Fermentation method for improving titer of abamectin B1a

Abstract

According to the fermentation method for improving the titer of the abamectin B1a, the rare earth elements are added into the abamectin fermentation culture medium for fermentation culture to produce the abamectin, so that the titer of the abamectin B1a is remarkably improved, and the fermentation method has a good industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibiotic fermentation, and specifically relates to a fermentation method for improving the titer of avermectin B1a. Background Art

[0002] Avermectin (AVM) is a type of sixteen-membered macrolide antibiotic produced by liquid fermentation of Streptomyces avermitilis. It is a brand-new pollution-free bio-agricultural and veterinary medicine. Due to its characteristics of high efficiency, high selectivity, low toxicity, pollution-free, and environmental compatibility, it is highly favored in the pesticide, veterinary medicine, and pharmaceutical industries.

[0003] The prior art has disclosed various methods for improving the titer and yield of avermectin B1a. For example, Liang Jianguang et al. replaced yeast powder with cottonseed cake powder and optimized the industrial complex fermentation medium of avermectin-producing strains by the response surface method. Under the optimized medium conditions, the shake-flask fermentation titer of avermectin B1a was 5235 μg / mL (Liang Jianguang, Chu Xiaohe, Chu Ju, etc. Optimization of the complex fermentation medium of avermectin B1a-producing strains by response surface method [J]. Chinese Journal of Pharmaceutical Industry, 2013, 44(10): 986-988+1004); the Chinese patent document with the publication number CN106609288B disclosed a method for improving the yield of avermectin B1a by optimizing the fermentation medium. After a large number of screenings, it was found that adding 0.1 g / L of calcium chloride to the Streptomyces avermitilis fermentation medium could increase the titer of avermectin B1a to 7200 μg / mL; Tian Pingping et al. used the atmospheric and room temperature plasma (ARTP) mutagenesis technology to screen high-yield strains of avermectin, and its yield increased by 23.4% compared with the original strain (Tian Pingping, Cao Peng, Chang Chuanyou, etc. Mutagenesis breeding of avermectin-producing bacteria by atmospheric and room temperature plasma and medium optimization [J]. Acta Microbiologica Sinica, 2017, 44(01): 150-160); Lv Miaomiao et al. used the double screening method of ultraviolet mutagenesis combined with different types of antibiotics to mutagenize and breed the original strain AW-8 of avermectin. Finally, the fermentation titer of the obtained high-yield strain R4 was 7317 μg / mL, which was 20.92% higher than that of the original strain (Lv Miaomiao, Yang Guangyao, Tian Pingya, etc. Compound mutagenesis of avermectin-producing bacteria [J]. Heilongjiang Agricultural Sciences, 2021(02): 61-65.). Although these methods have effectively improved the fermentation titer and yield of avermectin B1a, due to the increasingly wide application of avermectin, the current yield of avermectin B1a is still difficult to meet the market demand, and mutagenesis breeding has randomness and the mutagenized bacteria have instability in subculture. Therefore, regulating and optimizing the fermentation medium has become an important way to break through the bottleneck problems of the existing technology.

[0004] According to existing research, it has been found that a small amount of rare earth elements not only promote the growth of animals and plants, but also promote the growth of microbial cells and the accumulation of metabolites, and also have the function of improving the level of microbial fermentation to produce antibiotics in the field of antibiotics. However, there is no reported technical method for using rare earth elements to improve the titer of avermectin. Therefore, in the present invention, an appropriate amount of rare earth elements is added to the fermentation medium of Streptomyces avermitilis to enhance the fermentation titer of avermectin, thereby increasing the yield. Summary of the Invention

[0005] The purpose of the present invention is to provide a fermentation method for improving the titer of avermectin B1a. By inoculating Streptomyces avermitilis into a fermentation medium containing rare earth elements for fermentation culture to produce avermectin, the titer of avermectin B1a is effectively enhanced.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A fermentation method for using rare earth elements to improve the titer of avermectin, comprising the following steps: adding rare earth elements to the avermectin fermentation medium to obtain a fermentation medium containing rare earth elements, and then inoculating Streptomyces avermitilis into the fermentation medium containing rare earth elements for fermentation culture to produce avermectin.

[0008] Among them, the avermectin fermentation titer is the fermentation titer of the avermectin B1a component.

[0009] Among them, the rare earth elements are selected from lanthanum La 3+ , europium Eu 3+ or neodymium Nd 3+ .

[0010] Among them, the concentration of the rare earth element lanthanum La 3+ in the fermentation medium is 2 - 12 mg / L.

[0011] Among them, the concentration of the rare earth element europium Eu 3+ in the fermentation medium is 15 - 30 mg / L.

[0012] Among them, the concentration of the rare earth element neodymium Nd 3+ in the fermentation medium is 0.1 - 1.1 mg / L.

[0013] Among them, the composition of the fermentation medium is: corn starch 140 g / L, soybean cake powder 16 g / L, peanut cake powder 11 g / L, yeast powder 8 g / L, ammonium sulfate 0.2 g / L, sodium molybdate 0.02 g / L, manganese sulfate 0.0023 g / L, antifoam agent 4 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, sodium chloride 0.5 g / L, light calcium carbonate 2 g / L, cobalt chloride 0.004 g / L.

[0014] Among them, the fermentation culture conditions are as follows: the culture temperature is 28 ± 1 °C, the tank pressure is 0.05 ± 0.01 MPa, the aeration rate is 1:1, and the fermentation period is 315 ± 15 h.

[0015] The technical solution of the present invention has at least the following beneficial technical effects:

[0016] 1. The rare earth elements lanthanum La3+, europium Eu3+ or neodymium Nd3+ described in the present invention are widely sourced, and have good promoting effects on the material metabolism process of body cells and the activity of enzymes. The experimental method is simple and the repeatability is good.

[0017] 2. The present invention uses the rare earth elements lanthanum La3+, europium Eu3+ or neodymium Nd3+ for the liquid fermentation of avermectin. The fermentation titer of avermectin B1a can reach more than 7800 μg / mL, and the B1a / B1b ratio reaches more than 40, significantly improving the fermentation level of avermectin and having good industrial application prospects. Specific Embodiments

[0018] Through extensive and in-depth research, the inventors unexpectedly found that rare earth elements, especially lanthanum La3+, europium Eu3+ or neodymium Nd3+ have a positive effect on avermectin fermentation, significantly improving the fermentation titer of avermectin B1a.

[0019] The titer of avermectin B1a was determined by high performance liquid chromatography, and the method is as follows:

[0020] Collect 10 mL of the bacterial fermentation broth, place it in a 15 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, discard the supernatant, add methanol to 10 mL, ultrasonicate in the dark for 30 min, then centrifuge at 8000 rpm for 5 min. Take an appropriate amount of the supernatant with a syringe, filter it through a 0.22 μm filter membrane, and store it in a brown bottle, and store it in a -20 °C refrigerator for use.

[0021] Chromatographic conditions: chromatographic column: SB-C18 4.6 × 250 mm, 5 μm; mobile phase: methanol: ultrapure water (v / v) = 90:10; column temperature: 28 °C; flow rate: 0.8 mL / min; injection volume: 10 μl; detection wavelength: 245 nm.

[0022] The strain cultured in the avermectin fermentation medium described in the present invention 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 technical center of the company.

[0023] Example 1

[0024] Add different concentrations of La to the avermectin fermentation medium3+ (As shown in Table 1), after sterilization, a seed solution with a volume ratio of 8% was inoculated into a 50 L fermenter. The fermentation culture conditions were as follows: the culture temperature was controlled at 28 ± 1 °C, the tank pressure was maintained at 0.05 ± 0.01 MPa, the ventilation rate was 1:1, stirring started after 10 ± 1 h of fermentation, the stirring speed was 250 ± 30 rpm, and the fermentation cycle was approximately 300 - 360 h.

[0025] Table 1 Effects of Different Concentrations of La3+ on the Titer of Avermectin B1a and the Ratio of B1a / B1b

[0026]

[0027] Example 2

[0028] Different concentrations of Eu were added to the avermectin fermentation medium 3+ (As shown in Table 2), after sterilization, a seed solution with a volume ratio of 8% was inoculated into a 50 L fermenter. The fermentation culture conditions were as follows: the culture temperature was controlled at 28 ± 1 °C, the tank pressure was maintained at 0.05 ± 0.01 MPa, the ventilation rate was 1:1, stirring started after 10 ± 1 h of fermentation, the stirring speed was 250 ± 30 rpm, and the fermentation cycle was approximately 300 - 360 h.

[0029] Table 2 Effects of Different Concentrations of Eu3+ on the Titer of Avermectin B1a and the Ratio of B1a / B1b

[0030]

[0031] Example 3

[0032] Different concentrations of Nd were added to the avermectin fermentation medium 3+ (As shown in Table 3), after sterilization, a seed solution with a volume ratio of 8% was inoculated into a 50 L fermenter. The fermentation culture conditions were as follows: the culture temperature was controlled at 28 ± 1 °C, the tank pressure was maintained at 0.05 ± 0.01 MPa, the ventilation rate was 1:1, stirring started after 10 ± 1 h of fermentation, the stirring speed was 250 ± 30 rpm, and the fermentation cycle was approximately 300 - 360 h.

[0033] Table 3 Effects of Different Concentrations of Nd3+ on the Titer of Avermectin B1a and the Ratio of B1a / B1b

[0034]

[0035] Comparative Example 1

[0036] Without adding rare earth elements to the avermectin fermentation medium, after sterilization, a seed liquid with a volume ratio of 8% was inoculated into a 50 L fermenter. The fermentation culture conditions were as follows: the culture temperature was controlled at 28 ± 1 °C, the tank pressure was maintained at 0.05 ± 0.01 MPa, the ventilation rate was 1:1, stirring started after fermentation for 10 ± 1 h, the rotation speed was 250 ± 30 rpm, and the fermentation cycle was 315 h.

[0037] The final titer test results showed that the titer of avermectin B1a in the fermentation broth at the end of fermentation was 7089 μg / mL, and the B1a / B1b ratio was 39.36.

[0038] Comparative Example 2

[0039] Iridium Y3+ as a rare earth element was added to the avermectin fermentation medium at an addition concentration of 5 mg / L. After sterilization, a seed liquid with a volume ratio of 8% was inoculated into a 50 L fermenter. The fermentation culture conditions were as follows: the culture temperature was controlled at 28 ± 1 °C, the tank pressure was maintained at 0.05 ± 0.01 MPa, the ventilation rate was 1:1, stirring started after fermentation for 10 ± 1 h, the rotation speed was 250 ± 30 rpm, and the fermentation cycle was about 320 h.

[0040] The final titer test results showed that the titer of avermectin B1a in the fermentation broth at the end of fermentation was 6894 μg / mL, and the B1a / B1b ratio was 38.04.

[0041] Comparative Example 3

[0042] Erbium Er3+ as a rare earth element was added to the avermectin fermentation medium at an addition concentration of 5 mg / L. After sterilization, a seed liquid with a volume ratio of 8% was inoculated into a 50 L fermenter. The fermentation culture conditions were as follows: the culture temperature was controlled at 28 ± 1 °C, the tank pressure was maintained at 0.05 ± 0.01 MPa, the ventilation rate was 1:1, stirring started after fermentation for 10 ± 1 h, the rotation speed was 250 ± 30 rpm, and the fermentation cycle was about 330 h.

[0043] The final titer test results showed that the titer of avermectin B1a in the fermentation broth at the end of fermentation was 6746 μg / mL, and the B1a / B1b ratio was 37.53.

Claims

1. A fermentation method for improving the titer of avermectin by using rare earth elements, characterized in that, It includes the following steps: Add rare earth elements to the avermectin fermentation medium to obtain a fermentation medium containing rare earth elements, and inoculate Streptomyces avermitilis into the fermentation medium containing rare earth elements for fermentation culture to produce avermectin.

2. The fermentation method according to claim 1, characterized in that The fermentation titer of avermectin is the fermentation titer of avermectin B1a component.

3. The fermentation method according to claim 1, wherein The rare earth element is selected from lanthanum (La) 3+ , europium (Eu) 3+ or neodymium (Nd) 3+ .

4. A fermentation method for improving the titer of avermectin B1a according to claim 3, characterized in that, The rare earth element lanthanum (La) 3+ has a concentration of 2 - 12 mg / L in the fermentation medium.

5. A fermentation method for improving the titer of avermectin B1a according to claim 3, characterized in that, The rare earth element europium Eu 3+ has a concentration of 15-30 mg / L in the fermentation medium.

6. The fermentation method for improving the titer of avermectin B1a according to claim 3, wherein The rare earth element neodymium Nd 3+ has a concentration of 0.1 - 1.1 mg / L in the fermentation medium.

7. The fermentation method according to claim 1, wherein The composition of the fermentation medium is as follows: corn starch 140 g / L, soybean cake powder 16 g / L, peanut cake powder 11 g / L, yeast powder 8 g / L, ammonium sulfate 0.2 g / L, sodium molybdate 0.02 g / L, manganese sulfate 0.0023 g / L, antifoaming agent 4 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, sodium chloride 0.5 g / L, light calcium carbonate 2 g / L, cobalt chloride 0.004 g / L.

8. The fermentation method according to claim 1, wherein The fermentation culture conditions are as follows: the culture temperature is 28 ± 1 °C, the tank pressure is 0.05 ± 0.01 MPa, the ventilation rate is 1:1, and the fermentation period is 315 ± 15 h.

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