阅读说明：本技术 一种棘白菌素b合成培养基及应用 (Echinocandin B synthetic medium and application ) 是由 胡忠策 郎克鹏 牛坤 邹树平 柳志强 郑裕国 于 2020-12-11 设计创作，主要内容包括：本发明公开了一种棘白菌素B合成培养基及制备棘白菌素B的应用,所述培养基质量浓度组成为：油酸甲酯20-100g/L,淀粉10-60g/L,油酸10-30g/L,组氨酸10-30g/L,谷氨酸5-20g/L,L-苏氨酸2-5g/L,L-鸟氨酸盐酸盐5-8g/L,硝酸钾5-15g/L,K-2HPO-4·3H-2O 5-20g/L,MgSO-4·7H-2O 0.1-1g/L,MnSO-4·H-2O 0.1-0.5g/L,FeSO-4·7H-2O 0.01-0.1g/L,CaCl-20.1-0.5g/L,CuSO-4·5H-2O 0.5-1g/L,溶剂为水,pH6.5-7.0。本发明棘白菌素B产量达到了1950mg/L。(The invention discloses an echinocandin B synthetic culture medium and application for preparing echinocandin B, wherein the culture medium comprises the following components in mass concentration: 20-100g/L methyl oleate, 10-60g/L starch, 10-30g/L oleic acid, 10-30g/L histidine, 5-20g/L glutamic acid, 2-5 g/L-threonine, 5-8 g/L-ornithine hydrochloride, 5-15g/L potassium nitrate, K 2 HPO 4 ·3H 2 O 5‑20g/L，MgSO 4 ·7H 2 O 0.1‑1g/L，MnSO 4 ·H 2 O 0.1‑0.5g/L，FeSO 4 ·7H 2 O 0.01‑0.1g/L，CaCl 2 0.1‑0.5g/L，CuSO 4 ·5H 2 O0.5-1 g/L, water as solvent, and pH 6.5-7.0. The yield of echinocandin B reaches 1950mg/L。)

1. An echinocandin B synthetic culture medium is characterized in that the culture medium consists of the following components in mass concentration: 20-100g/L methyl oleate, 10-60g/L starch, 10-30g/L oleic acid, 10-30g/L histidine, 5-20g/L glutamic acid, 2-5 g/L-threonine, 5-8 g/L-ornithine hydrochloride, 5-15g/L potassium nitrate, K₂HPO₄·3H₂O 5-20g/L，MgSO₄·7H₂O0.1-1g/L，MnSO₄·H₂O 0.1-0.5g/L，FeSO₄·7H₂O 0.01-0.1g/L，CaCl₂ 0.1-0.5g/L，CuSO₄·5H₂O0.5-1 g/L, water as solvent, and pH 6.5-7.0.

2. The echinocandin B synthetic medium of claim 1, wherein the echinocandin B synthetic medium comprises the following mass concentrations: 70-90g/L methyl oleate, 15-20g/L starch, 15-20g/L oleic acid, 10-20g/L histidine, 8-12g/L glutamic acid, and L-threo3-4g/L of amino acid, 6-7g/L of L-ornithine hydrochloride and 8-12g/L of potassium nitrate, K₂HPO₄·3H₂O8-10g/L，MgSO₄·7H₂O 0.4-0.6g/L，MnSO₄·H₂O 0.1-0.2g/L，FeSO₄·7H₂O 0.05-0.1g/L，CaCl₂ 0.1-0.3g/L，CuSO₄·5H₂O0.5-0.6 g/L, water as solvent, and pH 6.5-7.0.

3. The echinocandin B synthetic medium of claim 1, wherein the echinocandin B synthetic medium comprises the following mass concentrations: 70g/L methyl oleate, 15g/L starch, 20g/L oleic acid, 10g/L histidine, 8g/L glutamic acid, 3.1 g/L-threonine, 6.1 g/L-ornithine hydrochloride, 10g/L potassium nitrate, K₂HPO₄·3H₂O 8g/L，MgSO₄·7H₂O 0.5g/L，MnSO₄·H₂O 0.2g/L，FeSO₄·7H₂O 0.05g/L，CaCl₂ 0.3g/L，CuSO₄·5H₂O0.6 g/L, water as solvent, pH 6.5-7.0.

4. Use of the echinocandin B synthesis medium of claim 1 for the synthesis of echinocandin B, characterized in that said use is: inoculating aspergillus nidulans to an echinocandin B synthetic culture medium, fermenting for 240 hours at the temperature of 25-30 ℃ and the rotational speed of 150-.

5. The use according to claim 4, wherein the Aspergillus nidulans is Aspergillus nidulans (Aspergillus nidulans) ZJB20027 deposited at China center for type culture Collection with the collection number of CCTCC NO: M2020495, with the collection date of 2020, 09, 14, address: china, wuhan university, zip code 430072.

6. The use of claim 4, wherein the Aspergillus nidulans is subjected to activation and seed expansion culture before fermentation, and the seed solution is inoculated to echinocandin B synthesis in an inoculum size of 10% by volume concentrationThe activation and seed amplification culture method comprises the following steps: inoculating Aspergillus nidulans into activated culture medium, and culturing at 25 deg.C in dark for 12-14 days to obtain activated Aspergillus nidulans; the final concentration composition of the activation medium is as follows: 3g/L potassium nitrate, 1g/L dipotassium hydrogen phosphate and MgSO₄·7H₂0.5g/L of O, 0.5g/L of potassium chloride, 0.01g/L of ferrous sulfate, 30g/L of cane sugar, 20g/L of agar, water as a solvent and natural pH; inoculating the activated aspergillus nidulans into a seed culture medium, and performing shaking table light-shielding culture at 25 ℃ and 160rpm for 3d to obtain a seed solution; the seed culture medium comprises the following components in percentage by mass: 8g/L of glutamic acid, 5g/L of potassium nitrate, 10g/L of glucose, 10g/L of glycerol and K₂HPO₄·3H₂O1 g/L，CaCO₃2g/L, solvent is water, and pH is 6.5-7.

7. The use according to claim 4, characterized in that the fermentation culture is carried out in a fermenter under the following conditions: the fermentation temperature is 25 ℃, the ventilation ratio is 1:1-1:2, the stirring speed is 200-.

(I) technical field

The invention relates to a culture medium, in particular to a synthetic culture medium for producing echinocandin B.

(II) background of the invention

In recent years, fungal infections have attracted increasing attention due to abuse of antibiotics, deterioration of the environment, and aging of the population. Fungal infections are infectious diseases of humans and animals caused by fungi. Among them, candida infection is the highest in deep infection in China, and reaches 91%. The mortality rate for invasive candida infections is reported to be 30%, and can be as high as 100% if not treated in time. Therefore, the development of antifungal infection diagnosis and treatment, especially antifungal drugs, has been a hot spot.

Antifungal drugs currently on the market mainly include polyenes, azoles, fluoropyrimidines and echinocandins. The problems of poor drug resistance, large toxic and side effects, narrow antibacterial spectrum and the like of alkenes and azoles generally exist, echinocandin antifungal drugs are low in toxicity, strong in antibacterial effect, free of antagonism with other drugs, free of liver metabolism and free of cross drug resistance, and the echinocandin antifungal drugs become important points of research in recent years.

Anidulafungin is an echinocandin antifungal drug used to treat invasive candida species. Anidulafungin has a structure of a cyclic hexapeptide plus a trityl chain, and is a semi-synthetic compound. Anidulafungin is obtained by removing linoleic acid side chain from echinocandin B through the action of acylase and then carrying out chemical modification, and is a key precursor for synthesizing anidulafungin.

The medium generally includes complex media, semi-synthetic media, and synthetic media. The composite culture medium has the advantages of rich nutrient components and good culture effect, but has the defects of complex components, poor quality stability of raw materials, and poor quality stability of produced products due to the influence of the production area and batches of the raw materials on the production of echinocandin B. At present, echinocandin B is produced by using a composite culture medium, such as soybean cake powder, peptone, peanut oil and the like, and no report of producing echinocandin B by using a synthetic culture medium exists in the field.

Therefore, in order to research the relationship between the fermentation conditions and the anabolism mechanism of the product, the development of a special synthetic medium suitable for producing echinocandin B by aspergillus nidulans has important practical significance.

Disclosure of the invention

The invention aims to provide a synthetic culture medium for high-yield echinocandin B, which solves the problems of poor stability and poor repeatability in the fermentation process of echinocandin B in the prior art.

The technical scheme adopted by the invention is as follows:

the invention provides an echinocandin B synthetic culture medium, which comprises the following components in percentage by mass: 20-100g/L methyl oleate, 10-60g/L starch, 10-30g/L oleic acid, 10-30g/L histidine, 5-20g/L glutamic acid, 2-5 g/L-threonine, 5-8 g/L-ornithine hydrochloride, 5-15g/L potassium nitrate, K₂HPO₄·3H₂O 5-20g/L，MgSO₄·7H₂O 0.1-1g/L，MnSO₄·H₂O 0.1-0.5g/L，FeSO₄·7H₂O 0.01-0.1g/L，CaCl₂ 0.1-0.5g/L，CuSO₄·5H₂O0.5-1 g/L, water as solvent, and pH 6.5-7.0.

Preferably, the mass concentration of the echinocandin B synthetic culture medium is as follows: 70-90g/L methyl oleate, 15-20g/L starch, 15-20g/L oleic acid, 10-20g/L histidine, 8-12g/L glutamic acid, 3-4 g/L-threonine, 6-7 g/L-ornithine hydrochloride, 8-12g/L potassium nitrate, K₂HPO₄·3H₂O 8-10g/L，MgSO₄·7H₂O 0.4-0.6g/L，MnSO₄·H₂O 0.1-0.2g/L，FeSO₄·7H₂O 0.05-0.1g/L，CaCl₂ 0.1-0.3g/L，CuSO₄·5H₂O0.5-0.6 g/L, water as solvent, and pH 6.5-7.0.

More preferably, the echinocandin B synthetic medium has the following mass concentration: 70g/L methyl oleate, 15g/L starch, 20g/L oleic acid, 10g/L histidine, 8g/L glutamic acid and 3.1 g/L-threonine6.1g/L of L-ornithine hydrochloride, 10g/L of potassium nitrate and K₂HPO₄·3H₂O 8g/L，MgSO₄·7H₂O 0.5g/L，MnSO₄·H₂O 0.2g/L，FeSO₄·7H₂O 0.05g/L，CaCl₂ 0.3g/L，CuSO₄·5H₂O0.6 g/L, water as solvent, pH 6.5-7.0.

The invention also provides an application of the echinocandin B synthetic culture medium in synthesizing echinocandin B, wherein the application comprises the following steps: inoculating aspergillus nidulans to an echinocandin B synthetic culture medium, fermenting for 240 hours at the temperature of 25-30 ℃ and the rotational speed of 150-.

The Aspergillus nidulans is Aspergillus nidulans (Aspergillus nidulans) ZJB20027, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020495, the preservation date is 2020, 09, 14 days, address: china, wuhan university, zip code 430072.

Before fermentation, the aspergillus nidulans is firstly activated and subjected to seed expansion culture, and then a seed solution is inoculated to an echinocandin B synthetic culture medium by an inoculum size of 10% of volume concentration, and the method specifically comprises the following steps: inoculating an activated culture medium to the aspergillus nidulans, and culturing for 12-14 days at 25 ℃ in a dark place to obtain activated aspergillus nidulans; the final concentration composition of the activation medium is as follows: potassium nitrate 3g/L, K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 0.5g/L，KCl 0.5g/L，FeSO₄·7H₂0.01g/L of O, 30g/L of cane sugar, 20g/L of agar and water as a solvent, wherein the pH value is natural; digging the activated Aspergillus nidulans with a sterile inoculating shovel to obtain a volume of about 1cm³Inoculating the fungus blocks into a 500mL triangular flask filled with 50mL seed culture medium, and performing shaking table light-tight culture at 25 ℃ and 160rpm for 3d to obtain a seed solution; the seed culture medium comprises the following components in percentage by mass: 8g/L of glutamic acid, 5g/L of potassium nitrate, 10g/L of glucose, 10g/L of glycerol and K₂HPO₄·3H₂O 1g/L，CaCO₃2g/L, solvent is water, and pH is 6.5-7.

Further, the fermentation culture is carried out in a fermentation tank, and the fermentation conditions are as follows: the fermentation temperature is 25 ℃, the ventilation ratio is 1:1-1:2, the stirring speed is 200-.

Compared with the existing culture medium, the invention has the following beneficial effects: at present, the production of echinocandin B has no report of a synthetic culture medium, but the culture medium has definite chemical components and good repeatability. Meanwhile, the preparation method is a common commercial chemical product and is simple to prepare. The yield of echinocandin B using the culture medium reaches 2000mg/L, which is obviously improved compared with the initial culture medium.

(IV) description of the drawings

FIG. 1 is an HPLC chart of echinocandin B standard.

FIG. 2 is an HPLC chart of fermentation broth echinocandin B.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

in order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Aspergillus nidulans (Aspergillus nidulans) CCTCC M2020495

Aspergillus nidulans (Aspergillus nidulans) CCTCC M2020495 was obtained by the following procedure

Performing ARTP mutagenesis treatment on Aspergillus nidulans (Aspergillus nidulans) CCTCC NO: M2012300 (disclosed in patent application CN 2013104776708), coating the plate, and culturing for 12-14d to obtain a mutagenesis treatment mutant strain, wherein the specific method comprises the following steps:

(1) aspergillus nidulans (Aspergillus nidulans) CCTCC M2012300 was inoculated into the activation medium and cultured at 25 ℃ for 12-14 days in the absence of light to obtain activated Aspergillus nidulans. The final concentration composition of the activation medium is as follows: potassium nitrate 3g/L, K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 0.5g/L，KCl 0.5g/L，FeSO₄·7H₂0.01g/L of O, 30g/L of cane sugar, 20g/L of agar and water as a solvent, wherein the pH value is natural; washing flat plate with dark green spores on surface 12-14d with sterile normal saline, placing in sterile triangular flask, blowing, vibrating, scattering spores and hyphae, filtering with sterile filter paper, preparing into monospore suspension, counting with blood counting plate, diluting monospore suspension with sterile normal saline, and controlling spore number at 10⁷-10⁹/mL。

(2) Wiping an operating room of an Atmospheric Room Temperature Plasma (ARTP) mutation breeding instrument (No-Sn-sourced Qingtian Wood Biotech Co., Ltd., model: ARTP-M) with alcohol cotton, and sterilizing by ultraviolet irradiation for 30 min. Pipette 5. mu.L of the prepared spore suspension into 5. mu.L of a 10% (v/v) aqueous glycerol solution, mix and spread evenly on the center of a sterile slide. The treated slide glass is clamped by sterile forceps and is placed on a hole site of an operation table of an ARTP system, and ARTP mutagenesis is carried out on the spores. The mutagenesis time was 280 s. Setting ARTP operation parameters as follows: carrier gas: high-purity helium gas with the flow rate set to be 10L/min; input RF power is 140W; the distance between the plasma torch nozzle and the slide glass is 4 mm; the temperature of the plasma jet was 25 ℃. Placing the processed slide glass in a 2mL EP tube containing 990 μ L sterile physiological saline, shaking uniformly, coating 100 μ L slide glass on a plate culture medium, and culturing at 25 deg.C in dark for 12-14 d. The plate culture medium has the following final concentration composition: potassium nitrate 3g/L, K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 0.5g/L，KCl 0.5g/L，FeSO₄·7H₂0.01g/L of O, 30g/L of cane sugar, 20g/L of agar and water as a solvent, wherein the pH value is natural;

(3) digging out the volume of about 1cm by using a sterile inoculating shovel³Inoculating the fungus blocks with different forms into a 500mL triangular flask filled with 50mL seed culture medium, and culturing for 3d at 25 ℃ and 160rpm in a shaking table in a dark place to obtain a seed solution. The mass concentration of the seed culture medium is (g/L): glutamic acid 8, potassium nitrate 5, glucose 10, glycerol 10 and K₂HPO₄·3H₂O 1，CaCO₃2, the solvent is water, and the pH value is 6.5-7.0.

(4) Concentrating the seed liquid by volumeInoculating the inoculum size of 10% to an echinocandin B screening synthetic culture medium, fermenting and culturing at the rotation speed of 150rpm and the temperature of 25 ℃ for 10 days, taking a fermentation liquid, and measuring the concentration of the echinocandin B product by a liquid phase. The mass concentration composition of the screened synthetic culture medium of echinocandin B is (g/L): 90 parts of methyl oleate, 20 parts of starch, 3.1 parts of L-threonine, 6.1 parts of L-ornithine hydrochloride, 10 parts of ammonium sulfate and K₂HPO₄·3H₂O 10，MgSO₄·7H₂O 0.8，MnSO₄·H₂O 0.2，FeSO₄·7H₂O 0.05，CaCl₂ 0.3，CuSO₄·5H₂O0.6, water as solvent and pH 6.5-7.0. Adding the components into a beaker, adding water to 1L, stirring uniformly, subpackaging into 500mL conical bottles with 50mL each, and sterilizing at 121 ℃ for 20 min.

(5) The strain with the highest echinocandin B yield is selected from nearly 2000 mutant strains to obtain a mutant strain ZJB20027, which is identified as Aspergillus nidulans (Aspergillus nidulans) ZJB20027 through 18S rDNA and is preserved in China center for type culture collection (CCTCC NO: M2020495), the preservation date is 2020, 9, 14 days, the address: wuhan university, Wuhan, China, zip code 430072.

And (3) measuring the concentration of a product: centrifuging 1mL of fermentation liquid at 4 ℃ and 12000 Xg for 5min, and removing supernatant; adding methanol to dilute 5 times, mixing uniformly, standing and extracting for 24 h. Centrifuging the extractive solution at 4 deg.C and 12000 Xg for 5min, collecting supernatant, filtering with 0.22 μm organic membrane, detecting echinocandin B peak area with liquid phase, and obtaining echinocandin B concentration according to echinocandin B standard curve.

Echinocandin B standard curve: dissolving echinocandin B standard substance in methanol to obtain standard substance solutions (0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, 1.0g/L) with different concentrations, and detecting with liquid phase. And (3) drawing a standard curve by taking the peak area as an ordinate and the concentration (g/L) of echinocandin B as an abscissa, wherein the standard curve y is 7.4415 x-9.8659.

Liquid phase detection conditions: the chromatographic column is a C18 column (4.6 mm. times.250 mm. times.5 μm); the mobile phase is methanol: acetonitrile: water 7: 1:2, v/v/v; the flow rate is 1 mL/min; the ultraviolet detection wavelength is 222 nm; the sample volume is 20 mu L; the column temperature was 40 ℃.

(6) And (3) strain identification: the strain identification is carried out by adopting an 18S rDNA method. After extracting the strain genome with a soil genome DNA extraction kit (purchased from MP biomedical corporation), PCR amplification was performed. The primers used are the fungus strain identification universal primers ITS1 and ITS 4. The ITS1 and ITS4 nucleic acid sequences are ITS1TCCGTAGGTGAACCTGCGG and ITS4 TCCTCCGCTTATTGATATGC, respectively.

25 μ L PCR reaction: 1 mu L of genome template; 5 × Buffer 12.5 μ L; dNTP 1 u L; 1 μ L of DNA polymerase; ITS 11 μ L; ITS 41 μ L; add double distilled water to 25. mu.L.

PCR cycling conditions: 94 ℃ for 4 min; 94 ℃ for 45 s; 45s at 55 ℃; 72 ℃ for 1 min; 30 cycles, 72 ℃ for 10 min.

The PCR product is sent to the Oncology Limited company for direct sequencing by using PCR primers, and then 18S rDNA sequencing results are submitted to NCBI database for BLAST comparison. The 18S rDNA sequence of the strain is 541bp, and the homology comparison of the 18S rDNA sequence and the gene sequence in GenBank shows that the homology of the strain and Aspergillus nidulans reaches 99.22%. I.e. the obtained strain is still aspergillus nidulans.

Sequencing result of general primer (SEQ ID NO.1)

GATTCGAGGTGCGGGCTGCCTCCGGGCGCCCACCTCCCACCCGTGACTACCTAACACTGTTGCTTCGGCGGGGAGCCCCCCAGGGGCGAGCCGCCGGGGACCACTGAACTTCATGCCTGAGAGTGATGCAGTCTGAGCCTGAATACAAATCAGTCAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAACTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGTCGTCGTCCCCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGTGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTCGATTAGGGCCGGCCGGGCGCCAGCCGGCGTCTCCAACCTTATTTTTCTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAG

Table 1: composition and mass concentration of different synthetic media

Adding each component of the culture medium in the table 1 into a beaker, adding water to 1L, uniformly stirring, subpackaging into 500mL conical bottles with 50mL each bottle, and sterilizing at 121 ℃ for 20min to obtain the synthetic culture medium of the fermented echinocandin B.

Comparative example 1

Aspergillus nidulans (Aspergillus nidulans) CCTCC M2012300 was inoculated into the activation medium and cultured at 25 ℃ for 12-14 days in the absence of light to obtain activated Aspergillus nidulans. The final concentration composition of the activation medium is as follows: potassium nitrate 3g/L, K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 0.5g/L，KCl 0.5g/L，FeSO₄·7H₂0.01g/L of O, 30g/L of cane sugar, 20g/L of agar and water as a solvent, wherein the pH value is natural;

digging the activated Aspergillus nidulans with a sterile inoculating shovel to obtain a volume of about 1cm³Inoculating the strain blocks into a 500mL triangular flask containing 50mL seed culture medium, and culturing for 3d at 25 ℃ and 160rpm in a shaking table in a dark place to obtain a seed solution. The mass concentration of the seed culture medium is (g/L): glutamic acid 8, potassium nitrate 5, glucose 10, glycerol 10 and K₂HPO₄·3H₂O 1，CaCO₃2, the solvent is water, and the pH value is 6.5-7.0.

Inoculating the seed solution into the culture medium 1 in the table 1 at the inoculation amount of 10% of the volume concentration, fermenting and culturing at 25 ℃ for 10 days at the rotation speed of 150rpm, taking the fermentation liquid, and determining that the yield of the echinocandin B reaches 450mg/L by adopting the method in the example 1.

Examples 2 to 8

Comparative example 1 under the same conditions, medium 1 was replaced with medium 2 to medium 8 in Table 1, and the yields of echinocandin B were 568mg/L, 1023mg/L, 1196mg/L, 1612mg/L, 1359mg/L, 1584mg/L and 1877mg/L, respectively, as shown in Table 2.

Example 9

Changing the culture medium of the comparative example 1 into a culture medium 5 in the table 1, inoculating the seed solution into a 5L fermentation tank filled with 3L fermentation echinocandin B synthetic culture medium according to the inoculation amount with the volume concentration of 10% for fermentation culture, and performing fermentation culture for 240h, wherein in the whole fermentation culture process, the fermentation temperature is controlled to be 25 ℃, the ventilation ratio is 1:1.8, and after the fermentation at 450rpm is finished, the yield of the echinocandin B reaches 1600 mg/L.

Example 10

Changing the culture medium of the comparative example 1 into the culture medium 8 in the table 1, inoculating the seed solution into a 5L fermentation tank filled with 3L fermentation echinocandin B synthetic culture medium according to the inoculation amount with the volume concentration of 10% for fermentation culture, and performing fermentation culture for 240h, wherein the fermentation temperature is controlled to be 25 ℃, the ventilation ratio is 1:1.5, 550rpm is controlled, and the dissolved oxygen is maintained to be more than 25% in the whole fermentation culture process. After the fermentation is finished, the yield of echinocandin B reaches 1950 mg/L.

Example 11

Changing the culture medium of the comparative example 1 into the culture medium 8 in the table 1, inoculating the seed solution into a 50L fermentation tank filled with 30L fermentation echinocandin B synthetic culture medium according to the inoculation amount with the volume concentration of 5% for fermentation culture, and performing fermentation culture for 240h, wherein in the whole fermentation culture process, the fermentation temperature is controlled to be 25 ℃, the ventilation ratio is 1:1.2, and the stirring speed is 250 rpm. After the fermentation is finished, the yield of echinocandin B reaches 1800 mg/L.

Table 2: yield of echinocandin B in different examples

In the control group, ammonium sulfate was used as an inorganic nitrogen source, and the yield of ECB was significantly lower than that of the experimental group. In an experimental group, ammonium sulfate is replaced by more proper potassium nitrate, and the addition amounts of methyl oleate and oleic acid are optimized, so that the yield of echinocandin B is remarkably improved. Subsequently, the yield of ECB is further improved by optimizing the addition amount of the screened glutamic acid and histidine, so that the yield of echinocandin B is improved from 450mg/L to 1800 mg/L. The subsequent culture in 5L and 50L fermentation tanks with optimized synthetic culture medium has echinocandin B yield over 1600 mg/L.

Sequence listing

<110> Zhejiang industrial university

<120> echinocandin B synthetic medium and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 541

<212> DNA

<213> Aspergillus nidulans (Aspergillus nidulans)

<400> 1

gattcgaggt gcgggctgcc tccgggcgcc cacctcccac ccgtgactac ctaacactgt 60

tgcttcggcg gggagccccc caggggcgag ccgccgggga ccactgaact tcatgcctga 120

gagtgatgca gtctgagcct gaatacaaat cagtcaaaac tttcaacaat ggatctcttg 180

gttccggcat cgatgaagaa cgcagcgaac tgcgataagt aatgtgaatt gcagaattca 240

gtgaatcatc gagtctttga acgcacattg cgccccctgg cattccgggg ggcatgcctg 300

tccgagcgtc attgctgccc tcaagcccgg cttgtgtgtt gggtcgtcgt cccccccggg 360

ggacgggccc gaaaggcagc ggcggcaccg tgtccggtcc tcgagcgtat ggggctttgt 420

cacccgctcg attagggccg gccgggcgcc agccggcgtc tccaacctta tttttctcag 480

gttgacctcg gatcaggtag ggatacccgc tgaacttaag catatcaata agcggaggaa 540

g 541