Fermentation culture medium for improving titer of abamectin and optimization method of fermentation culture medium

Abstract

According to the fermentation culture medium and the optimization method thereof, amino acid chelated cobalt with a proper concentration is added into a streptomyces avermitilis fermentation culture medium, so that trace elements are supplemented, and meanwhile, the fermentation culture medium has the dual functions of an amino acid enhancer, so that the titer of the abamectin is remarkably improved, and the yield of the abamectin is improved. And the method has a very good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibiotic fermentation, and particularly relates to a fermentation medium for improving the titer of avermectin and an optimization method thereof. Background Art

[0002] Avermectin (AVM) is a highly effective and low-toxic biological insecticide produced by Streptomyces avermitilis, and is one of the most concerned biological insecticidal and acaricidal agents today. Compared with traditional agricultural and veterinary drugs, avermectin has the characteristics of high efficiency, broad spectrum, long validity period, difficult to produce drug resistance, easy to degrade, no residue, etc., and is not easy to produce drug resistance, and has no cross-resistance with other insecticides. It degrades quickly in the soil, photolysis is rapid, safe for crops, and not easy to produce phytotoxicity. It is a highly effective and low-toxic agricultural and veterinary drug product recommended by the Ministry of Agriculture and Rural Affairs. Therefore, the development of avermectin has good application value and market prospects.

[0003] In the prior art (CN116478865A, a fermentation medium and fermentation method for increasing the production of avermectin), by adding fish meal to optimize the organic nitrogen source component in the basal medium to increase the yield of B1a, but the problem of this fermentation medium is that fish meal contains rich proteins and various amino acids. Except for the substances used in the biosynthesis of avermectin, the remaining substances are not easily absorbed and utilized, and usually precipitate in the fermentation broth in the form of impurities, indirectly affecting the production of avermectin. The fermentation titer of avermectin in the prior art is low, which not only increases the production cost, but also greatly increases the difficulty of subsequent separation and purification. Therefore, to fundamentally solve the problem of low fermentation titer of avermectin, it is necessary to optimize its fermentation medium with high quality. Summary of the Invention

[0004] Aiming at the problems in the prior art that the utilization rate of the added organic nitrogen source is low, and the excessive impurities in the fermentation broth lead to low fermentation titer of avermectin and great separation difficulty, etc., the present invention provides a fermentation medium for improving the titer of avermectin and an optimization method thereof. This process optimizes the formula of the avermectin fermentation medium, adds amino acid chelated cobalt to the basal medium, and this complex improves the stability of trace elements and promotes the absorption of Co 2+ by avermectin biosynthesis. At the same time, as an amino acid fortifier, it contains various target amino acids for avermectin biosynthesis, improves the biosynthesis ability of avermectin, enhances the toxin-producing ability of Streptomyces avermitilis cells, and makes the titer of avermectin increase by more than 5%.

[0005] The first aspect of the present invention relates to an avermectin fermentation medium, and the fermentation medium includes a basal medium of Streptomyces avermitilis and amino acid chelated cobalt.

[0006] Among them, the concentration of the amino acid chelated cobalt is 2-5 g / L.

[0007] Among them, the addition amount of the amino acid chelated cobalt is 0.2-0.4% of the volume of the fermentation broth.

[0008] Among them, the basic medium of the Streptomyces avermitilis includes: corn starch 180-185 g / L, amylase 0.05-0.06 g / L, medium-temperature soybean cake powder 25-30 g / L, yeast powder 3-6 g / L, calcium carbonate 2-3 g / L, antifoaming agent 5-7 g / L, solid base 0.3-0.5 g / L, ammonium sulfate 0.2-0.4 g / L, sodium molybdate 0.3-0.5 g / L, manganese sulfate 0.2-0.4 g / L.

[0009] Among them, the quality requirements of the fermentation medium after sterilization are: amino nitrogen 45-60 mg / 100 mL, total sugar 14-16 g / 100 mL, pH 6.5-6.8.

[0010] The second aspect of the present invention relates to a method for improving the titer of avermectin in the process of fermenting and producing avermectin by Streptomyces avermitilis, and the method includes: adding amino acid chelated cobalt with a concentration of 2-5 g / L to the basic medium of Streptomyces avermitilis.

[0011] Among them, the basic medium of the Streptomyces avermitilis includes: corn starch 180-185 g / L, amylase 0.05-0.06 g / L, medium-temperature soybean cake powder 25-30 g / L, yeast powder 3-6 g / L, calcium carbonate 2-3 g / L, antifoaming agent 5-7 g / L, solid base 0.3-0.5 g / L, ammonium sulfate 0.2-0.4 g / L, sodium molybdate 0.3-0.5 g / L, manganese sulfate 0.2-0.4 g / L.

[0012] Among them, the fermentation culture conditions are: the inoculation amount is 10%, the temperature is 27°C-29°C, the pH value is 6.4-6.8, the stirring speed is 150 rpm, the air volume is 0.4-0.8 m3 / h, and the culture period is 300-320 h.

[0013] The technical solution of the present invention has at least the following beneficial technical effects:

[0014] By optimizing the optimal ratio of nutrients in the fermentation medium and adding amino acid chelated cobalt to the basic medium, the present invention has a dual role of supplementing trace elements and acting as an amino acid fortifier, providing the required amino acids and Co2+ for the biosynthesis of avermectin, improving the metabolism of Streptomyces avermitilis cells, promoting the ability of avermectin biosynthesis, significantly improving the titer of avermectin, increasing the titer of avermectin by more than 4%, and being applicable to large-scale, stable and efficient fermentation production of avermectin. Specific embodiments

[0015] In the present invention, the strain cultured in the avermectin fermentation medium is generally known to those skilled in the art as Streptomyces avermitilis, preferably Streptomyces avermitilis ATCC 31267. The strain used in the present invention is provided by the company's technology center.

[0016] The detection method (HPLC method) for the avermectin titer in the examples and comparative examples of the present invention: ZORBAX EclipsePlus C18; mobile phase: 90% methanol + 10% phosphoric acid water; flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 10 μL; wavelength of the ultraviolet detector: 245 nm.

[0017] Example 1

[0018] After the Streptomyces avermitilis is matured through seed culture, it is transferred to a fermenter for fermentation culture. The fermentation medium formula is as follows: corn starch 180 g / L, amylase 0.05 g / L, medium-temperature soybean cake powder 25 g / L, yeast powder 3 g / L, calcium carbonate 2 g / L, antifoaming agent 5 g / L, solid base 0.3 g / L, ammonium sulfate 0.2 g / L, sodium molybdate 0.3 g / L, manganese sulfate 0.2 g / L, amino acid chelated cobalt 2 g / L. After the medium is sterilized, the amino nitrogen is 48 mg / 100 mL, the total sugar is 14 g / 100 mL, and the pH is 6.6. The fermenter is discharged at 317 h. When the fermentation ends, the detected titer is 8015 μg / mL.

[0019] Example 2

[0020] After the Streptomyces avermitilis is matured through seed culture, it is transferred to a fermenter for fermentation culture. The fermentation medium formula is as follows: corn starch 182 g / L, amylase 0.05 g / L, medium-temperature soybean cake powder 26 g / L, yeast powder 4 g / L, calcium carbonate 3 g / L, antifoaming agent 6 g / L, solid base 0.4 g / L, ammonium sulfate 0.3 g / L, sodium molybdate 0.3 g / L, manganese sulfate 0.3 g / L, amino acid chelated cobalt 3 g / L. After the medium is sterilized, the amino nitrogen is 47 mg / 100 mL, the total sugar is 15 g / 100 mL, and the pH is 6.6. The fermenter is discharged at 315 h. When the fermentation ends, the detected titer is 8150 μg / mL.

[0021] Example 3:

[0022] After the Streptomyces avermitilis is matured through seed culture, it is transferred to a fermenter for fermentation culture. The formula of the fermentation medium is as follows: corn starch 184 g / L, amylase 0.06 g / L, medium-temperature soybean cake powder 28 g / L, yeast powder 5 g / L, calcium carbonate 3 g / L, antifoaming agent 6 g / L, solid base 0.5 g / L, ammonium sulfate 0.4 g / L, sodium molybdate 0.4 g / L, manganese sulfate 0.4 g / L, amino acid chelated cobalt 4 g / L. After the medium is sterilized, the amino nitrogen is 52 mg / 100 mL, the total sugar is 15 g / 100 mL, and the pH is 6.8. The fermenter is discharged at 318 h, and the titer is detected to be 8036 μg / mL at the end of fermentation.

[0023] Example 4:

[0024] After the Streptomyces avermitilis is matured through seed culture, it is transferred to a fermenter for fermentation culture. The formula of the fermentation medium is as follows: corn starch 185 g / L, amylase 0.06 g / L, medium-temperature soybean cake powder 30 g / L, yeast powder 6 g / L, calcium carbonate 3 g / L, antifoaming agent 7 g / L, solid base 0.5 g / L, ammonium sulfate 0.4 g / L, sodium molybdate 0.5 g / L, manganese sulfate 0.5 g / L, amino acid chelated cobalt 5 g / L. After the medium is sterilized, the amino nitrogen is 50 mg / 100 mL, the total sugar is 15 g / 100 mL, and the pH is 6.7. The fermenter is discharged at 318 h, and the titer is detected to be 8085 μg / mL at the end of fermentation.

[0025] Comparative Example 1:

[0026] After the Streptomyces avermitilis is matured through seed culture, it is transferred to a fermenter for fermentation culture. The formula of the fermentation medium is as follows: corn starch 182 g / L, amylase 0.05 g / L, medium-temperature soybean cake powder 26 g / L, yeast powder 4 g / L, calcium carbonate 3 g / L, antifoaming agent 6 g / L, solid base 0.4 g / L, ammonium sulfate 0.3 g / L, sodium molybdate 0.3 g / L, manganese sulfate 0.3 g / L. After the medium is sterilized, the amino nitrogen is 47 mg / 100 mL, the total sugar is 15 g / 100 mL, and the pH is 6.6. The other culture conditions are the same as those in Example 1. The fermenter is discharged at 317 h, and the titer is detected to be 7580 μg / mL at the end of fermentation.

[0027] Comparative Example 2:

[0028] After the Streptomyces avermitilis was matured through seed culture, it was transferred to a fermenter for fermentation culture. The formula of the fermentation medium was as follows: corn starch 182 g / L, amylase 0.05 g / L, medium-temperature soybean cake powder 26 g / L, yeast powder 4 g / L, calcium carbonate 3 g / L, antifoaming agent 6 g / L, solid base 0.4 g / L, ammonium sulfate 0.3 g / L, sodium molybdate 0.3 g / L, manganese sulfate 0.3 g / L, cobalt chloride (non-chelated) 3 g / L. After the medium was sterilized, the amino nitrogen was 47 mg / 100 mL, the total sugar was 15 g / 100 mL, and the pH was 6.6. The remaining culture conditions were the same as those in Example 1. The fermenter was discharged at 316 h, and the titer was detected to be 7687 μg / mL at the end of fermentation.

[0029] Comparative Example 3:

[0030] After the Streptomyces avermitilis was matured through seed culture, it was transferred to a fermenter for fermentation culture. The formula of the fermentation medium was as follows: corn starch 182 g / L, amylase 0.05 g / L, medium-temperature soybean cake powder 26 g / L, yeast powder 4 g / L, calcium carbonate 3 g / L, antifoaming agent 6 g / L, solid base 0.4 g / L, ammonium sulfate 0.3 g / L, sodium molybdate 0.3 g / L, manganese sulfate 0.3 g / L, threonine (non-chelated) 3 g / L. After the medium was sterilized, the amino nitrogen was 47 mg / 100 mL, the total sugar was 15 g / 100 mL, and the pH was 6.6. The remaining culture conditions were the same as those in Example 1. The fermenter was discharged at 318 h, and the titer was detected to be 7628 μg / mL at the end of fermentation.

Claims

1. An avermectin fermentation medium, characterized in that, The fermentation medium includes the basal medium of Streptomyces avermitilis and cobalt amino acid chelate.

2. The avermectin fermentation medium according to claim 1, characterized in that The concentration of the cobalt amino acid chelate is 2 - 5 g / L.

3. The avermectin fermentation medium according to claim 1, wherein The addition amount of the cobalt amino acid chelate is 0.2 - 0.4% of the volume of the fermentation broth.

4. The avermectin fermentation medium according to claim 1, characterized in that The basal medium of Streptomyces avermitilis includes: corn starch 180 - 185 g / L, amylase 0.05 - 0.06 g / L, medium-temperature soybean cake powder 25 - 30 g / L, yeast powder 3 - 6 g / L, calcium carbonate 2 - 3 g / L, antifoaming agent 5 - 7 g / L, solid base 0.3 - 0.5 g / L, ammonium sulfate 0.2 - 0.4 g / L, sodium molybdate 0.3 - 0.5 g / L, manganese sulfate 0.2 - 0.4 g / L.

5. The avermectin fermentation medium according to claim 1, characterized in that The quality requirements of the fermentation medium after sterilization are: amino nitrogen 45 - 60 mg / 100 mL, total sugar 14 - 16 g / 100 mL, pH 6.5 - 6.

8.

6. A method for increasing the titer of avermectin during the fermentation of Streptomyces avermitilis to produce avermectin, the method comprising: Add cobalt amino acid chelate with a concentration of 2 - 5 g / L to the basal medium of Streptomyces avermitilis.

7. The method according to claim 6, characterized in that The basal medium of Streptomyces avermitilis includes: corn starch 180 - 185 g / L, amylase 0.05 - 0.06 g / L, medium-temperature soybean cake powder 25 - 30 g / L, yeast powder 3 - 6 g / L, calcium carbonate 2 - 3 g / L, antifoaming agent 5 - 7 g / L, solid base 0.3 - 0.5 g / L, ammonium sulfate 0.2 - 0.4 g / L, sodium molybdate 0.3 - 0.5 g / L, manganese sulfate 0.2 - 0.4 g / L.

8. The method according to claim 6, characterized in that The fermentation culture conditions are: inoculation amount 10%, temperature 27°C - 29°C, pH value 6.4 - 6.8, stirring speed 150 rpm, air volume 0.4 - 0.8 m3 / h, culture period 300 - 320 h.