阅读说明：本技术 一种提高春雷霉素发酵水平的生产方法 (Production method for improving fermentation level of kasugamycin ) 是由 王卫富 马文艳 潘忠成 王梦飞 李蒲民 于 2021-09-14 设计创作，主要内容包括：本发明涉及一种提高春雷霉素发酵水平的生产方法,通过调整种子培养配方中的关键速效和迟效氮源比例,有效控制发酵过程的补料速率和溶氧,综合提高种子的质量稳定性和发酵中前期的菌丝生长质量、发酵中后期的环境平衡,实现菌丝代谢活力和发酵水平稳定性的提高。通过对一二级种子的质量控制和发酵工艺的优化,生产大罐的发酵效价由11500-12500u/ml,最高突破到23500u/ml,平均达到17500-21500u/ml,平均效价提高了46-52%,产量提高65-70%,有效的降低了春雷霉素的生产成本。(The invention relates to a production method for improving kasugamycin fermentation level, which effectively controls the material supplementing rate and dissolved oxygen in the fermentation process by adjusting the proportion of key quick-acting and slow-acting nitrogen sources in a seed culture formula, comprehensively improves the quality stability of seeds, the growth quality of hyphae in the early stage of fermentation and the environmental balance in the middle and later stages of fermentation, and realizes the improvement of the hypha metabolic activity and the fermentation level stability. Through the quality control of the first-level and second-level seeds and the optimization of the fermentation process, the fermentation titer of the large production tank is from 11500-.)

1. A production method for improving the fermentation level of kasugamycin adopts a three-stage fermentation process, and is characterized by comprising the following steps:

1) burdening according to the basic formula of the kasugamycin seeds and the fermentation medium, and after water replenishing and volume fixing, the pH is natural;

2) after the first-level seed culture medium is sterilized, inoculating mature streptomyces parvus seeds, sampling and microscopic examination according to various culture conditions of a first-level seed tank in a culture period, monitoring various physicochemical indexes such as hypha morphological change, pH, bacterial concentration and the like, and transferring the seeds into a second-level seed tank in proportion when the seed indexes are qualified;

3) sampling and microscopic examination are carried out in the culture period according to various culture conditions of the second-level seeds, various physical and chemical indexes such as hypha morphological change, pH, bacterial concentration and the like are monitored, and the second-level seeds are transferred into a fermentation tank according to a proportion when the indexes of the second-level seeds are qualified;

4) according to various culture conditions of fermentation, ammonia and sugar are supplemented at a constant or non-constant rate, sugar is extracted according to process requirements, ammonia is controlled, pH is stabilized, dissolved oxygen is constant or regularly fluctuated within a set range, the dissolved oxygen is controlled to be relatively stable when the fermentation enters a production stabilization period, the fermentation titer and yield curve inflection point is monitored at the later stage of the fermentation, and the fermentation is carried out in a tank at a proper time.

2. The method of claim 1, wherein the seed medium is formulated as: 10.0-20.0 g/L of soybean cake powder, 1.0-5.0g/L of yeast powder, 0.5-1.0g/L of fish meal, 1.0-3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 10.0-30.0g/L of soybean oil and 0.01-0.03g/L of potassium dihydrogen phosphate/L, GPE defoaming agent.

3. The production method according to claim 1, wherein in the step 1), the ratio of each substance of the fermentation medium is as follows: 20.0-40.0 g/L of soybean cake powder, 10.0-30.0g/L of corn steep liquor or the same solid content of corn steep liquor dry powder, 1.0-5.0g/L of sodium chloride, 5.0-1.0g/L of vegetable peptone, 10.0-30.0g/L of soybean oil and 0.03-0.05g/L of potassium dihydrogen phosphate defoamer.

4. The production method according to claim 1, wherein in step 2), the mature streptomyces aureofaciens seeds are inoculated into shake flask seeds with microscopic hyphae spreading and branching to form a net, or test tube slants or eggplant-shaped bottles with microscopic spore maturation; in the step 2), the culture conditions of the first-level seed tank are as follows: the air quantity coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 60 percent, the rotating speed is 200-; first-level seed qualification index: the pH is 7.2-7.7, and the microscopic hyphae stretch out and branch to form a dispersed net.

5. The method for producing the compound of claim 1, wherein in the step 3), the culture conditions of the secondary seeds are as follows: the air quantity coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 10 percent, the rotating speed is 200-; the qualified index of the second-level seeds: the pH value is between 6.8 and 7.5, and the microscopic hypha branches are broken and are in a firm and dense net shape.

6. The production method according to claim 1, wherein in step 4), the culture conditions are fermented: air quantity coefficient is 0.5-0.8, temperature is 26.0-30.0 deg.C, dissolved oxygen is greater than 60%, rotation speed is 80-150 rpm, and pH value is controlled to 6.5-7.0 after ammonia is supplemented.

7. The production method of claim 1, wherein in the step 4), sugar and ammonia are supplemented in 24-36h before fermentation, and the feeding rate of industrial ammonia water is controlled to be 0.15-0.3kg/t.h in the whole process^-1The highest feeding rate of 45-55% liquid sugar is 1.4-1.8kg/t.h^-1。

8. The production method of claim 1, wherein in the step 4), the dissolved oxygen is controlled to be between 55 and 75 percent by using the feeding rate, the air volume and the rotating speed conditions in the middle stage of fermentation for 90 to 120 hours, and the dissolved oxygen is controlled to be between 75 and 85 percent after 140 hours in the later stage of fermentation.

9. The production method as claimed in claim 1, wherein in step 4), the fermentation yield rise per 8h in the late stage of fermentation is greater than 0.45kg/t, the fermentation period is selected to be prolonged to be less than 0.3kg/t, the tank is prepared, and the fermentation time is controlled to be between 210 and 230 h.

10. The fermentation material obtained by the production method according to any one of claims 1 to 9, which has a production titer of 17500 and 23500 u/ml.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a production method for improving the fermentation level of kasugamycin.

Background

Kasugamycin (kasugamycin) is a secondary metabolite produced by Streptomyces aureofaciens (Streptomyces kasugaensis), and belongs to the class of aminoglycoside antibiotics. Kasugamycin is widely applied to agriculture and has obvious control effects on rice blast, Chinese cabbage black rot, cucumber fusarium wilt and the like. Kasugamycin is a green, low-toxicity and low-residue agricultural antibiotic bactericide, meets the modern environmental protection requirement, is recommended as an AA-grade green food production material by the Chinese green food development center, is recommended as a pesticide for producing pollution-free agricultural products by the agricultural department, and is a first-pushed bactericide for vegetable standardization engineering by Shanghai markets. Along with the improvement of the awareness of people on the safety of pesticides, the kasugamycin has more and more extensive market prospect due to high-efficiency, broad-spectrum and pollution-free biological characteristics. It is widely used in agriculture because of its remarkable activity against plant pathogenic fungi. The pesticide has low toxicity, safety, high efficiency and environmental protection, is one of the green pesticides with popularization prospect at present, and is one of the pesticides with wide development prospect in the current crop pest control.

At present, three-level or four-level fermentation is adopted in kasugamycin production, the fermentation normal level is about 10000 mug/L under the existing process condition, the highest level does not exceed 14500 mug/L, the production process is controlled relatively coarsely, and key characterization parameters such as dissolved oxygen are not used as main control parameters. How to further improve the titer level of the streptomyces parvus in the fermentation process needs to be enhanced on the one hand for research on the basic metabolism aspect, and on the other hand, further innovation needs to be made on the theory of formula optimization and process control.

Chinese patent application CN107828702A discloses a kasugamycin fermentation medium and a fermentation method, wherein 2.0-2.5% of maltose, 3.5-4.0% of fish oil and 0.01-0.05% of inositol are added into a production medium, and the fermentation titer of kasugamycin in a laboratory can be improved to 16000 mug/L.

Chinese patent application CN109486881A discloses a fermentation medium and a fermentation process of kasugamycin, wherein maltose is added into a production medium: 0.8-1.5% of fish oil, 0.5-1.0% of kasugamycin fermentation filter residue and 50-60% of kasugamycin fermentation filter residue. By controlling the culture medium and the fermentation process, the kasugamycin fermentation filter residue is effectively utilized, and the kasugamycin fermentation unit is improved.

Chinese application patent CN201810817178.3 discloses a method for improving the biological value of kasugamycin, growth factors and trace element culture media are added into a basic culture medium for culture, the kasugamycin value is improved by 19.7 percent, and the yield of a single tank is improved by 10.8 percent.

The fermentation level of the streptomyces parvum can be effectively improved by changing the nutrient formula in the culture medium, but in the actual production process, hypha aging and material quality change are caused by the change of the nutrient, the production stability is poor, and the large-scale improvement of the fermentation yield cannot be completely realized. The invention realizes the control of metabolic flow on the basis of the existing production process, improves the production efficiency of hyphae to the maximum extent and realizes high breakthrough of fermentation level.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a production method for improving the fermentation level of kasugamycin, which starts with the improvement of seed vitality and hypha quality, changes the C/N ratio in seeds and a fermentation culture medium, utilizes the control theory of metabolic flow, adjusts the nutrient utilization efficiency of different periods of fermentation metabolism, improves the production efficiency of a single tank, and realizes the improvement of fermentation titer and the great breakthrough of production yield and production cost.

In order to realize the purpose of the invention, the invention adopts the following technical scheme: a production method for improving the fermentation level of kasugamycin adopts a three-stage fermentation process and comprises the following steps:

1) burdening according to the basic formula of the kasugamycin seeds and the fermentation medium, and after water replenishing and volume fixing, the pH is natural;

2) after the first-level seed culture medium is sterilized, inoculating mature streptomyces parvus seeds, sampling and microscopic examination according to various culture conditions of a first-level seed tank in a culture period, monitoring various physicochemical indexes such as hypha morphological change, pH, bacterial concentration and the like, and transferring the seeds into a second-level seed tank in proportion when the seed indexes are qualified;

3) sampling and microscopic examination are carried out in the culture period according to various culture conditions of the second-level seeds, various physical and chemical indexes such as hypha morphological change, pH, bacterial concentration and the like are monitored, and the second-level seeds are transferred into a fermentation tank according to a proportion when the indexes of the second-level seeds are qualified;

4) according to various culture conditions of fermentation, ammonia and sugar are supplemented at a constant or non-constant rate, sugar is extracted according to process requirements, ammonia is controlled, pH is stabilized, dissolved oxygen is constant or regularly fluctuated within a set range, the dissolved oxygen is controlled to be relatively stable when the fermentation enters a production stabilization period, the fermentation titer and yield curve inflection point is monitored at the later stage of the fermentation, and the fermentation is carried out in a tank at a proper time.

Further, the formula of the seed culture medium is as follows: 10.0-20.0 g/L of soybean cake powder, 1.0-5.0g/L of yeast powder, 0.5-1.0g/L of fish meal, 1.0-3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 10.0-30.0g/L of soybean oil and 0.01-0.03g/L of potassium dihydrogen phosphate/L, GPE defoaming agent.

Further, in the step 1), the ratio of each substance of the fermentation medium is as follows: 20.0-40.0 g/L of soybean cake powder, 10.0-30.0g/L of corn steep liquor or the same solid content of corn steep liquor dry powder, 1.0-5.0g/L of sodium chloride, 5.0-1.0g/L of vegetable peptone, 10.0-30.0g/L of soybean oil and 0.03-0.05g/L of potassium dihydrogen phosphate defoamer.

Further, in the step 2), mature streptomyces parvus seeds are inoculated to spread and branch to form shake flask seeds by microscopic examination hyphae, or test tube inclined planes or eggplant-shaped bottles mature by microscopic examination spores are used.

Further, in the step 2), the culture conditions of the primary seed tank are as follows: the air quantity coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 60 percent, the rotating speed is 200-450rmp, and the culture period is 24-48 h.

Further, in the step 2), the first-level seed qualification index is as follows: the pH is 7.2-7.7, and the microscopic hyphae stretch out and branch to form a dispersed net.

Further, in step 3), the culture conditions of the secondary seeds are as follows: the air volume coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 10 percent, the rotating speed is 200-350rmp, and the culture period is 18-36 h.

Further, in the step 3), the second-level seed qualification index is as follows: the pH value is between 6.8 and 7.5, and the microscopic hypha branches are broken and are in a firm and dense net shape.

Further, in the step 4), fermenting the culture conditions: air quantity coefficient is 0.5-0.8, temperature is 26.0-30.0 deg.C, dissolved oxygen is greater than 60%, rotation speed is 80-150 rpm, and pH value is controlled to 6.5-7.0 after ammonia is supplemented.

Further, in the step 4), sugar and ammonia are supplemented at the early stage of fermentation for 24-36h, and the feeding rate of the industrial ammonia water is controlled to be 0.15-0.3kg/t.h in the whole process^-1The highest feeding rate of 45-55% liquid sugar is 1.4-1.8kg/t.h^-1。

Further, in the step 4), dissolved oxygen is controlled to be between 55 and 75 percent by utilizing the conditions of feeding rate, air volume and rotating speed for 90 to 120 hours in the middle stage of fermentation, and the dissolved oxygen is controlled to be between 75 and 85 percent after 140 hours in the later stage of fermentation.

Further, in the step 4), the fermentation yield fluctuation is more than 0.45kg/t every 8h in the late fermentation period, the extended fermentation period is selected and is less than 0.3kg/t, the tank is prepared, and the fermentation time is controlled between 210 and 230 h.

The invention also protects the fermentation material obtained by the production method, and the production titer of the fermentation material is 17500-23500 u/ml.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention provides a production method for improving the fermentation level of kasugamycin, which starts from improving the seed vitality and the hypha quality, adjusts the nutrition metabolism efficiency in different fermentation periods by utilizing the control theory of metabolic flow and controlling dissolved oxygen, slows down the environmental deterioration in the middle and later fermentation periods, improves the production resistance rate in the later fermentation period and realizes the great breakthrough of the fermentation titer.

2) The production method provided by the invention realizes the stabilization of the metabolic activity of hyphae and the stability improvement of the fermentation yield, the average production fermentation titer is increased from 11500-12500u/ml to 17500-21500u/ml, the highest test breakthrough is 23500u/ml, the average production titer is increased by 46-52%, the yield of a production single tank is increased by 65-70%, and the production cost of kasugamycin is effectively reduced.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described with the following specific examples, but the present invention is by no means limited to these examples.

The method for detecting the kasugamycin content adopted by the invention is an HPLE method.

Example 1

1) The formula of the first-level seed culture medium is as follows: 10.0g/L of soybean cake powder, 5.0g/L of yeast powder, 1.0g/L of fish meal, 1.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 10.0g/L of soybean oil, 2.0g/L, GPE g/L of monopotassium phosphate as an antifoaming agent, and the pH value is 6.2-6.4 after water supplement and volume fixing.

2) The formula of the secondary seed culture medium is as follows: soybean cake powder 20.0g/L, yeast powder 1.0g/L, fish meal 0.5g/L, sodium chloride 3.0g/L, ammonium sulfate 3.0g/L, soybean oil 30.0g/L, potassium dihydrogen phosphate 2.0g/L, GPE defoaming agent 0.03g/L, water supplementing and volume fixing, and pH 6.2-6.4.

3) The fermentation medium formula comprises: 20.0g/L of soybean cake powder, 30.0g/L of corn steep liquor, 2.0g/L of sodium chloride, 5.0g/L of vegetable peptone, 30.0g/L of soybean oil and 0.5g/L, GPE g/L of potassium dihydrogen phosphate defoaming agent, and the pH value is 5.9-6.2 after water supplement and volume fixing.

4) After the first-level seed culture medium is sterilized, mature streptomyces parvus seeds are inoculated as shake flask seeds with microscopic hypha stretching and branching to form a net. The culture conditions are as follows: the air volume coefficient is 0.6, the temperature is 30.0-31.0 ℃, the rotating speed is 250-300rmp, the dissolved oxygen is natural, and the culture period is 24-30 h. Monitoring various physical and chemical indexes such as hypha morphological change, pH, bacterial concentration and the like, wherein the first-level seed qualification index is as follows: the pH is 7.2-7.3, the microscopic hyphae stretch out and branch to form a dispersed net shape, and the hyphae are transferred into a secondary seed tank according to the proportion of 10%.

5) The culture conditions of the secondary seeds are as follows: the air quantity coefficient is 0.6, the temperature is 29.0-30.0 ℃, the rotating speed is 250rmp, the dissolved oxygen is natural, and the culture period is 18-24 h. Monitoring various physical and chemical indexes such as hypha morphological change, pH, bacterial concentration and the like, and obtaining a second-level seed qualified index: when the pH value is between 6.8 and 7.0, the branches of the microscopic hyphae are broken and are in a firm and dense net shape, and the mixture is transferred into a fermentation tank according to the proportion of 12 percent.

6) Fermentation culture conditions: the air volume coefficient is 0.55, the temperature is 28.0 ℃, the dissolved oxygen is more than 70 percent, the rotating speed is 80 to 120rmp, and the pH value at the early stage is natural. The pH value is reduced to about 6.7 in the early stage of fermentation for 24-36h at a constant rate of 0.15-0.20kg/t.h^-1Adding industrial ammonia water, stabilizing fermentation pH at 6.5-6.7, adding 45-50% liquid sugar within 3-5 hr according to ammonia supplementing condition, and highest adding rate not more than 1.8kg/t.h^-1Controlling dissolved oxygen at 55-75% in the middle stage of fermentation for 90-120h, and controlling dissolved oxygen at the later stage of fermentation for 140hThe dissolved oxygen is controlled between 75-85%. And (3) in the later fermentation period, the fermentation yield rise per 8h is more than 0.45kg/t, the fermentation period is selected to be prolonged to be less than 0.3kg/t, the fermentation titer and yield curve inflection point is set to be between 210 and 230h, and the fermentation titer is set to be between 19500 and 23500 mu g/L at a proper time.

Example 2

1) The formula of the first-level seed culture medium is as follows: 15.0g/L of soybean cake powder, 3.0g/L of yeast powder, 0.5g/L of fish meal, 1.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 20.0g/L of soybean oil, 2.0g/L, GPE g/L of monopotassium phosphate as an antifoaming agent, and the pH value is 6.2-6.4 after water replenishing and volume fixing.

2) The formula of the secondary seed culture medium is as follows: 15.0g/L of soybean cake powder, 3.0g/L of yeast powder, 1.0g/L of fish meal, 3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 20.0g/L of soybean oil and 0.03g/L of potassium dihydrogen phosphate defoamer, and the pH value is 6.2-6.4 after water replenishing and volume fixing.

3) The fermentation medium formula comprises: 30.0g/L of soybean cake powder, 10.0g/L of corn steep liquor, 5.0g/L of sodium chloride, 3.0g/L of vegetable peptone, 20.0g/L of soybean oil and 0.03g/L of potassium dihydrogen phosphate/L, GPE defoaming agent, and the pH value is 5.9-6.2 after water replenishing and volume fixing.

4) After the first-level seed culture medium is sterilized, inoculating mature streptomyces parvus seeds as test tube inclined planes for microscopic examination of mature spores, and washing the thalli by adopting sterilized saline-free water or normal saline to form suspension. The culture conditions are as follows: the air volume coefficient is 0.8, the temperature is 29.0-30.0 ℃, the rotating speed is 300-350rmp, the dissolved oxygen is natural, the culture period is 40-48h, various physicochemical indexes such as hypha form change, pH, bacterial concentration and the like are monitored, and the first-level seed qualification index is as follows: the pH is 7.3-7.5, and the microscopic mycelia stretch out and branch to form a dispersed net shape, and are transferred into a secondary seed tank according to a proportion of 13%.

5) The culture conditions of the secondary seeds are as follows: the air quantity coefficient is 1.0, the temperature is 28.0-29.0 ℃, the rotating speed is 200rpm, the dissolved oxygen is more than 10 percent, and the culture period is 30-36 h. Monitoring various physical and chemical indexes such as hypha morphological change, pH, bacterial concentration and the like, and obtaining a second-level seed qualified index: the pH value is between 7.0 and 7.2, the microscopic hypha branches are broken and are in a firm and dense net shape, and the hypha branches are transferred into a fermentation tank according to the proportion of 10 percent.

6) Fermenting under various culture conditions: the air volume coefficient is 0.8, the temperature is 26.0 ℃, the dissolved oxygen is more than 65 percent,the rotation speed is 120-. In the early stage of fermentation for 24-36h, pH is reduced to 6.8-6.9, inflection point appears in the descending trend, 45-50% liquid sugar is fed in at the beginning, and the initial feeding rate is 0.2-0.3kg/t.h^-1While at the same time according to a constant rate of 0.15-0.20kg/t.h^-1Adding industrial ammonia water, stabilizing fermentation pH at 6.8-6.9, gradually increasing liquid sugar addition according to ammonia supplementing amount, and increasing highest addition rate not more than 1.8kg/t.h^-1The dissolved oxygen is controlled to be between 65 and 75 percent in the middle stage of fermentation for 90 to 120 hours, and the dissolved oxygen is controlled to be between 80 and 85 percent in the later stage of fermentation for 140 hours. And (3) in the later fermentation period, the fermentation yield rise per 8h is more than 0.45kg/t, the prolonged fermentation period is selected to be less than 0.3kg/t, the fermentation titer and yield curve inflection point is set to be between 210 and 230h, and the fermentation titer is set to be between 17500 and 19500 mug/L at a proper time.

Example 3

1) The formula of the first-level seed culture medium is as follows: soybean cake powder 20.0g/L, yeast powder 1.0g/L, fish meal 1.0g/L, sodium chloride 1.0g/L, ammonium sulfate 3.0g/L, soybean oil 15.0g/L, potassium dihydrogen phosphate 2.0g/L, GPE defoaming agent 0.03g/L, and after water replenishing and volume fixing, the pH value is 6.2-6.4.

2) The formula of the secondary seed culture medium is as follows: 10.0g/L of soybean cake powder, 5.0g/L of yeast powder, 0.5g/L of fish meal, 3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 10.0-30.0g/L of soybean oil, 2.0g/L, GPE g/L of monopotassium phosphate and 0.01-0.03g/L of antifoaming agent, and the pH value is 6.2-6.4 after constant volume and water supplement.

3) The fermentation medium formula comprises: 40.0g/L of soybean cake powder, 5.0g/L of corn steep liquor dry powder, 1.0g/L of sodium chloride, 1.0g/L of vegetable peptone, 15.0g/L of soybean oil and 0.05g/L of potassium dihydrogen phosphate/L, GPE defoaming agent, and the pH value is 5.9-6.2 after water replenishing and volume fixing.

4) After the first-level seed culture medium is sterilized, inoculating mature streptomyces parvus seeds as eggplant-shaped bottle seed slopes with mature microscopic spores, and washing thalli by adopting sterilized saline-free water or normal saline to form suspension. The culture conditions are as follows: the air volume coefficient is 1.0, the temperature is 28.0-29.0 ℃, the rotating speed is 400-: the pH is 7.5-7.7, the microscopic hyphae stretch out and branch to form a dispersed net shape, and the hyphae are transferred into a secondary seed tank according to the proportion of 10%.

5) The culture conditions of the secondary seeds are as follows: the air volume coefficient is 0.8, the temperature is 28.0-29.0 ℃, the rotating speed is 300-350rpm, the dissolved oxygen is natural, and the culture period is 24-30 h. Monitoring various physical and chemical indexes such as hypha morphological change, pH, bacterial concentration and the like, and obtaining a second-level seed qualified index: when the pH is between 7.0 and 7.2, the branches of the microscopic hyphae are broken and are in a firm and dense net shape, and the hyphae are transferred into a fermentation tank according to the proportion of 15 percent.

6) Fermenting under various culture conditions: the air volume coefficient is 0.65, the temperature is 30.0 ℃, the initial rotating speed is 105rpm, the dissolved oxygen is more than 65 percent, and the pH value at the early stage is natural. The pH rises to the highest point about 13h in the early stage of fermentation, the temperature is reduced to 29 ℃, the fermentation is carried out for 24-36h, the pH is reduced by 6.9-7.0, the inflection point appears in the trend of reduction, the temperature is reduced to 28 ℃, 45-50% of liquid sugar begins to be fed in a flowing manner, and the initial feeding rate is 0.2-0.3kg/t.h^-1After the pH is reduced to 6.8-6.9, the pH is controlled at a constant rate of 0.15-0.20kg/t.h^-1Feeding industrial ammonia water, stabilizing fermentation pH, gradually increasing liquid sugar feeding rate according to ammonia supplementing amount, with highest feeding rate not more than 1.8kg/t.h^-1Controlling the dissolved oxygen to be between 70 and 80 percent in the middle stage of fermentation for 90 to 120 hours, and controlling the dissolved oxygen to be about 80 percent after the dissolved oxygen rises excessively in the later stage of fermentation for 140 hours, reducing the rotating speed and the air volume. And (3) in the later stage of fermentation, the rise of the fermentation yield per 8h is more than 0.45kg/t, the fermentation period is prolonged to be less than 0.3kg/t, the fermentation titer and yield curve inflection point is selected, the fermentation period is before 210h, the fermentation tank is placed at a proper time, and the fermentation tank titer is 17500-.

Example 4 (production control Process)

1) The formula of the first-level seed culture medium is as follows: soybean cake powder 20.0g/L, yeast powder 1.0g/L, sodium chloride 1.0g/L, ammonium sulfate 3.0g/L, soybean oil 15.0g/L, GPE defoaming agent 0.01g/L, after water replenishing and volume fixing, the pH value is 6.2-6.4.

2) The formula of the secondary seed culture medium is as follows: 20.0g/L of soybean cake powder, 3.0g/L of corn steep liquor dry powder, 1.0g/L of sodium chloride, 3.0g/L of ammonium sulfate and 0.03g/L of soybean oil 10.0g/L, GPE defoaming agent, and the pH value is 6.2-6.4 after water supplementing and volume fixing.

3) The fermentation medium formula comprises: 30.0g/L of soybean cake powder, 1.0g/L of sodium chloride, 25.0g/L of soybean oil, 0.5g/L, GPE g/L of potassium dihydrogen phosphate defoaming agent, and after water replenishing and volume fixing, the pH value is 5.9-6.2.

4) After the first-level seed culture medium is sterilized, inoculating mature streptomyces parvus seeds as eggplant-shaped bottle seed slopes with mature microscopic spores, and washing thalli by sterilized physiological saline water to form suspension. The culture conditions are as follows: air volume coefficient is 1.0, temperature is 28.0 ℃, rotation speed is 450rmp, dissolved oxygen is natural, culture period is 33h, microscopic hypha is spread and branched to form a sparse net shape, pH is natural, and the hypha is transferred into a secondary seeding tank according to the proportion of 10%.

5) The culture conditions of the secondary seeds are as follows: air quantity coefficient is 0.8, temperature is 28.0 ℃, rotating speed is 350rpm, dissolved oxygen is natural, culture period is 24h, microscopic hypha branches are broken, the hypha branches are in a firm and dense net shape, pH is natural, and the hypha branches are transferred into a fermentation tank according to a proportion of 15%.

6) Fermenting under various culture conditions: the air volume coefficient is 0.7, the temperature is 28.0 ℃, the rotating speed is constant at 120-. After 24h of fermentation, the pH was lowered to 6.5-6.8, and a suitable control point was selected, initially at a constant rate of 0.10kg/t.h^-1Industrial ammonia water is fed in a flowing manner, and the medium term is improved to 0.15kg/t.h^-1The fermentation pH was constant. According to the actual ammonia supplementation condition, feeding 45-50% liquid sugar in the middle of 24-30h, fermenting for 60-85h, and increasing the liquid sugar to 1.5kg/t.h at the highest flow^-1. The fermentation period is 185-195h, the fermentation titer is 10500-12500 mug/L, and the tank is ready to be placed.

The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. Several alternatives or modifications to the described embodiments without departing from the inventive concept should be considered as falling within the scope of the present invention.