CN107201316A

CN107201316A - A kind of Aspergillus and its production lung read rhzomorph B0Method

Info

Publication number: CN107201316A
Application number: CN201710575306.3A
Authority: CN
Inventors: 李娜; 江沛; 郑玲辉
Original assignee: Hisun Pharmaceutical Hangzhou Co ltd; Zhejiang Hisun Pharmaceutical Co Ltd
Current assignee: Zhejiang Hisun Pharmaceutical Co Ltd
Priority date: 2017-07-14
Filing date: 2017-07-14
Publication date: 2017-09-26
Anticipated expiration: 2037-07-14
Also published as: CN107201316B

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

A kind of Aspergillus and its production lung read rhzomorph B0Method

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

CH₃

OH

B₀

H

OH

CH₃

OH

C₀

OH

H

OH

CH₃

OH

D₀

OH

CH₃

OH

E₀

H

OH

CH₃

OH

A₁

CH₃

OH

CH₃

H

OH

A₂

CH₃

OH

H

CH₃

OH

A₃

CH₃

OH

H

CH₃

H

A₄

CH₃

OH

H

CH₃

H

B₁

H

OH

CH₃

H

OH

B₂

H

oH

H

CH₃

OH

B₅

H

OH

H

CH₃

OH

B₆

H

oH

H

oH

CH₃

OH

D₂

OH

H

CH₃

OH

B₀Serine analogue

H

OH

H

OH

B₅Serine analogue

H

OH

H

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

Step (3) in the equivalent integers 1 of fermentative medium formula I；

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

(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

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.