original strain is in PDA slant mediums after 25 DEG C of cultures 13 days, aseptically with inoculation shovel by mycelium
Scrape, mycelium is ground in rub oral examination tube and is suspended in sterilized water, bacteria suspension is made, at NTG (nitrosoguanidine) mutagenesis
Reason.
NTG crystal 5mg are weighed, are dissolved in 5ml sterile Tris buffers (pH8.0), it is molten with pipette, extract 3ml NTG
Liquid is added to 2ml bacteria suspensions, is placed in 25 DEG C of culture mediums oscillation treatment 30min on rotary or reciprocal shaker.Remaining tool
Body step is as follows：
(1) mycelial preparation and culture
PDA solid mediums, 121 DEG C sterilize 30 minutes, are cooled to 50-60 DEG C and pour into flat board, by treated bacteria suspension
Through appropriate dilution, draw 0.1ml bacteria suspensions and be coated on PDA plate, the bacteria suspension for not making mutagenic treatment is also applied through appropriate dilution
PDA plate is distributed in as control, 25 DEG C of constant incubator cultures are placed in, after bacterium colony culture 13 days, mycelia is ripe.
(2) preparation and culture of seed liquor
Seed culture based formulas (g/L)：Cornstarch 10.0, glucose 30.0, soybean cake powder 20.0, peptone 8.0, flower
Raw cake powder 10.0, potassium dihydrogen phosphate 1.0, ammonium sulfate 0.8, calcium carbonate 1.0, pH6.5, shaking flask liquid amount are 20ml/250ml, 121
DEG C sterilizing 30 minutes.Each seed flask accesses 0.5 bacterium colony or so and is advisable, 22~28 DEG C of cultivation temperature, and culture humidity 40~
60%, bottle swingging machine amplitude 5cm, rotating speed 250rpm, cultivation cycle 88 hours.
(3) lung reads rhzomorph B0The preparation and culture of fermentation medium
Fermentative medium formula I (g/L)：Maltodextrin 100.0, glucose 5.0, soybean cake powder 20.0, peptone 5.0,
Cottonseed meal 30.0, potassium dihydrogen phosphate 5.0, ammonium sulfate 3.0, calcium chloride 4.0, zinc sulfate 1.0, L-PROLINE 15.0；PH7.0,
Shaking flask liquid amount is 30ml/250ml, and 121 DEG C sterilize 30 minutes.Seed liquor is accessed with the inoculum concentration of 10% (percent by volume)
Fermentation medium, 22~28 DEG C of cultivation temperature cultivates humidity 40~60%, bottle swingging machine amplitude 5cm, rotating speed 250rpm, culture week
239 hours phases.
1000 plants of the single bacterium colony after NTG mutagenesis is selected, shake flask fermentation is carried out, HPLC detection lungs read rhzomorph B0Production
Amount.Screening produces lung and reads rhzomorph B0The mutant strain of most high yield is bacterial strain HS-2158.Its main biological property is：When ripe
Bacterium colony be in blackish green, and produce yellow green spore, towering sturdy, slightly irregular, the diameter 0.8-1.2cm, without water-soluble of bacterium colony circle
Property pigment produce.
The strain idenfication of embodiment 2
Reference《Common mycology》、《Common bacteria system identification handbook》、《Molecular Cloning:A Laboratory guide》And《Middle traditional Chinese medicines
Allusion quotation》Relevant content in books such as (2005 editions XIH) is tested.
1. cultural characteristic：Using ISP1, ISP2, ISP3, ISP4, ISP5, Cha Shi, PDA, calcium malate, nutrition and Gao Shi
10 kinds of culture mediums of a number culture medium, after 28 DEG C are cultivated 7~10 days, observe mycelial color and pigment situation.The culture of bacterial strain
Feature is shown in Table 3.
Cultural characteristics of the bacterial strain HS-2158 of table 3 (CGMCC NO.13367) on 10 kinds of culture mediums
| Culture medium | Growing state | Matrix mycelia | Aerial hyphae | Can lysochrome |
| ISP1 | 2 | Olive-green | White | Nothing |
| ISP2 | 4 | Olive-green | Yellow green | Nothing |
| ISP3 | 4 | Olive-green | Yellow green | Nothing |
| ISP4 | 1 | Yellow green | White | Nothing |
| ISP5 | 1 | Pale pink | Pale pink | Nothing |
| Cha Shi | 2 | Breen | White | Nothing |
| PDA | 4 | Olive-green | Yellow green | Nothing |
| Calcium malate | 3 | Blackish green | White | Nothing |
| Nutrition | 3 | Brownish black | Brown | Nothing |
| Gause I | 1 | Breen | White | Nothing |
2. physiological and biochemical test：In addition to temperature is tested, it is 28 DEG C and cultivates 5~7 days.
A) utilization of carbon source：Using culture medium based on ISP9, the final concentration of various carbon sources is 1.0%, is shown in Table 4.
B) inorganic nitrogen-sourced utilization：Using culture medium based on ISP9, the concentration of potassium nitrate and ammonium sulfate is
1.0%, it is shown in Table 4.
C) Degrading experiment and NaCl tolerance tests use basal medium for GYEA (pH6.0), and Degrading experiment the results are shown in Table
5, NaCl tolerance experimental results are shown in Table 6.
D) oxidizing ferment and catalase test, pH experiments and humid test use PDA culture medium.Oxidizing ferment and peroxide
Change hydrogen enzyme test the results are shown in Table 7, pH result of the tests and be shown in Table 8, and humid test the results are shown in Table 9.
E) M.R, V.P are tested：Using《Common bacteria system identification handbook》Method.It the results are shown in Table 7.
Physiological and biochemical property：It is shown in Table 4~table 9.
The bacterial strain HS-2158 of table 4 (CGMCC NO.13367) carbon source and the utilization power of nitrogen source
The bacterial strain HS-2158 of table 5 (CGMCC NO.13367) Degrading experiment result
| Degradation product | Degradate concentrations | As a result * | Degradation product | Degradate concentrations | As a result |
| Adenine | 0.5% | 3 ,- | Casein | 1.0% | 4 ,- |
| Guanine | 0.5% | 3 ,- | Tyrosine | 1.0% | 3 ,- |
| Xanthine | 0.4% | 3 ,- | Tween-40 | 1.0% | 4 ,- |
| Hypoxanthine | 0.4% | 2 ,- | Tween-60 | 1.0% | 3 ,- |
| Xylan | 0.4% | 3 ,- | Tween-80 | 1.0% | 2 ,- |
The bacterial strain HS-2158 of table 6 (CGMCC NO.13367) is to NaCl tolerance
| NaCl concentration | 1% | 4% | 7% | 10% |
| Strain growth situation | 3 | 1 | 0 | 0 |
Physiological and biochemical property main the bacterial strain HS-2158 of table 7 (CGMCC NO.13367)
| Pilot project | As a result | Pilot project | As a result | Pilot project | As a result |
| Gelatin liquefaction | + | Hydrogen sulfide is produced | - | Catalase | - |
| Starch Hydrolysis | - | V.P is tested | - | Oxidizing ferment | - |
| Milk solidifies | - | M.R is tested | - | ||
| Milk is peptonized | - | Nitrate reduction | + |
The pH experiments of the bacterial strain HS-2158 of table 8 (CGMCC NO.13367) growths
| pH | 3.0 | 3.5 | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 |
| Growing state | 3 | 3 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 |
The humid test of the bacterial strain HS-2158 of table 9 (CGMCC NO.13367) growths
| Temperature (DEG C) | 7 | 14 | 28 | 37 | 45 |
| Growing state | 0 | 2 | 4 | 3 | 1 |
* note：In table 3-9：0, no growth；1, grow very weak；2, it can grow, there is a small amount of spore；3, well-grown has a large amount of
Spore；4, growth is best, there is abundant spore；+, it is positive；-, it is negative.
3. 26S rDNA sequence analyses：The new fresh thalli of PDA cultures is collected, STb gene mould is extracted using liquid nitrogen wall-breaking method
Plate, carries out 26S rDNA gene D1/D2 regions using Fungi Identification PCR Kit (TaKaRa) and expands, PCR
After product is detected through agarose electrophoresis, sequencing is directly carried out, the purifying of PCR primer gives birth to work bioengineering with sequencing by Shanghai
Technology Co., Ltd. is carried out.The 26S rDNA gene D1/D2 regional sequences warp that bacterial strain HS-2158 (CGMCC NO.13367) is surveyed
After check and correction, kind related to GenBank databases, the sequence of category carry out homologous sequence BLAST and compared, to determine point of the bacterial strain
Class status.
HS-2158 (CGMCC NO.13367) 26S rDNA D1/D2 sequences (see sequence table) are related to GenBank
Sequence carries out BLAST comparisons, the results are shown in Table 10 (the higher bacterial strain of homology is only listed in table).
The bacterial strain HS-2158 of table 10 (CGMCC NO.13367) and related strain homology
By the way that bacterial strain HS-2158 (CGMCC NO.13367) 26S rDNA D1/D2 regions are sequenced, itself and aspergillus are found
Bacterium (Glarea lozoyensis) has very high homology, and highest is up to 99%, while to bacterial strain HS-2158 (CGMCC
NO.13367 appearance features experiment) is carried out, it is found that the bacterial strain and Aspergillus (Glarea lozoyensis) classification relevant parameter are non-
Very close to, therefore bacterial strain HS-2158 (CGMCC NO.13367) is accredited as to Aspergillus (Glarea lozoyensis) bacterial strain.
The bacterial strain HS-2158 of embodiment 3 (CGMCC NO.13367) is sorbierite and malt in fermentation medium primary carbon source
During dextrin
Study on Fermentation
(1) PDA solid mediums, 121 DEG C sterilize 30 minutes, are cooled to 50-60 DEG C and pour into flat board, access 0.1ml bacterium are hanged
After liquid culture 13d, mycelia is ripe.
(2) step (2), cultivation cycle 87 hours in seed culture based formulas and condition of culture equivalent integers 1.
(3) lung reads rhzomorph B0The preparation and culture of fermentation medium.
Step (3) in the equivalent integers 1 of fermentative medium formula I；
Fermentative medium formula II (g/L)：Sorbierite 50.0, maltodextrin 50.0, glucose 5.0, soybean cake powder 20.0,
Peptone 5.0, cottonseed meal 30.0, potassium dihydrogen phosphate 5.0, ammonium sulfate 3.0, calcium chloride 4.0, zinc sulfate 1.0g, L-PROLINE
15.0, pH7.0；
Fermentative medium formula III (g/L)：Sorbierite 100.0, glucose 5.0, soybean cake powder 20.0, peptone 5.0, cotton
Seed cake powder 30.0, potassium dihydrogen phosphate 5.0, ammonium sulfate 3.0, calcium chloride 4.0, zinc sulfate 1.0g, L-PROLINE 15.0, pH7.0.
Shaking flask liquid amount is 30ml/250ml, and 121 DEG C sterilize 30 minutes.By seed liquor with 10% (percent by volume)
Inoculum concentration accesses fermentation medium, and 22~28 DEG C of cultivation temperature cultivates humidity 40~60%, bottle swingging machine amplitude 5cm, rotating speed
250rpm, cultivation cycle 240 hours.Viscosity and HPLC detections, three groups of fermentations the results are shown in Table 11.
Fermentation results of the table 11HS-2158 (CGMCC NO.13367) when different fermentations are formulated
FMⅠ：Fermentative medium formula I
FMⅡ：Fermentative medium formula II
FMⅢ：Fermentative medium formula III
As seen from table, when fermentation medium carbon source is maltodextrin or sorbierite, bacterial strain HS-2158 production lungs read rhzomorph B0's
Potency is higher, wherein the preferred sorbierite of carbon source, and fermentation titer is higher, and viscosity is lower.
The bacterial strain HS-2158 of embodiment 4 (CGMCC NO.13367) is maltodextrin and sorbierite in fermentation medium carbon source
When Study on Fermentation
(1) PDA solid mediums, 121 DEG C sterilize 30 minutes, are cooled to 50-60 DEG C and pour into flat board, access 0.1ml bacterium are hanged
After liquid culture 13d, mycelia is ripe.
(2) step (2), cultivation cycle 87 hours in seed culture based formulas and condition of culture equivalent integers 1.
(3) lung reads rhzomorph B0The preparation and culture of fermentation medium.
Step (3) in the equivalent integers 1 of fermentative medium formula I；
Fermentative medium formula III (g/L)：Sorbierite 100.0, glucose 5.0, soybean cake powder 20.0, peptone 5.0, cotton
Seed cake powder 30.0, potassium dihydrogen phosphate 5.0, ammonium sulfate 3.0, calcium chloride 4.0, zinc sulfate 1.0g, L-PROLINE 15.0, pH7.0.
Shaking flask liquid amount is 30ml/250ml, and 121 DEG C sterilize 30 minutes.By seed liquor with 10% (percent by volume)
Inoculum concentration accesses fermentation medium, and 22~28 DEG C of cultivation temperature cultivates humidity 40~60%, bottle swingging machine amplitude 5cm, rotating speed
250rpm, cultivation cycle 240 hours.Viscosity and HPLC detections, three groups of fermentations the results are shown in Table 12.
Fermentation comparative results of the table 12HS-2158 (CGMCC NO.13367) on FM I and FM III
FMⅠ：Fermentative medium formula I
FMⅢ：Fermentative medium formula III
As seen from table, no matter fermentation medium carbon source is sorbierite or maltodextrin, and bacterial strain HS-2158 of the present invention is produced
Lung read rhzomorph C0、E0、B5、B0The accessory substances such as serine analogs account for lung and read rhzomorph B0Degree close to and it is relatively low.
It is compared with disclosed patent WO00/08197, the patent specifically discloses addition 15g/L proline can be by
Lung reads rhzomorph C04.4% is reduced to, but lung reads rhzomorph E0Increase to 2.7%, addition trace element zinc makes lung read rhzomorph B0Serine
Analog and B5Content reduces 1% (B respectively0Serine analogs are 1.9%, B5For 3.9%), but lung reads rhzomorph E0Contain
Amount adds one times, and (lung reads rhzomorph E04.3%) and lung reads rhzomorph B for0Potency reduces 50%, and addition trace element cobalt can make
Lung reads rhzomorph E0Content is reduced to 1.8% from 2.2%, but lung reads rhzomorph B0Serine analogs and B5Content doubles (lung
Read rhzomorph B0Serine analogs are 6.0%, B57.2%) and lung reads rhzomorph B for0Potency reduces 25%.
Although reading rhzomorph C by comparing the lung for understanding all to contain in proline, this patent in fermenting0Percentage composition more
It is low；Lung reads rhzomorph E0、B5、B0The accessory substances such as serine analogs are compared with disclosed patent technique, and its percentage composition is also all more
It is low；In addition, it has been disclosed that patent reduces some impurity by adding proline, trace element zinc, nickel etc. respectively, and bacterium of the present invention
Strain HS-2158 can reduce lung simultaneously and read rhzomorph C0、E0、B5、B0The accessory substances such as serine analogs account for lung and read rhzomorph B0Percentage
Compare content.
The bacterial strain HS-2158 of embodiment 5 (CGMCC NO.13367) is in the sorbierite that fermentation medium carbon source is various concentrations
When Study on Fermentation
(1) step (1) in solid culture based formulas and condition of culture equivalent integers 3
(2) step (2), cultivation cycle 87 hours in seed culture based formulas and condition of culture equivalent integers 1.
(3) lung reads rhzomorph B0The preparation and culture of fermentation medium
Fermentation medium basic components IV (g/L)：Glucose 5.0, soybean cake powder 20.0, peptone 5.0, cottonseed meal
30.0th, potassium dihydrogen phosphate 5.0, ammonium sulfate 3.0, calcium chloride 4.0, zinc sulfate 1.0, L-PROLINE 15.0.In fermentation medium base
Sorbierite is added in plinth formula IV, sorbitol concentration is respectively 90.0g/L, 110.0g/L, 130.0g/L, 150.0g/L, is obtained
The fermentation medium of four groups of various concentrations sorbierites, pH7.0, shaking flask liquid amount is 30ml/250ml, and 121 DEG C sterilize 30 minutes.
Seed liquor is accessed in the fermentation medium of this four groups of various concentrations sorbierites with the inoculum concentration of 10% (percent by volume), cultivated
22~28 DEG C of temperature, culture humidity 40~60%, bottle swingging machine amplitude 5cm, rotating speed 250rpm, cultivation cycle is respectively 240,286
Hour.HPLC is detected, the results are shown in Table 12.
The sorbitol fermentation results contrast of the various concentrations of table 13
| Sorbitol concentration (g/L) | 90.0 | 110.0 | 130.0 | 150.0 |
| 240 hours HPLC B0(μg/ml) | 5613 | 6711 | 6102 | 5615 |
| 286 hours HPLC B0(μg/ml) | 4522 | 7194 | 7625 | 7013 |
Lab scale researchs of the bacterial strain HS-2158 of embodiment 6 (CGMCC NO.13367) on 50L fermentation tanks
(1) preparation of seeding tank seed liquor
10L seed culture medium (the proportioning be the same as Example 1 of seed culture medium, simultaneously addition is put into 15L seeding tanks
0.05% PPG is used as defoamer), sterilizing is used under the conditions of steam sterilizing, 121 DEG C 30 minutes, after cooling, accesses 200ml
Shake-flask seed liquid, 22~28 DEG C of cultivation temperature, 100~500rpm of speed of agitator, 0.5~1.0vvm of throughput, dissolved oxygen 20~
70%, cultivate 86 hours.
(2) preparation and culture of fermentation tank culture medium
The formula of fermentation medium is identical with fermentating formula III in previous embodiment 3, but to add 0.05%PPG as disappearing
Infusion, fermentation tank loading amount is 35L/50L, pH7.0, in steam sterilizing 30 minutes at 121 DEG C, after cooling, accesses about 3.5L seeds
Tank nutrient solution, 22~28 DEG C of fermentation temperature, 200~500rpm of speed of agitator, throughput is 0.5~1.0vvm, fermented and cultured 252
Hour, zymotic fluid carries out HPLC detections when tank is put in fermentation, measures lung and reads rhzomorph B0Fermentation titer is 7010 μ g/ml.C0Fermentation titer
156 μ g/ml, B5Fermentation titer 44 μ g/ml, E0Fermentation titer 32 μ g/ml, B0The μ g/ml of serine analogs fermentation titer 34.
Amplification cultures of the bacterial strain HS-2158 of embodiment 7 (CGMCC NO.13367) on 5000L fermentation tanks
(1) preparation of seeding tank seed liquor
300L seed culture medium (the proportioning be the same as Example 1 of seed culture medium, simultaneously addition is put into 500L seeding tanks
0.05% PPG is used as defoamer), sterilizing is used under the conditions of steam sterilizing, 121 DEG C 30 minutes, after cooling, accesses 1800ml
Shake-flask seed liquid, 22~28 DEG C of cultivation temperature, 50~200rpm of speed of agitator, 0.6~1.2vvm of throughput, dissolved oxygen 30~
70%, cultivate 98 hours.
(2) preparation and culture of fermentation tank culture medium
The formula of fermentation medium is identical with fermentating formula III in previous embodiment 3, but to add 0.05%PPG as disappearing
Infusion, fermentation tank loading amount is 3000L/5000L, pH7.0, in steam sterilizing 30 minutes at 121 DEG C, after cooling, accesses about 300L
Seed tank culture liquid, 22~28 DEG C of fermentation temperature, 300~500rpm of speed of agitator, throughput is 0.5~1.5vvm, fermentation training
Support 280 hours, zymotic fluid carries out HPLC detections when tank is put in fermentation, measures lung and reads rhzomorph B0Fermentation titer is 6845 μ g/ml.C0Hair
Ferment potency 138 μ g/ml, B5Fermentation titer 38 μ g/ml, E0Fermentation titer 34 μ g/ml, B0The μ g/ of serine analogs fermentation titer 29
ml。
Although the foregoing describing the embodiment of the present invention, it will be appreciated by those of skill in the art that these
It is merely illustrative of, on the premise of the principle and essence without departing substantially from the present invention, a variety of changes can be made to these embodiments
More or modification, therefore, protection scope of the present invention is defined by the appended claims.
SEQUENCE LISTING
<110>Haizheng Medicine Stock Co., Ltd., Zhejiang Prov
<120>A kind of method that Aspergillus and its production lung read rhzomorph B0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 999
<212> DNA
<213>Aspergillus（Glarea lozoyensis）
<400> 1
ggatctagac ggcagtgatt ccagctcggt accaccggga tcctctgcga gtgcaccagt 60
aacagctcaa atttgaaatc tggctctttt agggtccgag ttgtaatttg tagaagatgc 120
ttcgggtgta gctccggtct aagttccttg gaacaggacg tcatagaggg tgagaatccc 180
gtatgtgact ggtggctttc gcccatgtga agctctttcg acgagtcgag ttgtttggga 240
atgcagctct aaatgggtgg taaatttcat ctaaagctaa atattggcca gagaccgata 300
gcgcacaagt agagtgatcg aaagatgaaa agcactttgg aaagagagtt aaacagtacg 360
tgaaattgtt gaaagggaag cgcttgcaac cagatttgca ccccgtcgat catcccccgt 420
tctcggaggt gcactcggcg gggttcaggc cagcatcggt ttcggtggtg ggataaaggc 480
cttgggaacg tagctcctct cggggagtgt tatagccctc ggtgcaatgc cgcctaccgg 540
gaccgaggac cgcgcttcgg ctaggatgct ggcgtaatgg ttgtaagcga cccgtcttga 600
aacacggacc cctgtgtgaa attgttatcc gctcaatcgt cgacctgcag gcatgcaagc 660
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 720
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 780
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 840
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 900
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 960
cactcaaagg cggtaatacg gttatccaca gaatcaggg 999
Claims (10)
1. a kind of bacterial strain Glarea lozoyensis HS-2158, its deposit number is CGMCC NO.13367.
2. bacterial strain HS-2158 according to claim 1 reads rhzomorph B preparing lung0Or the application in its analog, the class
It is that lung reads rhzomorph A like thing0, C0, D0, E0, A1, A2, A3, A4, B1, B2, B5, B6, D2, B0Serine analogs, B5Serine is similar
Thing, preferably lung read rhzomorph C0、B5、E0、B0Serine analogs.
3. a kind of lung reads rhzomorph B0Preparation method, it is characterised in that this method is included bacterial strain as claimed in claim 1
Glarea lozoyensis HS-2158 carry out bacterium colony culture in solid medium, then carry out seed training in seed culture medium
Support, be inoculated in fermentation medium and carry out fermented and cultured.
4. preparation method according to claim 3, it is characterised in that the time of the bacterium colony culture is 5~15 days, preferably
7~12 days.
5. the preparation method according to claim any one of 3-4, it is characterised in that the time of the seed culture is 24~
120 hours, preferably 48~100 hours；The temperature of the seed culture is 20~30 DEG C, preferably 22~28 DEG C.
6. the preparation method according to claim any one of 3-5, it is characterised in that the time of the fermented and cultured is 96~
360 hours, preferably 216-340 hours；The temperature of the fermented and cultured is 20~30 DEG C, preferably 22~28 DEG C.
7. the preparation method according to claim any one of 3-6, it is characterised in that：
The concentration of described seed culture medium each component in the medium is：5.0~70.0g/L of carbon source, 5.0~60.0g/ of nitrogen source
L, 0~5.5g/L of inorganic salts, the pH of described seed culture medium is 5.0~7.0；And/or
The concentration of described fermentation medium each component in the medium is：5.0~170.0g/L of carbon source, nitrogen source 5.0~
65.0g/L, 0~21.0g/L of inorganic salts, 5.0~30.0g/L of amino acid, the pH of described fermentation medium is 5.0~7.5.
8. the preparation method according to claim any one of 3-7, it is characterised in that：
One or more of the carbon source of described seed culture medium in glucose, cornstarch, lactose, maltodextrin, institute
The one kind of the nitrogen source for the seed culture medium stated in soybean cake powder, cottonseed meal, peptone, groundnut meal, Dried Corn Steep Liquor Powder
Or it is several, the inorganic salts of described seed culture medium are selected from sodium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium nitrate, sulfuric acid
One or more in ammonium, calcium carbonate, zinc sulfate；And/or
The carbon source of described fermentation medium is selected from glucose, lactose, fructose, mannitol, sorbierite, maltodextrin, corn and formed sediment
One or more in powder, soya-bean oil, methyl oleate, the nitrogen source of described fermentation medium is selected from extraction from yeast powder, dusty yeast, ox
In meat medicinal extract, soy peptone, peptone, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, wheat bran
One or more, the inorganic salts of described fermentation medium are selected from ammonium chloride, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate,
Ammonium sulfate, calcium carbonate, ferrous sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, iron chloride, manganese sulfate, zinc sulfate, sulfuric acid
One or more in copper, the amino acid of described fermentation medium is selected from TYR, L-PROLINE, L-threonine, L- birds
One or more in propylhomoserin hydrochloride, Valine, L-arginine.
9. the preparation method according to claim 3~8, it is characterised in that：
Described seed culture medium contains 5.0~20.0g/L of cornstarch, 10.0~50.0g/L of glucose, soybean cake powder 10.0
~30.0g/L, 5.0~15.0g/L of peptone, 5.0~15.0g/L of groundnut meal, 0.5~2.0g/L of potassium dihydrogen phosphate, sulfuric acid
0.5~1.5g/L of ammonium, 0.5~2.0g/L of calcium carbonate, the pH of described seed culture medium is 6.0~7.0；And/or
Described fermentation medium contain 90.0~150.0g/L of sorbierite, 5.0~50.0g/L of glucose, soybean cake powder 5.0~
20.0g/L, 5.0~15.0g/L of peptone, 10.0~30.0g/L of cottonseed meal, 1.0~7.0g/L of potassium dihydrogen phosphate, ammonium sulfate
0.5~4.0g/L, 0.5~5.0g/L of calcium chloride, 0.8~5.0g/L of zinc sulfate, 5.0~30.0g/L of L-PROLINE, described hair
The pH of ferment culture medium is 5.0~7.0.
10. the preparation method according to claim 3~9, it is characterised in that：
Described seed culture medium contains cornstarch 10.0g/L, glucose 30.0g/L, soybean cake powder 20.0g/L, peptone
8.0g/L, groundnut meal 10.0g/L, potassium dihydrogen phosphate 1.0g/L, ammonium sulfate 0.8g/L, calcium carbonate 1.0g/L, described seed
The pH of culture medium is 6.5；And/or
Described fermentation medium contains sorbierite 100.0g/L, glucose 5.0g/L, soybean cake powder 20.0g/L, peptone
5.0g/L, cottonseed meal 30.0g/L, potassium dihydrogen phosphate 5.0g/L, ammonium sulfate 3.0g/L, calcium chloride 4.0g/L, zinc sulfate 1.0g/
L, L-PROLINE 15.0g/L, the pH of described fermentation medium is 7.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108265096A (en) * | 2016-12-30 | 2018-07-10 | 江苏恒瑞医药股份有限公司 | A kind of microbial fermentation prepares knob not Kangding B0Method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000008197A1 (en) * | 1998-08-07 | 2000-02-17 | Merck & Co., Inc. | Method for the production of an antibiotic agent |
| CN101928670A (en) * | 2009-09-24 | 2010-12-29 | 上海天伟生物制药有限公司 | High-yield bacterial strain of antibiotic, preparation method and usage thereof |
| CN103087928A (en) * | 2013-01-25 | 2013-05-08 | 杭州华东医药集团生物工程研究所有限公司 | The fungus Glarea lozoyensis and its application in regulating the microbial metabolite pneumocidine B0 |
| CN104531535A (en) * | 2014-10-17 | 2015-04-22 | 南京天凯生物技术股份有限公司 | A Gene Recombinant Strain for Producing Pneumocontin B0 and Its Breeding Method and Application |
-
2017
- 2017-07-14 CN CN201710575306.3A patent/CN107201316B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000008197A1 (en) * | 1998-08-07 | 2000-02-17 | Merck & Co., Inc. | Method for the production of an antibiotic agent |
| CN101928670A (en) * | 2009-09-24 | 2010-12-29 | 上海天伟生物制药有限公司 | High-yield bacterial strain of antibiotic, preparation method and usage thereof |
| CN103087928A (en) * | 2013-01-25 | 2013-05-08 | 杭州华东医药集团生物工程研究所有限公司 | The fungus Glarea lozoyensis and its application in regulating the microbial metabolite pneumocidine B0 |
| CN104531535A (en) * | 2014-10-17 | 2015-04-22 | 南京天凯生物技术股份有限公司 | A Gene Recombinant Strain for Producing Pneumocontin B0 and Its Breeding Method and Application |
Non-Patent Citations (3)
| Title |
|---|
| D.J. POLLARD等: "Rheological characterization of a fungal fermentation for the production of pneumocandins", 《BIOPROCESS BIOSYST ENG》 * |
| 宋萍等: "Glarea lozoyensis发酵生产棘白菌素类化合物纽莫康定的研究进展", 《中国抗生素杂志》 * |
| 杨渊等: "丝状真菌Glarea lozoyensis SIIA-F1108 产纽莫康定B0发酵条件的研究", 《微生物学通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108265096A (en) * | 2016-12-30 | 2018-07-10 | 江苏恒瑞医药股份有限公司 | A kind of microbial fermentation prepares knob not Kangding B0Method |
| CN108265096B (en) * | 2016-12-30 | 2021-11-16 | 江苏恒瑞医药股份有限公司 | Preparation of pneumocandin B by microbial fermentation0Method (2) |
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