CN112391432A - Fermentation medium and fermentation method for producing pingyangmycin - Google Patents

Fermentation medium and fermentation method for producing pingyangmycin Download PDF

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CN112391432A
CN112391432A CN201910763728.2A CN201910763728A CN112391432A CN 112391432 A CN112391432 A CN 112391432A CN 201910763728 A CN201910763728 A CN 201910763728A CN 112391432 A CN112391432 A CN 112391432A
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CN112391432B (en
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李继安
林惠敏
卢雪欢
张建斌
李亚军
邓旭
郭瑞玲
孟宪纬
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Shanghai Institute of Pharmaceutical Industry
China State Institute of Pharmaceutical Industry
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Abstract

The invention discloses a fermentation medium and a fermentation method for producing pingyangmycin. The fermentation medium comprises a carbon source, a nitrogen source, inorganic salt and water, wherein the content of the nitrogen source is 4.5-5.9%, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium. The fermentation culture medium of the invention is used for fermentation of pingyangmycin, which can greatly improve the yield of pingyangmycin (the yield can reach 2.47 times of the yield obtained by using the fermentation culture medium in the prior art), and simultaneously, the fermentation cost is obviously reduced, thereby laying a good foundation for the industrialized production of pingyangmycin.

Description

Fermentation medium and fermentation method for producing pingyangmycin
Technical Field
The invention belongs to the technical field of industrial microorganisms, and particularly relates to a fermentation medium and a fermentation method for producing pingyangmycin.
Background
Influence of organic nitrogen sources on the modification of fermentation components of spectinomycin in Shanghai third pharmaceutical factory (Yu anxiety, longevity, plum blossom)]Microbiological reports, 1980,7(4):14-17.) in 1976 that the addition of corn steep liquor significantly increased the fermentation titer of pingyangmycin, which was then found to be related to the presence of spermidine in the corn steep liquor. The conditions influencing the fermentation of pingyangmycin are studied by a shaking test, and the fermentation conditions are 0.5 percent of glucose, 7 percent of maltose, 0.3 percent of NaCl0.3 percent of NaNO3 0.2％，KH2PO4 0.1％、ZnSO4·7H2O 0.05％、CuSO4The shake flask fermentation was carried out under the conditions of 0.01%, pH6.5, 150ml/750ml triangular flask, and the results indicated that feeding of corn steep liquor during the fermentation (1.5% of corn steep liquor fed at 48 hours from the beginning of the fermentation, and 1.5% fed every 24 hours thereafter) promoted the formation of component A5, making A5 the major component, increasing from 14.8% to 56.8%, regardless of the strain used. In addition, researches show that in experiments of proportioning other organic nitrogen sources in the basic culture medium, other formulas are unchanged, corn steep liquor is supplemented under the conditions of 2.5% of peanut cake powder and 2.5% of cicada pupa powder, and the yield can be improved by 3 times. The pingyangmycin detection method in the document uses thin-layer chromatography, and compared with the existing liquid phase detection method, the thin-layer chromatography method has low accuracy and larger interference, and the referential significance of the pingyangmycin fermentation titer is not large.
In precursor experiments, it was found by Gaoqian (Fujii, A et al [ J ] Antibot.27: 73,1979) et al that the addition of spermidine at the beginning of the fermentation resulted in 100% A5 of the bleomycin-producing strain, followed by sterile filtration of spermidine hydrochloride and addition of 0.36mg/ml to the fermentation medium before inoculation, resulting in a 466% increase in A5 over the control. And the main component is A5. However, the precursor used in the method is expensive and has high production cost, so that the production requirement cannot be met.
Disclosure of Invention
The invention aims to overcome the defects of high production cost, difficult expanded production and the like of fermentation of pingyangmycin in the prior art and provide a fermentation medium and a fermentation method for producing pingyangmycin.
The invention adopts the following technical scheme to solve the technical problems:
the invention provides a fermentation medium for producing pingyangmycin, which comprises a carbon source, a nitrogen source, inorganic salt and water, wherein the content of the nitrogen source is 4.5-5.9%, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium.
Preferably, the content of the corn steep liquor is 1.2-2.0%, such as 1.4%, 1.6% or 1.8%; the percentage is the mass percentage of the fermentation medium.
Wherein, the content of the soybean cake powder is preferably 1.1 to 3.5%, more preferably 1.7 to 2.9%, further more preferably 1.9 to 2.7%, such as 2.1%, 2.3% or 2.5%; the percentage is the mass percentage of the fermentation medium.
The soybean cake powder is preferably cold-pressed soybean cake powder.
The nitrogen source as described above preferably further comprises one or more selected from the group consisting of cottonseed meal, peanut meal, albumen powder, feather meal, bran, medium temperature soybean meal, yeast powder and hot-pressed soybean meal, preferably peanut meal and yeast powder.
The content of the peanut cake is preferably 0.6-2.4%, preferably 0.8-1.6%, such as 1.0%, 1.2% or 1.4%, and the percentage is the mass percentage of the fermentation medium.
Preferably, the total content of the peanut meal cake and the cold-pressed soybean cake powder is 3.5 percent, and the percentage is the mass percentage of the fermentation medium.
In the present invention, the content of the carbon source may be conventional in the art; preferably, the content of the carbon source is 3.8-9.8%, and the percentage is the mass percentage of the fermentation medium.
The carbon source preferably comprises glucose and starch, and the content of the glucose is 0.3-0.8%, preferably 0.5%; the content of the starch is 3.5-9%, preferably 6%, and the percentage is the mass percentage of the fermentation medium; the starch is preferably alpha-amylase liquefied starch or alpha-amylase gelatinized starch.
The inorganic salt described in the present invention preferably includes NaNO3、KH2PO4、ZnSO4·7H2O and CuSO4·5H2O; wherein:
the NaNO3The content of (C) is preferably 0.2%, and the KH is2PO4The content of (b) is preferably 0.1%, and the ZnSO4·7H2The content of O is preferably 0.05%, and the CuSO is4·5H2The content of O is preferably 0.01 percent, and the percentage is the mass percentage of the fermentation medium.
In a preferred embodiment of the invention, the fermentation medium comprises 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), NaNO30.2％、KH2PO4 0.1％、ZnSO4·7H20.05% of O and CuSO4·5H20.01 percent of O, wherein the percentage is the mass percentage of the fermentation medium.
The invention provides an application of the fermentation medium in fermentation production of pingyangmycin.
The invention provides a fermentation method of pingyangmycin, which comprises the following steps:
(1) inoculating seed solution of Streptomyces pingyangensis (Streptomyces pingyangensis n.sp) into the fermentation culture medium, and performing liquid fermentation culture to obtain fermentation broth;
(2) separating the pingyangmycin from the fermentation liquor.
The Streptomyces verticillatus Pingyang variety in the step (1) is preferably the Streptomyces verticillatus Pingyang variety with the number of CPCC 200554.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the fermentation culture medium of the invention is used for fermentation of pingyangmycin, which can greatly improve the yield of pingyangmycin, the titer can be improved from the original 13 mu g/ml to 32.11 mu g/ml (namely, the yield can reach 2.47 times of the yield obtained by using the fermentation culture medium in the prior art), and simultaneously, the fermentation cost is obviously reduced, and a good foundation is laid for the industrial production of pingyangmycin.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
First, experimental material
1. Instrument for measuring the position of a moving object
Figure BDA0002171219930000041
2. Reagent
Figure BDA0002171219930000042
Figure BDA0002171219930000051
3. Bacterial strain
The invention adopts the strain Streptomyces pingyangensis n.sp purchased from China pharmaceutical microorganism strain preservation management center (CPCC) and the strain number is CPCC 200554.
4. Culture medium
A first culture medium: slant medium (g/L): soluble starch (20g), KNO3(1g)，K2HPO4(0.5g)，MgSO4·7H2O(0.5g)，NaCl(0.5g)，FeSO4·7H2O (0.01g), corn steep liquor (0.5g), agar 20g, and pH of 7.4-7.6
And (2) culture medium II: seed fermentation culture medium: 3% of corn steep liquor, 2% of soybean cake powder, 0.2% of pomegranate flower yeast powder, 1% of glucose, 1% of maltodextrin and KH2PO4 0.1％，ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％；pH 6.5。
And (3) culture medium III: basic fermentation medium: 0.75% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of durian yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO40.1％,ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％；pH 6.5。
Second, the method
1. Culture method
The invention comprises the following processes:
and (3) sterilization conditions: 121 ℃ for 20min
Under the aseptic condition, a freeze-dried tube in which the altemaria verticillata sun-exposed variety is preserved is inoculated into an eggplant bottle filled with a slant culture medium by using a sterilized inoculating needle for culture, the surface light white thalli can be seen on the next day of slant culture, the color is deepened on the third day or the fourth day, a small amount of white spores are seen to be generated, the culture is continued until a large amount of white spores are seen to be generated on the surface of the culture medium, and the culture medium body is changed into ginger yellow due to the generation of the altemaria verticillata sun-exposed variety pigment. The slant can be used for passage or inoculation.
Preparing a seed culture medium according to a seed culture medium formula, slightly scratching a slant culture medium with spores by using a sterilization inoculating shovel under an aseptic condition, digging a slant culture medium which is as thin as possible and is full of spores and about 0.8cm multiplied by 1.5cm, inoculating the slant culture medium into the killed seed culture medium, shaking at 29 ℃ for 24-28 hours at 240rpm, changing the color of a culture in a shaking bottle from dark brown to yellow turbid, shaking, having a flowing sense, having high viscosity and uniformly sliding down adherent walls. The hyphae are clustered and spread to the periphery, are long and free of foreign bacteria, and have the pH value of 6.9-7.0.
Inoculating the cultured shake flask seeds into a sterilized fermentation medium, wherein the inoculation amount is 3ml/35ml, the shaking table is 29 ℃, the rotation speed is 240rpm, and the days are 8-9. The fermentation shake flask becomes thick and deepens color in about 24 hours, a layer of white spore layer appears on the upper wall of the shake flask in 3-4 days, the viscosity of the shake flask is not reduced in 8-9 days, microscopic examination shows that hyphae are broken more, dense vacuoles are generated in the hyphae, the hyphae are thin and weak, and the condition that the hyphae enter the later stage of secondary metabolism and the pH value is 7.0-8.0 is shown.
In the invention, the conditions except the nitrogen source in the three formulas of the culture medium are not changed, and multiple shake flask fermentation experiments are carried out by increasing the types of the nitrogen source and adjusting the proportion and the total amount of the corresponding nitrogen source, so that the fermentation culture medium formula with higher yield is finally obtained.
2. Detection method
Centrifuging the fermentation liquor at 12000rpm, taking the supernatant, adding methanol for precipitating protein impurities according to the ratio of the supernatant to the methanol being 1:3, centrifuging at 12000rpm, taking the supernatant, and measuring the fermentation unit by using a high performance liquid chromatography.
1) Mobile phase: mobile phase a ═ hexane sodium sulfonate 7.53g/L, disodium EDTA 3.72g/L, acetic acid 0.08mol/L, pH adjusted to 4.3 with pure ammonia, mobile phase B ═ methanol-acetonitrile (70-30)
2) Liquid phase conditions: a chromatographic column: agilent Eclipse Plus C18(4.6 mm. times.250 mm, 5 μm);
the flow rate is 1.0 mL/min; injecting 20 mu L of sample; the detection wavelength is 254 nm; the column temperature is 40 ℃; gradient elution.
Unless otherwise specified, "%" referred to in the following examples is a mass percentage of a specific component based on the total mass of the medium.
The starch (alpha-amylase liquefaction) in the following examples can be replaced by alpha-amylase gelatinized starch, and the effect of the culture medium containing the starch and the starch is basically the same under the condition that other components are kept unchanged.
Example 1
Taking the basic fermentation medium (the third culture medium) as a reference, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salts and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 0.75% to 1%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 1
Culture medium III Example 1
Corn steep liquor dosage (%) 0.75 1
Relative potency 1.00 1.04
The optimized formula comprises: 1% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H20.05% of O and CuSO4·5H2O 0.01％。
The titer obtained by fermentation using the above medium III was 13. mu.g/ml.
Example 2
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1% to 1.2%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 2
Corn steep liquor dosage (%) 1 1.2
Relative potency 1.00 1.12
The optimized formula comprises: 1.2% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 3
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.2% to 1.4%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 3
Corn steep liquor dosage (%) 1.2 1.4
Relative potency 1 1.09
The optimized formula comprises: 1.4% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 4
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.4% to 1.6%, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 4
Corn steep liquor dosage (%) 1.4 1.6
Relative potency 1 1.18
The optimized formula comprises: 1.6% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 5
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.6% to 1.8%, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 5
Corn steep liquor dosage (%) 1.6 1.8
Relative potency 1 1.12
The optimized formula comprises: 1.8% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO40.1％、ZnSO4·7H2O 0.05％、CuSO4·5H2O 0.01％。
Example 6
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.8% to 2.0%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 6
Corn steep liquor dosage (%) 1.8 2.0
Relative potency 1 1.17
The optimized formula comprises: 2.0% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H2O 0.05％、CuSO4·5H2O 0.01％。
Example 7
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 2.0% to 2.2%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 7
Corn steep liquor dosage (%) 2.0 2.2
Relative potency 1 1.13
The optimized formula comprises: 2.2% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 8
Taking the optimized culture medium formula as a reference, under the condition that components such as a carbon source, inorganic salts and the like are kept unchanged, under the condition that the contents of corn steep liquor and the pomegranate flower yeast powder are unchanged, the content of cold-pressed soybean cake powder in the basic culture medium is changed from 3.5 percent to 2.5 percent, the rest 1 percent of cold-pressed soybean cake powder is subjected to substitution experiments by adopting cottonseed powder, peanut cake powder, pomegranate flower yeast powder, protein powder, feather meal, bran, medium-temperature soybean cake powder and hot-pressed soybean cake powder, and the mixture is subjected to shake flask fermentation for 8 days and a high-efficiency liquid phase fermentation unit is adopted.
TABLE 8
Figure BDA0002171219930000091
The optimized formula comprises: 2.2% of corn steep liquor, 2.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO40.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 9
Taking the optimized culture medium formula as a reference, performing an experiment for adjusting the content ratio of the cold-pressed soybean cake powder to the peanut cake powder under the condition that the total content of the cold-pressed soybean cake powder and the peanut cake powder is 3.5% under the condition that the components such as carbon source, inorganic salt, durian yeast powder and the like are kept unchanged, performing shake-flask fermentation for 8 days, and measuring a fermentation unit by using a high performance liquid phase.
TABLE 9
Peanut cake powder (%) 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
Relative potency 0.97 0.98 1.00 1.01 1.03 0.98 0.95 0.92 0.86 0.78
According to the results of the above experiments, the optimized formula is as follows: 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO40.1％、ZnSO4·7H2O 0.05％、CuSO4·5H2O 0.01％。
The titer obtained after the final fermentation using this optimized formulation was 32.11. mu.g/ml.

Claims (10)

1. A fermentation medium for producing pingyangmycin comprises a carbon source, a nitrogen source, inorganic salt and water, and is characterized in that the content of the nitrogen source is 4.5-5.9%, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium.
2. The fermentation medium of claim 1, wherein the corn steep liquor is present in an amount of 1.2 to 2.0%, such as 1.4%, 1.6% or 1.8%; the percentage is the mass percentage of the fermentation medium.
3. The fermentation medium of claim 1, wherein the soybean meal is present in an amount of 1.1 to 3.5%, preferably 1.7 to 2.9%, more preferably 1.9 to 2.7%, such as 2.1%, 2.3% or 2.5%; the percentage is the mass percentage of the fermentation medium;
and/or the soybean cake powder is cold-pressed soybean cake powder.
4. The fermentation medium of claim 3, wherein the nitrogen source further comprises one or more selected from the group consisting of cottonseed meal, peanut meal, protein meal, feather meal, bran, medium temperature soybean meal, yeast meal, and hot pressed soybean meal, preferably peanut meal and yeast meal.
5. The fermentation medium of claim 4, wherein the peanut meal cake is present in an amount of 0.6 to 2.4%, preferably 0.8 to 1.6%, such as 1.0%, 1.2% or 1.4%, by weight of the fermentation medium; preferably, the total content of the peanut meal cake and the cold-pressed soybean cake powder is 3.5 percent, and the percentage is the mass percentage of the fermentation medium.
6. The fermentation medium of claim 1, wherein the carbon source is present in an amount of 3.8 to 9.8% by weight of the fermentation medium;
preferably, the carbon source comprises glucose and starch, and the content of the glucose is 0.3-0.8%, preferably 0.5%; the content of the starch is 3.5-9%, preferably 6%, and the percentage is the mass percentage of the fermentation medium;
more preferably, the starch is alpha-amylase liquefied starch or alpha-amylase gelatinized starch.
7. The fermentation medium of claim 1, wherein the inorganic salt comprises NaNO3、KH2PO4、ZnSO4·7H2O and CuSO4·5H2O; the NaNO3Preferably 0.2%, of saidKH2PO4The content of (b) is preferably 0.1%, and the ZnSO4·7H2The content of O is preferably 0.05%, and the CuSO is4·5H2The content of O is preferably 0.01 percent, and the percentage is the mass percentage of the fermentation medium.
8. The fermentation medium of any one of claims 1 to 7, wherein the fermentation medium comprises 2.2% corn steep liquor, 2.1% cold-pressed soybean meal, 0.2% yeast powder, 1.4% peanut meal, 0.5% glucose, 6% starch (alpha-amylase liquefied), NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H20.05% of O and CuSO4·5H20.01 percent of O, wherein the percentage is the mass percentage of the fermentation medium.
9. Use of a fermentation medium according to any one of claims 1 to 8 for the fermentative production of pingyangmycin.
10. A fermentation method of pingyangmycin is characterized by comprising the following steps:
(1) inoculating seed liquid of Streptomyces pingyangensis (Streptomyces pingyangensis n.sp) into the fermentation medium of any one of claims 1-8, and performing liquid fermentation culture to obtain fermentation liquid; the streptomyces verticillata Pingyang variety is preferably the streptomyces verticillata Pingyang variety with the serial number of CPCC 200554;
(2) separating the pingyangmycin from the fermentation liquor.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114381386A (en) * 2021-11-21 2022-04-22 河北兴柏农业科技有限公司 Culture medium for producing abamectin by fermentation
CN116179527A (en) * 2022-12-28 2023-05-30 山东福洋生物科技股份有限公司 Feed medium and feed method for producing D-psicose-3 epimerase

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101607986A (en) * 2008-06-20 2009-12-23 中国医学科学院医药生物技术研究所 A kind of method of preparing boningmycin by chemical semisynthesis
CN102747115A (en) * 2011-04-19 2012-10-24 上海医药工业研究院 Fermentation medium for fermentation production of antibiotic calicheamicin gamma1 I, fermentation method suitable for the fermentation medium and use of the fermentation medium
US20180057799A1 (en) * 2016-08-26 2018-03-01 Qingdao Yaodong Group Streptomyces sp. strain and method for preparing sulfide oxidase preparation from the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101607986A (en) * 2008-06-20 2009-12-23 中国医学科学院医药生物技术研究所 A kind of method of preparing boningmycin by chemical semisynthesis
CN102747115A (en) * 2011-04-19 2012-10-24 上海医药工业研究院 Fermentation medium for fermentation production of antibiotic calicheamicin gamma1 I, fermentation method suitable for the fermentation medium and use of the fermentation medium
US20180057799A1 (en) * 2016-08-26 2018-03-01 Qingdao Yaodong Group Streptomyces sp. strain and method for preparing sulfide oxidase preparation from the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
虞悝等: "平阳霉素发酵中若干问题的探讨", 《中国抗生素杂志》 *
顾觉奋: "《抗生素》", 30 September 2001, 上海科学技术出版社 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114381386A (en) * 2021-11-21 2022-04-22 河北兴柏农业科技有限公司 Culture medium for producing abamectin by fermentation
CN114381386B (en) * 2021-11-21 2023-12-15 河北兴柏农业科技股份有限公司 Culture medium for producing avermectin through fermentation
CN116179527A (en) * 2022-12-28 2023-05-30 山东福洋生物科技股份有限公司 Feed medium and feed method for producing D-psicose-3 epimerase

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