WO2018166427A1 - 一种伊维菌素B1b产生菌及其应用 - Google Patents

一种伊维菌素B1b产生菌及其应用 Download PDF

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WO2018166427A1
WO2018166427A1 PCT/CN2018/078773 CN2018078773W WO2018166427A1 WO 2018166427 A1 WO2018166427 A1 WO 2018166427A1 CN 2018078773 W CN2018078773 W CN 2018078773W WO 2018166427 A1 WO2018166427 A1 WO 2018166427A1
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ivermectin
strain
medium
powder
cake powder
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PCT/CN2018/078773
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English (en)
French (fr)
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黄隽
李娜
林甲壇
李美红
周军
常宗明
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浙江海正药业股份有限公司
台州市劢康生物科技有限公司
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Priority to EP18768702.5A priority Critical patent/EP3613842B1/en
Priority to CN201880017729.6A priority patent/CN110832066B/zh
Priority to US16/494,262 priority patent/US20200131552A1/en
Publication of WO2018166427A1 publication Critical patent/WO2018166427A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/445—The saccharide radical is condensed with a heterocyclic radical, e.g. everninomycin, papulacandin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/14—Ectoparasiticides, e.g. scabicides
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C12P19/623—Avermectin; Milbemycin; Ivermectin; C-076
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the invention belongs to the field of biomedicine, and relates to a strain of Streptomyces avermitilis with high yield of ivermectin B1b and a method for producing ivermectin B1b by using the strain.
  • Ivermectin has an effective killing effect on both internal and external parasites, especially nematodes and arthropods. It has stronger permeability and safety for mammalian body tissues and has a longer duration. It is especially suitable for the control of parasites in muscles, organs and special tissues that are difficult to reach with general oral repellents.
  • the nematode and ectoparasite have special curative effects and are recognized as multi-component antibiotics with broad spectrum, high efficiency, safety, little residue and no drug resistance.
  • the production cost of the process is high, and the yield of ivermectin B1b is only 15%; and the chemical synthesis of ivermectin B1b from avermectin B1, the impurities are many, the purification process is more complicated, it is difficult to isolate a large amount of ivermectin B1b; the other is to use genetic engineering bacteria to produce Iraq A oxytocin, such as CN200510074936.X, discloses that a gene encoding a gene encoding the avermectin polyketide DH2-KR2 domain of S. avermitilis is replaced with a coding gene for the polyketide synthase DH-ER-KR domain.
  • the method for engineering bacteria of ivermectin the yield of ivermectin B1a is about 1-4 ⁇ g / mL, but the yield of ivermectin B1b is not mentioned;
  • CN200910089970.2 discloses the coding of Streptomyces avermitilis A method for increasing the utilization efficiency of maltose by introducing a malEFG gene of a maltose transporter into a recombinant vector in which a maltose transporter overexpresses is introduced into a Streptomyces avermitilis by an expression vector, and the maltose transport system gene is overexpressed by the recombinant strain, and the method uses Y.
  • the gene was replaced with the milA1 gene of Streptomyces milbemycinicus to obtain recombinant Streptomyces MA220.
  • the recombinant Streptomyces sp. MA220 fermentation product has a very small amount of ivermectin B1b component in addition to the main components of tianweimycin A (TEVA) and tianweimycin B (TEVB).
  • TEVA tianweimycin A
  • TEVB tianweimycin B
  • the yield of ivermectin in the prior art is low, and the product is either a mixture of ivermectin B1a and B1b, or ivermectin B1a, and the production of a single component ivermectin B1b.
  • the present inventors unexpectedly screened a high-yield strain for directly fermenting ivermectin B1b, and further provided a method for producing ivermectin B1b using the strain.
  • ivermectin B1b can be effectively utilized, and on the other hand, it can lay a foundation for further research on the biological activity of ivermectin B1b and its structural modification.
  • One of the objects of the present invention is to provide a novel high-yield strain capable of producing ivermectin B1b to solve the above problems.
  • One aspect of the present invention provides a strain of ivermectin B1b high-yielding microorganism.
  • the strain is Streptomyces avermitilis strain C63-51, which was deposited on December 22, 2016 in the General Microbiology Center of China Microbial Culture Collection Management Committee (Address: No. 1 Beichen West Road, Chaoyang District, Beijing, China) Institute of Microbiology, Academy of Sciences), deposited under the symbol CGMCC No. 13370, and registered in the book, proving survival.
  • Another aspect of the present invention is to provide use of S. avermitilis strain C63-51 in the production of ivermectin B1b or a pharmaceutical composition containing ivermectin B1b.
  • Another aspect of the present invention provides a method for producing ivermectin B1b using S. avermitilis strain C63-51, which comprises culturing S. avermitilis strain C63-51 with an assimilable carbon source and/or Fermentation is carried out in a medium of nitrogen source.
  • the invention also relates to a composition comprising the S. avermitilis strain C63-51 of the invention.
  • the composition may be a strain composition comprising a strain other than the strain C63-51; preferably, the other strain may be a strain for producing ivermectin B1b, such as Streptomyces avermitilis, a water-absorbent chain Mold, genetically engineered bacteria MA220, etc., especially genetically engineered bacteria MA220.
  • the invention further relates to the use of the above composition in the manufacture of ivermectin B1b or a pharmaceutical composition comprising ivermectin B1b.
  • the present invention is also directed to a pesticidal composition comprising ivermectin B1b prepared by the method of the present invention.
  • the above assimilable carbon source is sucrose, glucose, amylase, fructose, rhamnose, raffinose, xylose, arabinose, industrial molasses, lactose, galactose, maltose, trehalose, xylan, paste
  • sucrose glucose, amylase, fructose, rhamnose, raffinose, xylose, arabinose, industrial molasses, lactose, galactose, maltose, trehalose, xylan, paste
  • the assimilable nitrogen source is beef extract, yeast extract, yeast extract, yeast powder, peptone, tryptone, gluten powder, cottonseed cake powder, peanut cake powder, soybean cake powder, corn syrup dry powder
  • the assimilable nitrogen source is beef extract, yeast extract, yeast extract, yeast powder, peptone, tryptone, gluten powder, cottonseed cake powder, peanut cake powder, soybean cake powder, corn syrup dry powder
  • bran urea, ammonium salt, and nitrate.
  • the medium further comprises an inorganic salt, which is MnSO 4 , Na 2 MoO 4 , CoCl 2 , CaCO 3 , ZnSO 4 , FeSO 4 , (NH 4 ) 2 SO 4 , FeCl 3 , KNO 3
  • an inorganic salt which is MnSO 4 , Na 2 MoO 4 , CoCl 2 , CaCO 3 , ZnSO 4 , FeSO 4 , (NH 4 ) 2 SO 4 , FeCl 3 , KNO 3
  • KH 2 PO 4 , K 2 HPO 4 , MgCl 2 , MgSO 4 , NaCl, CuSO 4 , NiSO 4 , KCl an inorganic salt
  • the culture medium comprises corn starch 120-160 g/L, amylase 0.2 g/L, soybean cake powder 10-40 g/L, peanut cake powder 0-10 g/L, and yeast powder 5-20 g/L, ( NH 4 ) 2 SO 4 0 to 4 g/L, MnSO 4 0.024 g/L, Na 2 MoO 4 0.024 g/L, CoCl 2 0.01 to 0.04 g/L, and CaCO 3 3 to 7 g/L.
  • the medium contains corn starch 140g/L, amylase 0.2g/L, soybean cake powder 20g/L, peanut cake powder 5g/L, yeast powder 10g/L, (NH 4 ) 2 SO 4 2g/ L, MnSO 4 0.024 g / L, Na 2 MoO 4 0.024 g / L, CoCl 2 0.02 g / L, CaCO 3 7 g / L.
  • the temperature of the fermentation is from 20 ° C to 40 ° C, more preferably from 25 ° C to 30 ° C, most preferably 28 ° C.
  • the pH conditions of the fermentation are controlled in the fermentation process of the invention, the pH being from 6.0 to 8.0, more preferably from 6.5 to 7.5, most preferably from 7.0 to 7.2.
  • the fermentation time of the process of the invention may range from 288 to 312 hours; the aeration of the process of the invention may range from 0.6 to 1.1 vvm.
  • "aeration amount” means the amount of filter-sterilized sterile air per unit volume of culture fluid per minute.
  • the "genetically engineered bacteria MA220”, “MA220 strain”, “Tianweimycin producing strain MA220”, “recombinant Streptomyces MA220”, and “starting strain MA220” have the same meanings.
  • ivermectin B1b can be detected by the following HPLC method:
  • the column is a C18 column, 5 ⁇ m, 4.6 ⁇ 250 mm;
  • Injection volume 10 ⁇ l.
  • the S. avermitilis strain C63-51 screened by the present invention can be directly used for the fermentative production of ivermectin B1b, and avoids complicated separation and purification and chemical reduction compared with chemical preparation of ivermectin. Process steps, resulting in a significant reduction in production costs.
  • the yield of ivermectin B1a in CN200510074936.X is about 1-4 mg/L, but the yield of ivermectin B1b is not mentioned; the yield of ivermectin in CN200910089970.2 About 35mg / L, and the product is a mixture of ivermectin B1a and B1b, it is more difficult to isolate ivermectin B1b; WO2015135242A1 and WO2015135467A1 starting strain MA220 production ivermectin B1b titer is 200mg / L; The ivermectin B1b shake flask fermentation unit of the present invention is up to 6500 mg/L, and the potency can reach 4000 mg/L in the 50 L fermentor, and the yield of the ivermectin B1b of the invention is far.
  • Figure 1 Comparison of fermentation products of S. avermitilis strain C63-51 and starting strain MA220: A is the HPLC chromatogram of the fermentation broth produced in Example 1 for the starting strain MA220, and B is the S. avermitilis strain C63-51 in the examples. HPLC chromatogram of the fermentation broth produced in 1.
  • the reagents and instruments used are all commonly used in the art, and can be purchased from chemical or biological products/preparation companies; the methods used in the following examples are conventional in the art. Methods, those skilled in the art can undoubtedly know the operation of these experiments and obtain corresponding results according to the prior art or the operation manual provided by the manufacturer.
  • NVG Nitrosoguanidine
  • the avermectin producing strain MA220 (the preparation method thereof is described in WO2015135242A1 and WO2015135467A1) is the starting strain. Remove the fresh slant of the strain strain MA220, prepare a spore suspension with 0.1 mol/L phosphate buffer solution of pH 6.0, treat the spore suspension with NTG at a final concentration of 1 mg/ml, shake at 34 ° C for 1 h, and use physiological The spores were washed three times with saline, and then diluted and applied to YMS solid medium (yeast extract 4 g/L, glucose 4 g/L, malt extract 10 g/L, trace element solution 5 ml/L, agar 20 g/L) ), cultured at 28 ° C for 4 days.
  • YMS solid medium yeast extract 4 g/L, glucose 4 g/L, malt extract 10 g/L, trace element solution 5 ml/L, agar 20 g/L
  • a single colony with large colony diameter and sporulation-rich colonies was picked and transferred to YMS solid plate medium, and cultured at 28 ° C for 7 days. According to the fermentation verification method described below, about 600 strains of mutagenized strains were screened to obtain mutant strain 58-46, and the ivermectin B1b titer was 1600 mg/L.
  • the mutant strain 58-46 which was induced by the above NTG was used as a starting strain, and the mature spores on the fresh slope were picked, and the spore suspension was prepared by washing with physiological saline. 10 ⁇ l of spore suspension was added to a sterile slide and placed in a plasma chamber for irradiation.
  • the irradiation conditions were set as follows: the helium gas was generated gas, the flow rate was 12.5 L/min, the distance between the emission port and the sample carrier was 2 mm, the irradiation power was 100 W, and the irradiation time was 30 s.
  • the sample slides were taken out with sterile forceps, placed in a 2 ml sterile EP tube containing physiological saline, shaken vigorously for 2 min using a vortex mixer, and the sample on the slide was thoroughly washed and suspended in physiological saline. . After dilution by gradient, it was applied to a YMS solid medium plate and cultured at 28 ° C for 4 d. A single colony with large colony diameter and sporulation-rich colonies was picked and transferred to YMS solid plate medium, and cultured at 28 ° C for 7 days.
  • a total of 600 strains of mutagenized strains were screened, and a high-yield strain of ivermectin B1b was obtained, which was named as Streptomyces avermitilis strain C63-51.
  • the titer of the avermectin B1b from the starting strain MA220 was 200 mg/L, and the impurities were more.
  • the titer of the ivermectin B1b produced by the Streptomyces avermitilis strain C63-51 was increased to 3000 mg/L, and the impurities were less.
  • Fig. 1 wherein A is an HPLC chromatogram of the fermentation broth of the starting strain MA220, and B is an HPLC chromatogram of the fermentation broth of the S. avermitilis strain C63-51.
  • the fermentation verification method is as follows:
  • the cultured spores were scraped with a inoculating shovel to a seed culture medium of about 1 cm 2 of the lawn, and the culture temperature was 28 ° C, 250 rpm, and shaking culture was carried out for 40 hours.
  • the culture solution had a pH of 6.86 and a mycelial concentration of 35% by volume.
  • the inoculum amount of the seed liquid was 6% by volume.
  • the culture temperature was 28 ° C, 250 rpm, and shaking culture was carried out for 12 days. At this time, the culture solution had a pH of 6.60 and a mycelial concentration of 33% by volume.
  • ISP2 4 Light Brown Cinnamon Light yellow brown ISP3 3 light yellow Yellow white no ISP4 3 yellow light yellow Light yellow brown Gaoshi No.1 4 Beige milky yellow Glucose aspartate 4 Yellow orange Yellow orange Light yellow brown LB 2 Beige Beige yellow YMS 4 yellow yellow yellow yellow
  • the fresh cells of the test strain C63-51 were collected, and the total DNA template was extracted by the lysozyme-modified Pitcher method (Letters in Applied Microbiology, 1989, 8: 151-156), and 16S rDNA was carried out using universal primers (27F and 1495R). After gene amplification, the PCR product was purified and directly sequenced, and sequencing was performed by Nanjing Jinsui Biotechnology Co., Ltd. The 16S rDNA sequence was compared with the sequence of the related gene and genus in the GenBank database to determine the taxonomic status of the strain.
  • Strain C63-51 (CGMCC NO.13370) was sequenced by 16S rDNA region and found to have 100% homology with Streptomyces avermitilis, and the apparent characteristics of strain C63-51 (CGMCC NO.13370). The strain was found to be very similar to the classification parameters of Streptomyces avermitilis, so strain C63-51 (CGMCC NO. 13370) was identified as Streptomyces avermitilis.
  • Example 1 Using the fermentation medium as the base medium of Example 1, adjusting the concentration of each component in the formulation, optimizing the fermentation medium, and optimizing the formula to (g/L): corn starch 120-160, amylase 0.2, soybean cake powder 10-40 , peanut cake powder 0 ⁇ 10, yeast powder 5 ⁇ 20, (NH 4 ) 2 SO 4 0 ⁇ 4, MnSO 4 0.024, Na 2 MoO 4 0.024, CoCl 2 0.01 ⁇ 0.04, CaCO 3 3 ⁇ 7, pH 7.2, Sterilize at 121 ° C for 20 minutes. The inoculum amount of the seed liquid was 6% by volume. The culture temperature was 28 ° C, 250 rpm, and shaking culture was carried out for 12 days.
  • the filtrate was used directly for HPLC analysis.
  • the titer of the fermentation broth of different fermentation media was determined as described above, and the preferred fermentation medium formulation was determined according to the titer of the fermentation broth.
  • the most preferred fermentation medium formulation is (g/L): corn starch 140, amylase 0.2, soybean cake powder 20, peanut cake powder 5, yeast powder 10, (NH 4 ) 2 SO 4 2, MnSO 4 0.024, Na 2 MoO 4 0.024, CoCl 2 0.02, CaCO 3 7, pH 7.2, sterilized at 121 ° C for 20 minutes.
  • the culture temperature was 28 ° C, 250 rpm, and shaking culture was carried out for 12 days. At this time, the culture solution had a pH of 6.65 and a mycelial concentration of 37% by volume.
  • the potency of ivermectin B1b produced by S. avermitilis strain C63-51 was increased to 6500 mg/L, which was 2 times higher than the initial formulation.
  • the slope was made of YMS medium (g/L): yeast extract 4, glucose 4, malt extract 10, trace element solution 5 ml/L, agar 20, distilled water 1000 ml, pH 7.2, sterilized at 121 ° C for 20 minutes. Cool to 50-60 ° C pendulum slope, access strain C63-51, cultured at 28 ° C for 7d.
  • the cultured spores were scraped with a inoculating shovel to a seed culture medium of about 1 cm 2 of the lawn, and the culture temperature was 28 ° C, 250 rpm, and shaking culture was carried out for 40 hours.
  • the culture solution had a pH of 6.86 and a mycelial concentration of 35% by volume.
  • 10L of seed culture medium was put into 15L seed tank, sterilization was carried out by steam sterilization, and it was carried out for 30 minutes at 121 °C, and 300 ml shake flask seed solution was added, the culture temperature was 28 ⁇ 1 ° C, the stirring speed was 100-250 rpm, and the air was ventilated. The amount was 1.0 vvm and cultured for 18 hours. At this time, the culture solution had a pH of 6.80 and a hyphal concentration of 30% by volume.
  • the stirring speed was 101-308 rpm, the aeration amount was 0.6-1.1 vvm, the fermentation was cultured for 12 days, and the tank was placed, and the fermentation unit of ivermectin B1b was measured to be 4000 mg/L.
  • the obtained fermentation broth was filtered with a filter cloth to obtain a filter cake, and the filter cake was extracted twice with ethanol to obtain an ethanol extract.
  • the ethanol extract was concentrated to dryness in vacuo and extracted with ethyl acetate to give an extract containing ivermectin B1b.
  • the extract was mixed with silica gel, it was applied to a silica gel column and eluted with a petroleum ether/acetone gradient of 90:10, 80:20, 70:30, 60:40 in a volume ratio, and the eluted fraction was collected in stages, TLC.
  • Liquid phase system Agilent 1100 semi-preparative high pressure liquid chromatography
  • Streptomyces avermitilis C63-51 is a high-yield strain of ivermectin B1b, and the shake flask fermentation unit reaches 6500 mg/L, and the 50 L tank fermentation unit reaches 4000 mg/L.

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Abstract

提供了一种伊维菌素B1b高产的除虫链霉菌(Streptomyces avermitilis)菌株C63-51以及利用该菌株生产伊维菌素B1b的方法。利用上述菌株和方法，可以实现单一伊维菌素B1b的高效生产。

Description

一种伊维菌素B1b产生菌及其应用 技术领域
本发明属于生物医药领域，涉及一种伊维菌素B1b高产的除虫链霉菌(Streptomyces avermitilis)菌株及利用该菌株生产伊维菌素B1b的方法。
背景技术
伊维菌素对体内外寄生虫特别是线虫和节肢动物均具有高效驱杀作用。对于哺乳动物的机体组织有更强的渗透性和安全性，持效期更长，特别适用于一般口服驱虫剂难以到达的肌肉、器官和特殊组织中的寄生虫防治，对家畜的胃肠道线虫、外寄生虫有着特殊的疗效，是人们公认的具有广谱、高效、安全、残留少、无抗药性的多组分抗生素。
迄今为止，国内外报道的伊维菌素生产方法主要有两种：一种是用化学法对阿维菌素B1进行加氢选择性还原得到伊维菌素。该化学方法需要从阿维链霉菌的发酵液中分离、提纯阿维菌素B1后，将其还原成伊维菌素，该工艺的生产成本较高，其中伊维菌素B1b的产量仅有15％；且从阿维菌素B1化学合成伊维菌素B1b的工艺，杂质多，提纯工艺比较复杂，很难分离得到大量的伊维菌素B1b；另一种是使用基因工程菌生产伊维菌素，例如CN200510074936.X公开了将阿维链霉菌的阿维菌素聚酮合酶DH2-KR2结构域的编码基因替换为聚酮合酶DH-ER-KR结构域的编码基因得到产伊维菌素的工程菌的方法，其伊维菌素B1a的产量约为1-4μg/mL，但未提及伊维菌素B1b的产量；CN200910089970.2公开了将阿维链霉菌中编码麦芽糖转运蛋白的malEFG基因通过表达载体引入阿维链霉菌中获得麦芽糖转运蛋白过表达的重组菌，通过该重组菌过量表达麦芽糖转运系统基因来提高麦芽糖的利用效率的方法，该方法将伊维菌素的产量由约10μg/mL提高到约35μg/mL；PCT国际申请WO2015135242A1和WO2015135467A1公布了基因工程菌MA220的构建过程，基因工程菌MA220由除虫链霉菌MA-4680(Streptomyces avermitilis MA-4680)通过两步基因改造获得：首先通过PCR打靶(PCR targeting)技术将MA4680aveD基因失活，得到不产阿维菌素A组分只产阿维菌素B组分的基因工程菌AD28；然后通过基因重组技术将aveA1基因替换为吸水链霉菌(Streptomyces milbemycinicus)的milA1基因，得到重组链霉菌MA220。重组链霉菌MA220发酵产物中除主要组分天维菌素A(TEVA)和天维菌素B(TEVB)外，有极少量的伊维菌素B1b组分。上述通过基因工程构建新菌种生产伊维菌素的方法或者对实验条件和技术人员的技术水平要求较高，或者基因工程菌发酵产量不令人满意，基本上还停留在实验室阶段，不利于形成产业化。
综上，现有技术中伊维菌素的产量低，而且，产物或者均为伊维菌素B1a和B1b的混合物，或者为伊维菌素B1a，单一组分伊维菌素B1b的生产则尚未有关注。因此，现有技术中迫切需要寻找能够获得令人满意产量的伊维菌素尤其是伊维菌素B1b的新的微生物菌株及其制备方法。
本发明人意外地筛选得到直接发酵生产伊维菌素B1b的高产菌株，并进一步提供了利用该菌株生产伊维菌素B1b的方法。这一方面可以有效利用伊维菌素B1b，另一方面也可以为今后进一步研究伊维菌素B1b生物学活性和对其进行结构改造打下基础。
发明内容
本发明的目的之一是提供一种能够产生伊维菌素B1b的新的高产菌株，以解决上述问题。
本发明的一个方面在于提供一种伊维菌素B1b高产微生物菌株。该菌株为除虫链霉菌(Streptomyces avermitilis)菌株C63-51，于2016年12月22日保存于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院，中国科学院微生物研究所)，保藏编号为CGMCC NO.13370，并登记入册，证明存活。
本发明的另一方面是提供除虫链霉菌菌株C63-51在生产伊维菌素B1b或含有伊维菌素B1b的药物组合物中的应用。
本发明的另一方面是提供一种利用除虫链霉菌菌株C63-51生产伊维菌素B1b的方法，该方法包括将除虫链霉菌菌株C63-51在含有可同化的碳源和/或氮源的培养基里进行发酵。
本发明还涉及一种组合物，其包含本发明的除虫链霉菌菌株C63-51。所述组合物可以是菌株组合物，其包含除菌株C63-51以外的其他菌株；优选地，所述其他菌株可以是用于生产伊维菌素B1b的菌株，例如阿维链霉菌、吸水链霉菌、基因工程菌MA220等，尤其是基因工程菌MA220。
本发明还涉及上述组合物在生产伊维菌素B1b或含有伊维菌素B1b的药物组合物中的应用。
在另一方面，本发明还涉及一种杀虫组合物，其包含由本发明的方法制得 的伊维菌素B1b。
优选地，上述可同化的碳源为蔗糖、葡萄糖、淀粉酶、果糖、鼠李糖、棉籽糖、木糖、阿拉伯糖、工业糖蜜、乳糖、半乳糖、麦芽糖、海藻糖、木聚糖、糊精、玉米淀粉、山梨醇、水杨苷、肌醇、甘露醇、甘油、甘氨酸、菊粉中的一种或几种。
优选地，上述可同化的氮源为牛肉浸膏、酵母浸膏、酵母抽提物、酵母粉、蛋白胨、胰蛋白胨、麸质粉、棉籽饼粉、花生饼粉、黄豆饼粉、玉米浆干粉、麸皮、尿素、铵盐、硝酸盐中的一种或几种。
优选地，所述培养基还包括无机盐，所述无机盐为MnSO 4，Na 2MoO 4，CoCl 2，CaCO 3，ZnSO 4，FeSO 4，(NH 4) 2SO 4，FeCl 3，KNO 3，KH 2PO 4，K 2HPO 4，MgCl 2，MgSO 4，NaCl，CuSO 4，NiSO 4，KCl中的一种或几种。
优选地，所述培养基含有玉米淀粉120～160g/L，淀粉酶0.2g/L，黄豆饼粉10～40g/L，花生饼粉0～10g/L，酵母粉5～20g/L，(NH 4) 2SO 4 0～4g/L，MnSO 4 0.024g/L，Na 2MoO 4 0.024g/L，CoCl 2 0.01～0.04g/L，CaCO 3 3～7g/L。
优选地，所述培养基含有玉米淀粉140g/L，淀粉酶0.2g/L，黄豆饼粉20g/L，花生饼粉5g/L，酵母粉10g/L，(NH 4) 2SO 4 2g/L，MnSO 4 0.024g/L，Na 2MoO 4 0.024g/L，CoCl 2 0.02g/L，CaCO 3 7g/L。
优选地，所述发酵的温度为20℃-40℃，更优选为25℃-30℃，最优选为28℃。优选地，在本发明的发酵方法中对发酵的pH条件进行控制，所述pH为6.0-8.0，更优选为6.5-7.5，最优选为7.0-7.2。本发明方法的发酵时间可以为288-312小时；本发明方法的通气量可以为0.6-1.1vvm。
在本发明中，“通气量”表示每分钟内通过单位体积培养液的经过滤除菌的无菌空气的量。
在本发明中，所述“除虫链霉菌C63-51”、“菌C63-51”、“诱变菌C63-51”具有相同的含义。
在本发明中，所述“基因工程菌MA220”、“MA220菌株”、“天维菌素产生菌MA220”、“重组链霉菌MA220”、“出发菌株MA220”具有相同的含义。
本发明中，伊维菌素B1b可以通过以下HPLC方法进行检测：
色谱柱为C18柱，5μm，4.6×250mm；
流动相为甲醇∶乙腈∶水＝81∶7∶12(体积比)；
流速：1ml/min；
检测波长：240nm；
进样量：10μl。
本发明与现有技术相比至少具有以下优点：
1、本发明筛选得到的除虫链霉菌菌株C63-51可直接用于伊维菌素B1b的发酵生产，与用化学法制备伊维菌素相比，避免了复杂的分离提纯和化学还原的工艺步骤，使生产成本大幅降低。
2、在现有技术中，例如CN200510074936.X中伊维菌素B1a的产量约为1-4mg/L，但未提及伊维菌素B1b的产量；CN200910089970.2中伊维菌素的产量约为35mg/L，且产物为伊维菌素B1a和B1b的混合物，较难分离出伊维菌素B1b；WO2015135242A1和WO2015135467A1中出发菌株MA220生产伊维菌素B1b的效价为200mg/L；而本发明除虫链霉菌C63-51生产伊维菌素B1b摇瓶发酵单位可达6500mg/L，在50L发酵罐中效价可达4000mg/L，本发明伊维菌素B1b的产量远远高于现有技术；且本发明除虫链霉菌C63-51发酵液中伊维菌素B1b含量超过60％，杂质少，发酵单位高，提纯工艺简单。
附图说明
图1：除虫链霉菌菌株C63-51和出发菌株MA220发酵产物对比图：A为出发菌株MA220在实施例1中产生的发酵液HPLC图谱，B为除虫链霉菌菌株C63-51在实施例1中产生的发酵液HPLC图谱。
图2：实施例5中制备得到的伊维菌素B1b的质谱图。
具体实施方式
以下各实施例中，如无特殊说明，所使用的试剂、仪器都是本领域常用试 剂、仪器，可以从化学或生物制品/制剂公司购买得到；以下实施例中使用的方法都是本领域常规方法，本领域技术人员根据现有技术或生产商提供的操作手册可以毫无疑义地知道这些实验的操作过程并获得相应结果。
实施例1 除虫链霉菌C63-51的筛选
1、MA220菌株的亚硝基胍(NTG)诱变：
以天维菌素产生菌MA220(其制备方法参见WO2015135242A1和WO2015135467A1)为出发菌株。取出发菌株MA220的新鲜斜面，用0.1mol/L，pH6.0的磷酸盐缓冲液制成孢子悬液，用终浓度为1mg/ml的NTG处理孢子悬液，于34℃震荡1h，用生理盐水离心洗涤孢子3次，经稀释后涂布于YMS固体培养基(酵母抽提物4g/L，葡萄糖4g/L，麦芽抽提物10g/L，微量元素溶液5ml/L，琼脂20g/L)上，28℃培养4d。挑取菌落直径较大，产孢丰富的单菌落转接到YMS固体平板培养基上，28℃培养7d。按如下所述的发酵验证方法，共筛选诱变菌600株左右，得到突变菌株58-46，其伊维菌素B1b效价为1600mg/L。
2、突变菌株58-46的常压室温等离子体(ARTP)诱变：
以上述NTG诱变得到的突变菌株58-46为出发菌株，挑取新鲜斜面上的成熟孢子，用生理盐水洗涤制备孢子悬液。取10μl孢子悬液滴加在无菌的载片上，放于等离子体室照射。照射条件设定为：以氦气为发生气，流量为12.5L/min，发射口与盛样品载片间距离2mm，照射功率100W，照射时间30s。照射完毕后，用无菌镊子将样品载片取出，放置于装有生理盐水的2ml无菌EP管中，利用漩涡混合器剧烈震荡2min，将载片上的样品彻底洗下并悬浮于生理盐水中。经梯度稀释后，涂布于YMS固体培养基平板，28℃培养4d。挑取菌落直径较大，产孢丰富的单菌落转接到YMS固体平板培养基上，28℃培养7d。按如下所述的发酵验证方法，共筛选诱变菌600株，获得一株伊维菌素B1b的高产菌株，命名为除虫链霉菌菌株C63-51。出发菌株MA220生产伊维菌素B1b的效价为200mg/L，杂质较多；除虫链霉菌菌株C63-51生产伊维菌素B1b的效价提高到 3000mg/L，杂质较少。结果如图1所示，其中A是出发菌株MA220的发酵液HPLC图谱，B是除虫链霉菌菌株C63-51的发酵液HPLC图谱。
3、发酵验证方法如下：
(1)种子液的制备与培养
种子培养基配方(g/L)：玉米淀粉25，黄豆饼粉8，花生饼粉10，酵母粉9.5，CoCl 2 0.03，pH7.2，经121℃灭菌20分钟。将培养好的孢子用接种铲刮取约1cm 2的菌苔转接种子培养基中，培养温度28℃，250rpm，摇床振荡培养40小时。此时培养液pH 6.86，菌丝浓度为35％(体积百分比)。
(2)发酵培养基的制备与培养
发酵培养基配方(g/L)：玉米淀粉120，淀粉酶0.2，黄豆饼粉10，酵母粉5，MnSO 4 0.024，Na 2MoO 4 0.024，CoCl 2 0.01，CaCO 3 3，pH 7.2，经121℃灭菌20分钟。种子液接种量为6％(体积百分比)。培养温度28℃，250rpm，摇床振荡培养12天。此时培养液pH 6.60，菌丝浓度为33％(体积百分比)。
(3)高液相色谱方法(HPLC)测定发酵液的效价
发酵液萃取：取发酵液1ml，加入4ml无水甲醇浸泡，超声1h后，过滤。滤液直接用于HPLC分析。HPLC分析条件为：色谱柱：C18Hypersil ODS24.6×250×5(大连依利特)，流动相：甲醇∶乙腈∶水＝81∶7∶12(体积比)，流速：1ml/min，吸收波长：240nm。
实施例2 除虫链霉菌C63-51的形态学和培养学特征
参照《链霉菌鉴定手册》、《放线菌的分类与鉴定》、《常见细菌系统鉴定手册》等书中的有关内容进行实验。菌株的形态学和培养学特征实验采用ISP2、ISP3、ISP4、高氏一号、葡萄糖天冬素、LB和YMS 7种培养基，28℃培养5～7天后，观察菌丝体的颜色及色素情况。结果见表1。
表1 菌株C63-51在7种不同培养基上的培养特征
培养基 生长情况 基质菌丝 气生菌丝 可溶性色素
ISP2 4 浅棕色 肉桂色 浅黄褐色
ISP3 3 浅黄色 黄白色 无
ISP4 3 黄色 浅黄色 浅黄棕色
高氏一号 4 米色 乳白色 黄色
葡萄糖天冬素 4 黄橙色 黄橙色 浅黄褐色
LB 2 米色 米色 黄色
YMS 4 黄色 黄色 黄色
实施例3除虫链霉菌C63-51的生理生化特征
(1)碳源的利用：采用ISP9作为基础培养基，各种碳源的终浓度均为1.0％(质量百分浓度)。其结果见表2。
(2)氮源的利用：采用配方为KH 2PO 4 1.36g/L，Na 2HPO 4 2.13g/L，MgSO 4 0.2g/L，CaCl 2 0.005g/L，葡萄糖10g/L，FeSO 4 0.0005g/L的培养基作为基础培养基，硝酸钾和硫酸铵的浓度均为0.1％(质量百分浓度)。其结果见表2。
表2 菌株C63-51的碳源和氮源的利用情况
Figure PCTCN2018078773-appb-000001
(3)降解试验和NaCl耐受试验：降解试验采用基础培养基为GYEA(pH 6.8)，各种降解试验结果见表3；NaCl耐受试验结果为：NaCl耐受性较好，在7％的NaCl存在下仍能生长。
表3 菌株C63-51的降解试验结果
降解物 降解物浓度 结果 降解物 降解物浓度 结果
腺嘌呤 0.5％ 4，+ 酪氨酸 1.0％ 4，+
鸟嘌呤 0.5％ 4，- Tween-40 1.0％ 3，-
次黄嘌呤 0.4％ 4，- Tween-80 1.0％ 4，-
酪蛋白 1.0％ 4，+      
(4)生理生化试验、pH试验和温度试验：生理生化试验结果见表4。pH试验和温度试验均采用高氏一号培养基进行。其结果为：菌株在14℃到37℃之间均能生长，最适生长温度为28℃；pH 6.0～8.0均能生长，最适宜的范围为6.5～7.5。
表4 菌株C63-51的主要生理生化特征
试验项目 结果 试验项目 结果 试验项目 结果
明胶液化 - 牛奶胨化 + 纤维素利用 -
淀粉水解 + 硝酸盐还原 +    
牛奶凝固 + 硫化氢产生 +    
注：表1-4中的数字和符号分别代表：
0：无生长；1：生长很弱；2：能生长；3：生长良好；4：生长最好；
+：阳性；-：阴性。
实施例4 16S rDNA序列分析以及菌种鉴定
收集待测菌株C63-51的新鲜菌体，采用溶菌酶法改进的Pitcher法(Letters in Applied Microbiology,1989,8∶151-156)提取总DNA模板，采用通用引物(27F和1495R)进行16S rDNA基因扩增，PCR产物经检测纯化后，直接进行序列测定，测序由南京金斯瑞生物技术有限公司进行。所测的16S rDNA序列经校对后与GenBank数据库中相关种、属的序列进行同源序列BLAST比较，以确定该菌株的分类地位。
菌株C63-51(CGMCC NO.13370)16S rDNA序列与GenBank中相关序列进行BLAST比较，结果见表5(表中只列出同源性较高的菌株)。
表5 菌株C63-51和相关菌株的同源性
Figure PCTCN2018078773-appb-000002
菌株C63-51(CGMCC NO.13370)通过16S rDNA区域测序发现其和除虫链霉菌(Streptomyces avermitilis)有100％的同源性，同时对菌株C63-51(CGMCC NO.13370)进行表观特征试验，发现该菌株和除虫链霉菌(Streptomyces avermitilis)分类相关参数非常接近，故将菌株C63-51(CGMCC NO.13370)鉴定为除虫链霉菌(Streptomyces avermitilis)。
实施例5 发酵培养基优化
(1)发酵培养基的优化与培养
以实施例1发酵培养基为基础培养基，调整配方中各组分浓度，优化发酵培养基，配方优化为(g/L)：玉米淀粉120～160，淀粉酶0.2，黄豆饼粉10～40，花生饼粉0～10，酵母粉5～20，(NH 4) 2SO 4 0～4，MnSO 4 0.024，Na 2MoO 4 0.024，CoCl 2 0.01～0.04，CaCO 3 3～7，pH 7.2，经121℃灭菌20分钟。种子液接种量为6％(体积百分比)。培养温度28℃，250rpm，摇床振荡培养12天。
(2)高液相色谱方法(HPLC)测定发酵液的效价
发酵液萃取：取发酵液1ml，加入4ml无水甲醇浸泡，超声1h后，过滤。
滤液直接用于HPLC分析。HPLC分析条件为：色谱柱：C18Hypersil ODS24.6×250×5(大连依利特)，流动相：甲醇∶乙腈∶水＝81∶7∶12(体积比)，流速：1ml/min，吸收波长：240nm。
按上述方法测定不同发酵培养基的发酵液效价，根据发酵液效价确定优选的发酵培养基配方。最为优选的发酵培养基配方为(g/L)：玉米淀粉140，淀粉酶0.2，黄豆饼粉20，花生饼粉5，酵母粉10，(NH 4) 2SO 4 2，MnSO 4 0.024，Na 2MoO 4 0.024，CoCl 2 0.02，CaCO 3 7，pH7.2，经121℃灭菌20分钟。培养温度28℃，250rpm，摇床振荡培养12天。此时培养液pH 6.65，菌丝浓度为37％(体积百分比)。除虫链霉菌菌株C63-51生产伊维菌素B1b的效价提高到6500mg/L，比初始配方提高了2倍。
实施例6 发酵罐生产伊维菌素B1b
(1)斜面菌体的制备与培养
斜面采用YMS培养基(g/L)：酵母抽提物4，葡萄糖4，麦芽抽提物10，微量元素溶液5ml/L，琼脂20，蒸馏水1000ml，pH 7.2，经121℃灭菌20分钟，冷却到50-60℃摆斜面，接入菌株C63-51，28℃培养7d。
(2)种子液的制备与培养
种子培养基配方(g/L)：玉米淀粉25，黄豆饼粉8，花生饼粉10，酵母粉9.5，CoCl 2 0.03，pH7.2，经121℃灭菌20分钟。将培养好的孢子用接种铲刮取约1cm 2的菌苔转接种子培养基中，培养温度28℃，250rpm，摇床振荡培养40小时。此时培养液pH 6.86，菌丝浓度为35％(体积百分比)。
(3)种子罐种子液的制备
在15L种子罐中投入10L的种子培养基，灭菌采用蒸汽灭菌，121℃条件下进行30分钟，接入300ml摇瓶种子液，培养温度为28±1℃，搅拌转速100-250rpm，通气量1.0vvm，培养18h，此时培养液pH 6.80，菌丝浓度30％(体积百分比)。
(4)发酵罐培养基的配制与培养
发酵培养基配方(g/L)：玉米淀粉140，淀粉酶0.2，黄豆饼粉20，花生饼粉5，酵母粉10，(NH 4) 2SO 4 2，MnSO 4 0.024，Na 2MoO 4 0.024，CoCl 2 0.02，CaCO 37，发酵罐体积50L，投料体积为30L，pH 7.2，于121℃条件下蒸汽灭菌30min，冷却后，接入约3L种子罐培养液，发酵温度28±1℃，搅拌转速101-308rpm， 通气量0.6-1.1vvm，发酵培养12d，放罐，测得伊维菌素B1b发酵单位为4000mg/L。
(5)发酵液萃取
将得到的发酵液用滤布过滤得到滤饼，滤饼用乙醇提取两次，合并得到乙醇提取液。乙醇提取液经过真空浓缩至干后用乙酸乙酯萃取，即得到含有伊维菌素B1b的浸膏。该浸膏用硅胶拌样后，上硅胶柱，用体积比为90∶10，80∶20，70∶30，60∶40的石油醚/丙酮梯度洗脱，分段收集洗脱流分，TLC检测，得到含伊维菌素B1b的组分，该组分经真空浓缩至干得含伊维菌素B1b的样品，该样品在以下条件下进行反向色谱分离：
液相系统：Agilent 1100半制备高压液相色谱仪
色谱柱：ZORBAXEclipse XDB-C18(250mm×9.4mm)
洗脱剂：甲醇∶乙腈∶水＝46∶46∶8(体积比)
流速：1.5ml/min
检测波长：λ＝240nm
收集保留时间为26.79min的峰得到化合物，将该化合物做质谱分析，图谱见图2。结果表明其分子量为860，确定为伊维菌素B1b，结构如下：
Figure PCTCN2018078773-appb-000003
综上所述，本发明除虫链霉菌C63-51为伊维菌素B1b的高产菌株，其摇瓶发酵单位达到6500mg/L，50L罐的发酵单位达到4000mg/L。

Claims (14)

  1. 一种除虫链霉菌(Streptomyces avermitilis)菌株C63-51，其于2016年12月22日以保藏编号CGMCC NO.13370保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)。
  2. 一种组合物，其包含权利要求1所述的除虫链霉菌菌株C63-51。
  3. 权利要求1所述的除虫链霉菌菌株C63-51或权利要求2所述的组合物在生产伊维菌素B1b或含有伊维菌素B1b的药物组合物中的应用。
  4. 一种生产伊维菌素B1b的方法，该方法包括将权利要求1所述的除虫链霉菌菌株C63-51在含有可同化的碳源和/或氮源的培养基里进行发酵。
  5. 根据权利要求4所述的方法，其中所述可同化的碳源为蔗糖、葡萄糖、淀粉酶、果糖、鼠李糖、棉籽糖、木糖、阿拉伯糖、工业糖蜜、乳糖、半乳糖、麦芽糖、海藻糖、木聚糖、糊精、玉米淀粉、山梨醇、水杨苷、肌醇、甘露醇、甘油、甘氨酸、菊粉中的一种或几种。
  6. 根据权利要求4或5所述的方法，其中所述可同化的氮源为牛肉浸膏、酵母浸膏、酵母抽提物、酵母粉、蛋白胨、胰蛋白胨、麸质粉、棉籽饼粉、花生饼粉、黄豆饼粉、玉米浆干粉、麸皮、尿素、铵盐、硝酸盐中的一种或几种。
  7. 根据权利要求4-6中任一项所述的方法，其中所述培养基还包含无机盐，所述无机盐为MnSO 4，Na 2MoO 4，CoCl 2，CaCO 3，ZnSO 4，FeSO 4，(NH 4) 2SO 4，FeCl 3，KNO 3，KH 2PO 4，K 2HPO 4，MgCl 2，MgSO 4，NaCl，CuSO 4，NiSO 4，KCl中的一种或几种。
  8. 根据权利要求4-7中任一项所述的方法，其中所述培养基含有玉米淀粉120～160g/L，淀粉酶0.2g/L，黄豆饼粉10～40g/L，花生饼粉0～10g/L，酵母粉5～20g/L，(NH 4) 2SO 4 0～4g/L，MnSO 4 0.024g/L，Na 2MoO 4 0.024g/L，CoCl 20.01～0.04g/L，CaCO 3 3～7g/L。
  9. 根据权利要求4-8中任一项所述的方法，其中所述培养基含有玉米淀粉140g/L，淀粉酶0.2g/L，黄豆饼粉20g/L，花生饼粉5g/L，酵母粉10g/L， (NH 4) 2SO 4 2g/L，MnSO 4 0.024g/L，Na 2MoO 4 0.024g/L，CoCl 2 0.02g/L，CaCO 37g/L。
  10. 根据权利要求4-9中任一项所述的方法，其中所述发酵的温度为20℃-40℃。
  11. 根据权利要求4-10中任一项所述的方法，其中所述培养基的pH为6.0-8.0。
  12. 根据权利要求4-11中任一项所述的方法，其中发酵时间为288-312小时；和/或通气量为0.6-1.1vvm。
  13. 根据权利要求4-10中任一项所述的方法，其中所述发酵的温度为25℃-30℃，优选为28℃；所述培养基的pH为6.5-7.5，优选为7.0-7.2。
  14. 一种杀虫组合物，其包含由权利要求4-13中任一项的方法制得的伊维菌素B1b。
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