CN106337073B - Fermentation medium and method for increasing output of nikkomycin - Google Patents
Fermentation medium and method for increasing output of nikkomycin Download PDFInfo
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- CN106337073B CN106337073B CN201510409028.5A CN201510409028A CN106337073B CN 106337073 B CN106337073 B CN 106337073B CN 201510409028 A CN201510409028 A CN 201510409028A CN 106337073 B CN106337073 B CN 106337073B
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Abstract
The invention relates to the field of microorganisms, and particularly provides a streptomyces circulans fermentation medium and a method for preparing nikkomycin by using the medium. The method improves the yield of nikkomycin by adding a proper amount of threonine into the fermentation culture medium, further optimizing the types and the amounts of specific components of the fermentation culture medium and controlling other culture conditions. The yield of the nikkomycin can reach 42800 mu g/mL, and the method has the advantages of simple scheme and low cost and is suitable for industrial large-scale production.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly provides a streptomyces circulans fermentation medium and a method for preparing nikkomycin by using the medium.
Background
Nikkomycin (nikkomycin) is a nucleoside peptide antibiotic, and can effectively inhibit the synthesis of chitin, one of the skeleton components of fungal cell wall, so as to effectively inhibit the growth of fungi, insects and insects. The nikkomycin fermentation liquor contains more than twenty components, wherein nikkomycin Z and X are main bioactive components. Nikkomycin is easy to degrade in nature and has no residue; is relatively nontoxic to mammals, bees, plants, etc. As an inhibitor of fungal cell wall chitin synthetase, nikkomycin has wide application prospect in preventing and treating animal and plant and human fungal infectious diseases. In agriculture, nikkomycin has a great market for the control of plant insects and certain fungal diseases.
At present, the method for preparing nikkomycin in the prior art has the problems of low yield and large raw material consumption.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a streptomyces circulans fermentation medium and a method for preparing nikkomycin by using the medium.
The Streptomyces circulans BJX005 used in the invention has been preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of Xilu 1 of Beijing Kogyang, institute of microbiology, Chinese academy of sciences, zip code 100101) at 2.5.2015, the preservation number is CGMCC No.10523, and the Streptomyces circulans and Streptomyces chromogenes are classified and named.
The streptomyces chromogenes BJX005 strain is coiled, and the spore silk is hooked or the coil is rarely looped. The spore is spherical and elliptical.
The invention provides a method for preparing nikkomycin by utilizing streptomyces circineochromogenes BJX005 with the preservation number of CGMCC No.10523, which specifically comprises the following steps:
step (1): inoculating a seed solution of Streptomyces circulans (BJX) 005 with the preservation number of CGMCC No.10523 into a fermentation culture medium containing threonine with the mass concentration of 0.0001-0.0005% for fermentation culture to obtain a fermentation liquid;
step (2): extracting nikkomycin from the fermentation broth.
The addition of a proper amount of threonine can generate good synergistic effect with other components of the fermentation medium, thereby effectively improving the yield of nikkomycin.
The fermentation medium in the step (1) comprises the following components: 2.5-3% of corn starch, 2-3% of soybean cake powder, 0.2-0.3% of yeast powder, 0.4-0.6% of sodium chloride, 0.04-0.05% of ammonium sulfate, 0.45-0.5% of calcium carbonate, 0.02-0.025% of an antifoaming agent, 0.0001-0.0005% of threonine and the balance of water by mass percentage; adjusting the pH value to 6.3-7.5, preferably 6.8.
Further, the present invention preferably selects that the fermentation medium in step (1) comprises the following components: according to the mass percentage, 3 percent of corn starch, 3 percent of soybean cake powder, 0.2 percent of yeast powder, 0.6 percent of sodium chloride, 0.04 percent of ammonium sulfate, 0.5 percent of calcium carbonate, 0.02 percent of defoaming agent, 0.0003 percent of threonine and the balance of distilled water; the pH was adjusted to 6.8.
The inoculation amount of the seed liquid of the streptomyces chromogenes circulans in the step (1) is 4-10% (v/v).
The range selection of the inoculation amount can ensure sufficient dissolved oxygen, reasonably control the fermentation culture time and increase the fermentation efficiency.
The fermentation culture in the step (1) is shaking culture, the rotation speed of the shaking culture is 200-220 r/min, the fermentation time is 60-80 h, and the fermentation temperature is 25-28 ℃.
As an overall technical solution, the present invention provides a more targeted solution, specifically as follows:
the invention provides a method for producing nikkomycin by fermenting streptomyces circinelloides BJX005, which comprises the following steps:
step (1): inoculating the seed liquid of the streptomyces chromagens into a fermentation culture medium by an inoculation amount of 4-10% for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: according to the mass percentage, 3 percent of corn starch, 3 percent of soybean cake powder, 0.2 percent of yeast powder, 0.6 percent of sodium chloride, 0.04 percent of ammonium sulfate, 0.5 percent of calcium carbonate, 0.02 percent of polysiloxane antifoaming agent, 0.0003 percent of threonine and the balance of water; adjusting the pH to 6.8;
step (2): extracting nikkomycin from the fermentation broth.
The streptomyces circulans BJX005 strain adopted by the invention has good genetic stability, and the capability of producing nikkomycin is kept at the original level after continuous passage for 5 times. According to the invention, the proper amount of threonine is added into the fermentation medium, the types and the use amounts of all the components of the fermentation medium are optimized, and other culture conditions are controlled, so that all the components of the fermentation medium generate good synergistic effect, and the yield of nikkomycin is obviously improved. The yield of the nikkomycin can reach 42800 mu g/mL, the scheme is simple, and the cost is low; therefore, the method for preparing nikkomycin is suitable for popularization and application in the industrial production of nikkomycin.
The invention also provides a fermentation medium, which comprises the following components in percentage by mass: 2.5-3% of corn starch, 2-3% of soybean cake powder, 0.2-0.3% of yeast powder, 0.4-0.6% of sodium chloride, 0.04-0.05% of ammonium sulfate, 0.45-0.5% of calcium carbonate, 0.02-0.025% of defoaming agent, 0.0001-0.0005% of threonine and the balance of water. The fermentation medium can be applied to the production of nikkomycin.
The products of the invention are all sold in the market, the concentration is mass concentration, and the inoculation amount is volume ratio.
Detailed Description
The preservation number used by the invention is CGMCC No.10523 Streptomyces chromogenes (BJX 005), which has been preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu No.1 of North Cheng of the sunward area in Beijing, institute of microbiology in China, zip code 100101) 2 month and 5 days 2015, the preservation number is CGMCC No.10523, and the product is classified and named as Streptomyces chromogenes (Streptomyces and chromogenes).
The products in the examples are all commercially available products, wherein threonine is purchased from bio-engineering of huge dragon, Henan, Inc., polysiloxane defoamer DF-805, from Defeng defoamer factory, Dongguan, Guangdong; the alternaria alternate is used as an indicator bacterium for biological detection, and the strain is obtained by collection and separation in a tobacco field.
Example 1
The embodiment specifically provides a method for preparing nikkomycin by fermenting streptomyces coiorchromogenes BJX005, which comprises the following steps:
step (1): inoculating the seed liquid of the streptomyces chromagens into a fermentation culture medium by an inoculation amount of 4% for fermentation culture to obtain a fermentation liquid; the culture medium comprises the following components: according to the mass percentage, 3 percent of corn starch, 3 percent of soybean cake powder, 0.2 percent of yeast powder, 0.6 percent of sodium chloride, 0.04 percent of ammonium sulfate, 0.5 percent of calcium carbonate, 0.0003 percent of polysiloxane antifoaming agent DF-8050.02 percent, 0.0003 percent of threonine and the balance of water; adjusting the pH to 6.8; the fermentation culture is shaking culture, the rotation speed of the shaking culture is 220r/min, the fermentation time is 70h, and the fermentation temperature is 28 ℃;
step (2): extracting nikkomycin from the fermentation broth.
The content of nikkomycin prepared in the embodiment is 42800 mug/mL by detection.
Example 2
This example specifically provides a method for preparing nikkomycin by fermenting Streptomyces circulans BJX 005. Comprises the following steps:
step (1): inoculating the seed liquid of the streptomyces chromagens into a fermentation culture medium by 10% of inoculation amount for fermentation culture to obtain fermentation liquid; the fermentation medium comprises the following components: according to mass percentage, 2.5 percent of corn starch, 2 percent of soybean cake powder, 0.3 percent of yeast powder, 0.4 percent of sodium chloride, 0.05 percent of ammonium sulfate, 0.45 percent of calcium carbonate, 0.025 percent of polysiloxane antifoaming agent SXP-101-1 (purchased from Huarun chemical Co., Ltd. of Highua, Anhui), 0.0001 percent of threonine and the balance of water; adjusting the pH to 7.5; the fermentation culture is shaking culture, the rotation speed of the shaking culture is 200r/min, the fermentation time is 80h, and the fermentation temperature is 25 ℃;
step (2): extracting nikkomycin from the fermentation broth.
The content of the dactinomycin prepared in the embodiment is 41400 mu g/mL through detection.
Example 3
This example specifically provides a method for preparing nikkomycin by fermenting Streptomyces circulans BJX 005. Comprises the following steps:
step (1): inoculating the seed liquid of the streptomyces chromagens into a fermentation culture medium by 6% of inoculation amount for fermentation culture to obtain fermentation liquid; the fermentation medium comprises the following components: according to the mass percentage, 2.5 percent of corn starch, 2.5 percent of soybean cake powder, 0.25 percent of yeast powder, 0.5 percent of sodium chloride, 0.05 percent of ammonium sulfate, 0.45 percent of calcium carbonate, 0.025 percent of GPE type polyether defoamer namely foam enemy (purchased from the south-Ji Australian chemical industry Co., Ltd.), 0.0005 percent of threonine and the balance of water; adjusting the pH to 6.3; the fermentation culture is shaking culture, the rotation speed of the shaking culture is 210 r/min, the fermentation time is 60h, and the fermentation temperature is 27 ℃;
step (2): extracting nikkomycin from the fermentation broth.
The content of the dactinomycin prepared in the embodiment is 42000 mu g/mL by detection.
Example 4
This example provides a fermentation medium, which comprises the following components: the soybean cake flour is prepared from 3% of corn starch, 3% of soybean cake flour, 0.3% of yeast powder, 0.6% of sodium chloride, 0.05% of ammonium sulfate, 0.5% of calcium carbonate, 0.025% of an antifoaming agent, 0.0005% of threonine and the balance of water.
Example 5
This example provides a fermentation medium, which comprises the following components: according to the mass percentage, the corn starch is 2.5 percent, the soybean cake powder is 2 percent, the yeast powder is 0.2 percent, the sodium chloride is 0.4 percent, the ammonium sulfate is 0.04 percent, the calcium carbonate is 0.45 percent, the defoaming agent is 0.02 percent, the threonine is 0.0001 percent, and the balance is water.
Comparative example 1
The only difference compared to example 1 is that the fermentation medium of this example does not contain threonine, and the conditions are otherwise the same.
The content of nikkomycin prepared in the embodiment is 40000 mug/mL by detection.
Comparative example 2
The only difference from example 1 is that the amount of threonine used in step (2) of this example was controlled so that the concentration thereof in the fermentation medium was 0.01% by mass.
The content of the nikkomycin prepared in the embodiment is 39000 mu g/mL by detection.
Experiments show that: the fermentation medium component contains threonine, and the dosage of the threonine is controlled so that the yield of the nikkomycin can be effectively improved under the comprehensive action of the threonine and other fermentation medium components when the mass concentration of the threonine in the fermentation medium is 0.0001-0.0005%.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for preparing nikkomycin by using streptomyces ansochromogenes is characterized by comprising the following steps:
step (1): inoculating a seed solution of Streptomyces circulans (BJX) 005 with the preservation number of CGMCC No.10523 into a fermentation culture medium containing threonine with the mass concentration of 0.0001-0.0005% for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: 2.5-3% of corn starch, 2-3% of soybean cake powder, 0.2-0.3% of yeast powder, 0.4-0.6% of sodium chloride, 0.04-0.05% of ammonium sulfate, 0.45-0.5% of calcium carbonate, 0.02-0.025% of an antifoaming agent, 0.0001-0.0005% of threonine and the balance of water by mass percentage; adjusting the pH value to 6.3-7.5;
step (2): extracting nikkomycin from the fermentation broth.
2. The method of claim 1, wherein the fermentation medium in step (1) has a pH of 6.8.
3. The method of claim 2, wherein the fermentation medium in step (1) is composed of: according to the mass percentage, 3 percent of corn starch, 3 percent of soybean cake powder, 0.2 percent of yeast powder, 0.6 percent of sodium chloride, 0.04 percent of ammonium sulfate, 0.5 percent of calcium carbonate, 0.02 percent of defoaming agent, 0.0003 percent of threonine and the balance of distilled water; the pH was adjusted to 6.8.
4. The method according to any one of claims 1 to 3, wherein the inoculum size of the Streptomyces circochromogenes seed solution in step (1) is 4-10% (v/v).
5. The method according to claim 1, wherein the fermentation culture in step (1) is shaking culture, the rotation speed of the shaking culture is 200-220 r/min, the fermentation time is 60-80 h, and the fermentation temperature is 25-28 ℃.
6. The method according to claim 4, wherein the fermentation culture in the step (1) is shaking culture, the rotation speed of the shaking culture is 200-220 r/min, the fermentation time is 60-80 h, and the fermentation temperature is 25-28 ℃.
7. The method of claim 1, comprising the steps of:
step (1): inoculating the seed liquid of the streptomyces circulans BJX005 into a fermentation culture medium in an inoculation amount of 4-10% (v/v) for fermentation culture to obtain a fermentation liquid; the fermentation medium comprises the following components: according to the mass percentage, 3 percent of corn starch, 3 percent of soybean cake powder, 0.2 percent of yeast powder, 0.6 percent of sodium chloride, 0.04 percent of ammonium sulfate, 0.5 percent of calcium carbonate, 0.02 percent of polysiloxane antifoaming agent, 0.0003 percent of threonine and the balance of water; adjusting the pH to 6.8;
step (2): extracting nikkomycin from the fermentation broth.
8. The application of the fermentation medium in producing nikkomycin by Streptomyces chromagens (BJX 005) with the preservation number of CGMCC No. 10523; the fermentation medium comprises the following components: 2.5-3% of corn starch, 2-3% of soybean cake powder, 0.2-0.3% of yeast powder, 0.4-0.6% of sodium chloride, 0.04-0.05% of ammonium sulfate, 0.45-0.5% of calcium carbonate, 0.02-0.025% of defoaming agent, 0.0001-0.0005% of threonine and the balance of water.
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| CN1406275A (en) * | 2000-12-30 | 2003-03-26 | 第一制糖株式会社 | Microorganism producing L-lysine and processes for producing L-lysine usi ng the same |
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| CN1071700A (en) * | 1991-10-18 | 1993-05-05 | 江西农业大学 | Method for producing Nanchang mycin by fermenting Nanchang streptomycete |
| CN1195054C (en) * | 2001-09-06 | 2005-03-30 | 中国科学院微生物研究所 | Nikemycin Z component engineering bacterium and its application |
| CN1262641C (en) * | 2003-05-07 | 2006-07-05 | 中国科学院微生物研究所 | High-yield engineering bacterium of nicomycin X component and its application |
| CN100347302C (en) * | 2004-08-11 | 2007-11-07 | 中国科学院微生物研究所 | Nikkomycin biological synthesis regulating gene and its coded protein and engineering bacterium for expression |
| CN101591635B (en) * | 2009-07-02 | 2011-02-02 | 中国科学院微生物研究所 | Recombinant streptomyces ansochromogenes, preparation method thereof and use thereof |
| CN101591634B (en) * | 2009-07-02 | 2011-02-02 | 中国科学院微生物研究所 | Method for producing nikkomycin Z, special culture medium of nikkomycin Z and special engineering bacteria of nikkomycin Z |
| CN106148214B (en) * | 2015-03-23 | 2019-08-30 | 牡丹江佰佳信生物科技有限公司 | A kind of streptomyces ansochromogenes and the method for preparing Nikemycin |
| CN106148459B (en) * | 2015-04-27 | 2020-06-09 | 牡丹江佰佳信生物科技有限公司 | Fermentation medium and fermentation method of nikkomycin |
| CN106701867B (en) * | 2015-07-13 | 2020-10-23 | 牡丹江佰佳信生物科技有限公司 | Method for improving output of nikkomycin |
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Denomination of invention: A Fermentation Medium and Method for Improving Nicomycin Production Effective date of registration: 20230522 Granted publication date: 20200207 Pledgee: Mudanjiang New Area Branch of Longjiang Bank Co.,Ltd. Pledgor: MUDANJIANG BIOSEEN BIOLOGICAL CO.,LTD. Registration number: Y2023230000041 |
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