阅读说明：本技术 一种三七黑斑病防治用多抗霉素的发酵工艺 (Fermentation process of polyoxin for preventing and treating panax notoginseng black spot ) 是由 王亮 祁晨娟 潘忠成 李蒲民 于 2019-10-14 设计创作，主要内容包括：本发明涉及一种三七黑斑病防治用多抗霉素的发酵工艺,该工艺通过三七黑斑病病原菌为指示菌,筛选出可高效抑制三七黑斑病病原菌的多抗霉素产生菌——金色产色链霉菌,进一步优化培养基组分,添加无机盐对金色产色链霉菌的代谢网络进行扰动,强化目标产物生成途径,促进了产物的生成。同时对多抗霉素发酵工艺进行优化,采用定量补加饴糖,自动控制pH值滴加浓氨水的方法进行补料分批发酵方法,大幅提高了发酵单位及生产产量,降低生产成本。(The invention relates to a fermentation process of polyoxin for preventing and treating panax notoginseng black spot, which screens a polyoxin producing strain, namely streptomyces aureochromogenes, capable of efficiently inhibiting pathogenic bacteria of panax notoginseng black spot by taking pathogenic bacteria of panax notoginseng black spot as indicator bacteria, further optimizes the components of a culture medium, and adds inorganic salt to disturb the metabolic network of the streptomyces aureochromogenes, thereby strengthening the generation path of a target product and promoting the generation of the product. Meanwhile, the fermentation process of the polyoxin is optimized, and a method of quantitatively adding maltose and automatically controlling the pH value and dripping concentrated ammonia water is adopted to carry out fed-batch fermentation, so that the fermentation unit and the production yield are greatly improved, and the production cost is reduced.)

1. A fermentation process of polyoxin for preventing and treating panax notoginseng black spot is characterized by comprising the steps of screening out streptomyces aureochromogenes which is a polyoxin producing strain for efficiently inhibiting pathogenic bacteria of panax notoginseng black spot, and culturing, fermenting and fed-batch fermenting the streptomyces aureochromogenes to improve the biomass of the polyoxin.

2. Fermentation process according to claim 1, characterized in that it comprises the following steps:

(1) breeding golden color producing streptomyces: selecting Streptomyces aureochromogenes by using panax notoginseng black spot pathogenic bacteria as indicator bacteria and using a bacteriostatic circle method;

(2) culturing streptomyces aureochromogenes: inoculating the streptomyces aureochromogenes strain screened in the step (1) into a culture medium for culture, culturing for 8-10 days at 26-28 ℃, selecting a colony inclined plane with full growth, selecting an inoculating strain of a strain of;

(3) the fermentation process comprises the following steps: transferring a seed liquid culture medium and a fermentation culture medium with the volume ratio of 10-15:100, inoculating the culture medium and the fermentation culture medium into a fermentation tank for fermentation culture, wherein the liquid loading volume of the fermentation tank is 60-70%, the tank pressure is controlled to be 0.03-0.05Mpa, the fermentation temperature is 26-28 ℃, the aeration ratio is 1:0.5-0.6, the stirring rotation speed is 120-150rpm, and the fermentation time is 30-40 h;

(4) the fed-batch fermentation process comprises the following steps: in the fermentation process, the concentration of reducing sugar is kept between 1.0 and 2.0 percent, the pH value is controlled between 6.0 and 6.5, and the fermentation culture is carried out for 120-140h to obtain the fermentation liquid.

3. The fermentation process according to claim 2, wherein in the step (1), the bacteriostatic circle method is a double-layer culture medium bacteriostatic circle method which utilizes a glass large disc with a cover and is 30cm x 30cm, and the streptomyces aureochromogenes screened by the invention is a multi-antibiotic producing bacterium which can effectively inhibit the pathogenic bacteria of the panax notoginseng black spot.

4. The fermentation process according to claim 2, wherein in the step (2), the culture medium is a plate culture medium prepared from 0.2-0.5% of maltose (AR), 0.3-0.5% of yeast extract (BR), 1.0-1.5% of corn flour (IR), 1.8-2.0% of agar and distilled water and having a pH value of 7.0-7.2.

5. The fermentation process of claim 2, wherein in step (2), the seed liquid medium comprises: low-temperature soybean cake powder 1.5-2.0%, beer yeast powder 0.4-0.6%, corn flour 1.5-2.0%, NaCl 0.1-0.3%, KH₂PO₄ 0.1-0.3%、CaCO₃ 0.3-0.5%, a-high temperature resistant amylase 1000ppm, and natural pH.

6. The fermentation process of claim 2, wherein in step (3), the fermentation medium comprises: low-temperature soybean cake powder 2.0-2.5%, beer yeast powder 0.4-0.6%, fish meal 0.3-0.5%, corn flour 6.0-8.0%, NaCl0.3-0.5%, KH₂PO₄ 0.1-0.3%、CaCO₃0.3-0.5%, ammonium sulfate 0.5-1.0%, a-high temperature resistant amylase 1000ppm, defoaming agent 0.01-0.05%, and natural pH.

7. The fermentation process of claim 2, wherein maltose is quantitatively added, and concentrated ammonia water is added to control pH value for fed-batch fermentation; the mass concentration of the maltose is 45%.

8. The fermentation process of claim 2, wherein the final concentration of ammonium sulfate is 0.8%.

9. The fermentation process according to claim 2, wherein in the step (3), the tank pressure is controlled to be 0.03MPa and the tank temperature is controlled to be 26 ℃.

Technical Field

The invention belongs to the technical field of fermentation engineering, and relates to a fermentation process of polyoxin for preventing and treating panax notoginseng black spot.

Background

Polyoxin (Polyoxin), also known as Polyoxin, Polyoxin and pleiotomycin, is a broad-spectrum agricultural antibiotic of peptide pyrimidine nucleosides produced by modern bioengineering technology. The polyoxin has systemic and therapeutic effects and high target bioselectivity, and can specifically act on common fungal diseases of various crops. The polyoxin has the most prominent effect of inhibiting twenty-five fungal diseases such as flagellates, ascomycetes, adelomycetes and the like. The results of a large number of field experiments at home and abroad show that the polyoxin has excellent control on various diseases such as gray mold, damping off, powdery mildew, anthracnose, stem blight, black spot and the like of melons, fruits and vegetables, and has obvious effect on crop diseases such as rice sheath blight, barley (wheat) powdery mildew and the like. The bactericide has particularly obvious control effects on panax notoginseng black spot, grass toxicity gray mold, apple alternaria leaf spot, pear black spot and tobacco brown spot, and is a first-choice bactericide for controlling the diseases. The biological activity of polyoxin is an important characteristic, the polyoxin is specially used for plant pathogenic bacteria, is safe to people and livestock, has no irritation to skin and eyes, no phytotoxicity, no natural enemy insect killing, no accumulation effect, no three-cause effect and extremely high safety. The polyoxin also has the capability of promoting the growth and defense of crops by adding trace amount, and the yield is increased by 15-25% generally on average. The polyoxin has good adaptability and compatibility with nature, can be degraded quickly after being applied, has no residue, does not pollute the environment, has no cross drug resistance, has strong stability and the like, and becomes one of the first-line medicaments of domestic biological pesticides.

Through the development at home and abroad, people can find that although many researches are carried out on the fermentation production of polyoxin, the problems of low capability of original strains for producing polyoxin, long fermentation period, many intermediate products, high-concentration product and substrate inhibition, complex fermentation liquor separation and extraction and the like still exist. At present, the yield of polyoxin produced by a domestic fermentation method is about 2500 mu/ml, but the fermentation time is about 150h, the side products such as polyoxin analogues and the like in a reaction system are more, and the utilization rate of sugar and the product concentration are further improved. Research and development of a polyoxin high-density culture method become keys for improving the yield of a target product, reducing the production cost and breaking through international market monopoly.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a fermentation process of polyoxin for preventing and treating panax notoginseng black spot, which improves the biomass of polyoxin by screening out streptomyces aureochromogenes which is a polyoxin-producing bacterium for efficiently inhibiting pathogenic bacteria of panax notoginseng black spot, and culturing, fermenting and fed-batch fermenting the streptomyces aureochromogenes, further improves the fermentation level of the polyoxin-producing bacterium on the polyoxin, improves the fermentation unit and yield, reduces the production cost of the polyoxin, and provides an effective way for large-scale production and application and popularization of biological pesticides.

More specifically, the invention adopts the following technical scheme: a fermentation process of polyoxin for preventing and treating panax notoginseng black spot comprises the following steps:

(1) breeding golden color producing streptomyces: selecting Streptomyces aureochromogenes by using panax notoginseng black spot pathogenic bacteria as indicator bacteria and using a bacteriostatic circle method;

(2) culturing streptomyces aureochromogenes: inoculating the streptomyces aureochromogenes strain screened in the step (1) into a culture medium for culture, culturing for 8-10 days at 26-28 ℃, selecting a colony inclined plane with full growth, selecting an inoculating strain of a strain of;

(3) the fermentation process comprises the following steps: transferring a seed liquid culture medium and a fermentation culture medium with the volume ratio of 10-15:100, inoculating the culture medium and the fermentation culture medium into a fermentation tank for fermentation culture, wherein the liquid loading volume of the fermentation tank is 60-70%, the tank pressure is controlled to be 0.03-0.05Mpa, the fermentation temperature is 26-28 ℃, the aeration ratio is 1:0.5-0.6, the stirring rotation speed is 120-150rpm, and the fermentation time is 30-40 h;

(4) the fed-batch fermentation process comprises the following steps: in the fermentation process, the concentration of reducing sugar is kept between 1.0 and 2.0 percent, the pH value is controlled between 6.0 and 6.5, and the fermentation culture is carried out for 120-140h to obtain the fermentation liquid.

In a preferred embodiment of the present invention, in step (1), the bacteriostatic ring method is a double-layer culture medium bacteriostatic ring method using a glass plate with a cover of 30cm × 30 cm. The screened streptomyces aureochromogenes is a polyoxin producing strain capable of efficiently inhibiting the pathogenic bacteria of the panax notoginseng black spot.

In a preferred embodiment of the present invention, in the step (2), the culture medium is a plate culture medium prepared from 0.2-0.5% of maltose (AR), 0.3-0.5% of yeast extract (BR), 1.0-1.5% of corn flour (IR), 1.8-2.0% of agar and distilled water, and having a pH of 7.0-7.2.

In a preferred embodiment of the present invention, in step (2), the seed liquid culture medium comprises: low-temperature soybean cake powder 1.5-2.0%, beer yeast powder 0.4-0.6%, corn flour 1.5-2.0%, NaCl 0.1-0.3%, KH₂PO₄ 0.1-0.3%、CaCO₃ 0.3-0.5%, a-high temperature resistant amylase 1000ppm, and natural pH.

In a preferred embodiment of the present invention, in step (3), the fermentation medium comprises: low-temperature soybean cake powder 2.0-2.5%, beer yeast powder 0.4-0.6%, fish meal 0.3-0.5%, corn flour 6.0-8.0%, NaCl0.3-0.5%, KH₂PO₄ 0.1-0.3%、CaCO₃0.3-0.5%, ammonium sulfate 0.5-1.0%, a-high temperature resistant amylase 1000ppm, defoaming agent 0.01-0.05%, and natural pH.

In the preferred embodiment of the invention, the method of quantitatively adding maltose and dropwise adding strong ammonia water to control the pH value is adopted for fed-batch fermentation.

In a preferred embodiment of the invention, the final concentration of ammonium sulfate is 0.8%.

In a preferred embodiment of the present invention, in the step (3), the pot pressure is controlled to 0.03MPa and the pot temperature is controlled to 26 ℃.

In a preferred embodiment of the present invention, the mass concentration of the maltose is 45%.

The invention also protects the streptomyces aureochromogenes which is fermented and cultured by adopting the fermentation process to obtain the polyoxin. Compared with the prior art, the invention has the beneficial effects that:

according to the invention, the pathogenic bacteria of the panax notoginseng black spot are taken as indicator bacteria, the streptomyces aureochromogenes which is a polyoxin producing bacteria capable of efficiently inhibiting the pathogenic bacteria of the panax notoginseng black spot is screened, the components of the culture medium are further optimized, inorganic salt is added to disturb the metabolic network of the streptomyces aureochromogenes, the generation way of a target product is strengthened, and the generation of the product is promoted. Meanwhile, the fermentation process of the polyoxin is optimized, and a method of quantitatively adding maltose and automatically controlling the pH value and dripping concentrated ammonia water is adopted to carry out fed-batch fermentation, so that the fermentation unit and the production yield are greatly improved, and the production cost is reduced.

By the bred streptomyces aureochromogenes and the fermentation process, the content of polyoxin in the fermentation liquor can be obviously improved, generally, the average titer of polyoxin in the fermentation liquor is not lower than 4000 mu/mL, the prevention and treatment effect on the black spot of panax notoginseng is improved, and the average yield of panax notoginseng is increased by 15-25%.

The invention greatly reduces the production cost of the polyoxin preparation and has great significance for increasing the agricultural production and the income of farmers.

Detailed Description

The following describes embodiments of the present invention in detail, and the embodiments are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific processes are given, but the scope of the present invention is not limited to the following embodiments.