CN113652458B - Production method for improving fermentation level of kasugamycin - Google Patents

Production method for improving fermentation level of kasugamycin Download PDF

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CN113652458B
CN113652458B CN202111073621.9A CN202111073621A CN113652458B CN 113652458 B CN113652458 B CN 113652458B CN 202111073621 A CN202111073621 A CN 202111073621A CN 113652458 B CN113652458 B CN 113652458B
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王卫富
马文艳
潘忠成
王梦飞
李蒲民
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Shaanxi Microbe Biotechnology Co ltd
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Abstract

The invention relates to a production method for improving the fermentation level of kasugamycin, which is characterized in that the quality stability of seeds, the growth quality of mycelium at the middle and earlier stages of fermentation and the environmental balance at the middle and later stages of fermentation are comprehensively improved by adjusting the key quick-acting and slow-acting nitrogen source proportion in a seed culture formula to effectively control the feeding rate and dissolved oxygen in the fermentation process, so that the metabolic activity of mycelium and the stability of the fermentation level are improved. By optimizing the quality control and fermentation process of the secondary seeds, the fermentation titer of the production large tank is changed from 11500-12500u/ml to 23500u/ml at the highest, the average titer is 17500-21500u/ml, the average titer is improved by 46-52%, the yield is improved by 65-70%, and the production cost of kasugamycin is effectively reduced.

Description

Production method for improving fermentation level of kasugamycin
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 and belongs to the class of aminoglycoside antibiotics. The kasugamycin is widely applied to agriculture and has obvious prevention and treatment effects on rice blast, cabbage black rot, cucumber fusarium wilt and the like. Kasugamycin is used as an agricultural antibiotic bactericide with green, low toxicity and low residue, meets the modern environmental protection requirement, is recommended as AA-grade green food production data by China green food development center, is listed as a recommended pesticide for pollution-free agricultural product production by the department of agriculture, and is listed as a first-push bactericide for vegetable standardization engineering by Shanghai. With the improvement of people's safety consciousness of pesticides, kasugamycin has high efficiency, broad spectrum and nuisanceless biological characteristics, and has shown wider and wider market prospect. 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 friendliness, is one of the green pesticides with the most popularization prospect at present, and is one of pesticides with wide development prospect in the current crop pest control.
At present, the kasugamycin production adopts three-level or four-level fermentation, the normal fermentation level is about 10000 mu g/L under the existing process conditions, the highest fermentation level is not more than 14500 mu g/L, the production process is controlled relatively roughly, and key characterization parameters such as dissolved oxygen and the like are not used as main control parameters. How to further improve the titer level of streptomyces parvulus in the fermentation process, on one hand, the research on basic metabolism needs to be enhanced, and on the other hand, further innovation is made on the aspects of formula optimization and process control theory.
Chinese patent application CN107828702A discloses a kasugamycin fermentation medium and a fermentation method, wherein maltose 2.0-2.5%, fish oil 3.5-4.0% and inositol 0.01-0.05% are added in the production medium, so that the kasugamycin fermentation titer of a laboratory can be improved to 16000 mug/L.
Chinese patent application CN109486881A is a fermentation medium and a fermentation process of kasugamycin, maltose is added into a production medium: 0.8 to 1.5 percent, 0.5 to 1.0 percent of fish oil and 50 to 60 percent of kasugamycin fermentation filter residue. By controlling the culture medium and the fermentation process, the fermentation filter residues of the kasugamycin are effectively utilized, and the kasugamycin fermentation unit is improved.
Chinese application patent CN201810817178.3 discloses a method for improving the biological titer of kasugamycin, which comprises the steps of adding a growth factor and a trace element culture medium into a basic culture medium for culture, wherein the kasugamycin titer is improved by 19.7%, and the yield of a single pot is improved by 10.8%.
The fermentation level of streptomyces parvulus can be effectively improved by changing the nutrition formula in the culture medium, but in the actual production process, mycelium aging and material quality change are caused by nutrition change, so that the production stability is poor, and the great improvement of the fermentation yield cannot be completely realized. The invention realizes the control of metabolic flow based on the existing production process, improves the production efficiency of hyphae to the maximum extent, and realizes the 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 from improving the activity of seeds and the quality of hyphae, changes the C/N ratio in the seeds and a fermentation medium, adjusts the nutrition utilization efficiency of fermentation metabolism in different periods by utilizing the control theory of metabolic flow, 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 achieve the aim 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) Proportioning according to basic formulas of kasugamycin seeds and a fermentation medium, supplementing water, fixing the volume, and naturally regulating the pH;
2) Inoculating mature Streptomyces parvus seeds after the primary seed culture medium is sterilized, sampling and microscopic examination are carried out in a culture period according to various culture conditions of a primary seed tank, various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like are monitored, and the seeds are transferred into a secondary seed tank according to a proportion after the indexes of the seeds are qualified;
3) Sampling and microscopic examination are carried out in a culture period according to each culture condition of the secondary seeds, each physicochemical index such as the change of mycelium form, pH, fungus concentration and the like is monitored, and when the seed index is qualified, the seeds are proportionally moved into a fermentation tank;
4) According to various culture conditions of fermentation, ammonia and sugar are supplemented at a constant or non-constant rate, sugar is extracted and ammonia is controlled according to process requirements, stable pH and dissolved oxygen constantly or regularly fluctuate within a set range, the dissolved oxygen is controlled to be relatively stable when fermentation enters a production stability period, fermentation titer and yield curve inflection points are monitored in the later period of fermentation, and a tank is selected at a proper time.
Further, the formula of the seed culture medium is as follows: 10.0 to 20.0g/L of soybean cake powder, 1.0 to 5.0g/L of yeast powder, 0.5 to 1.0g/L of fish meal, 1.0 to 3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 10.0 to 30.0g/L of soybean oil, 2.0g/L, GPE of monopotassium phosphate and 0.01 to 0.03g/L of defoamer.
Further, in the step 1), the ratio of each substance of the fermentation medium is as follows: 20.0 to 40.0g/L of soybean cake powder, 10.0 to 30.0g/L of corn steep liquor or corn steep liquor dry powder, 1.0 to 5.0g/L of sodium chloride, 5.0 to 1.0g/L of plant peptone, 10.0 to 30.0g/L of soybean oil, 0.5 to 1.0g/L, GPE g/L of potassium dihydrogen phosphate and 0.03 to 0.05g/L of defoamer.
Further, in step 2), inoculating mature Streptomyces parvulus seeds as microscopic hyphae stretching branching net-forming shake flask seeds, or using microscopic spore mature test tube inclined planes or eggplant-shaped bottles.
Further, in step 2), the culture conditions of the primary seed tank: the air volume coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 60%, the rotating speed is 200-450rmp, and the culture period is 24-48h.
Further, in step 2), the first-level seed qualification index: the pH value is between 7.2 and 7.7, and the microscopic hyphae are stretched and branched to form an evacuation net shape.
Further, in step 3), each culture condition of the secondary seeds: the air volume coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 10%, the rotating speed is 200-350rmp, and the culture period is 18-36h.
Further, in step 3), the second-level seed qualification index: the pH value is between 6.8 and 7.5, and the hypha branches are microscopic to be broken and are in a firm and dense net shape.
Further, in step 4), each culture condition is fermented: the air quantity coefficient is 0.5-0.8, the temperature is 26.0-30.0 ℃, the dissolved oxygen is more than 60%, the rotating speed is 80-150 rpm, and the pH value is controlled to be 6.5-7.0 after ammonia is supplemented.
Further, in the step 4), the sugar and the ammonia are supplemented in the early fermentation period of 24-36 hours, and the whole process of the industrial ammonia water flow acceleration rate is controlled to be 0.15-0.3kg/t.h -1 45-55% liquid sugar maximum feeding rate 1.4-1.8kg/t.h -1 。
Further, in the step 4), the dissolved oxygen is controlled between 55 and 75 percent by utilizing the conditions of the feeding rate, the air quantity and the rotating speed in the middle fermentation period of 90 to 120 hours, and the dissolved oxygen is controlled between 75 and 85 percent after 140 hours in the later fermentation period.
Further, in the step 4), the fermentation yield fluctuation is more than 0.45kg/t every 8 hours in the later fermentation period, the fermentation period is prolonged and is lower than 0.3kg/t, the tank is ready to be put, and the fermentation time is controlled between 210 and 230 hours.
The invention also provides the fermented material obtained by the production method, and the production titer of the fermented material is 17500-23500u/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 activity and mycelium quality of seeds, adjusts the nutrition metabolism efficiency of different fermentation periods through dissolved oxygen control by utilizing the control theory of metabolic flow, slows down the environmental deterioration in the middle and later stages of fermentation, improves the yield-resistance rate in the later stages of fermentation, and realizes the great breakthrough of fermentation titer.
2) The production method provided by the invention realizes the stability of the metabolic activity of hyphae and the stability improvement of the fermentation yield, improves the average titer of production fermentation from 11500-12500u/ml to 17500-21500u/ml, breaks through 23500u/ml the highest test, improves the average titer of production by 46-52%, improves the yield of a single production tank by 65-70%, and effectively reduces the production cost of kasugamycin.
Detailed Description
The present invention will be described in more detail with reference to the following examples, but the present invention is not 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 primary seed culture medium comprises: 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 of potassium dihydrogen phosphate and 0.01g/L of defoamer, and the pH value is 6.2-6.4 after water supplementing and volume fixing.
2) The formula of the secondary seed culture medium comprises: 20.0g/L of soybean cake powder, 1.0g/L of yeast powder, 0.5g/L of fish meal, 3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 30.0g/L of soybean oil, 2.0g/L, GPE of potassium dihydrogen phosphate and 0.03g/L of defoamer, and the pH value is 6.2-6.4 after water supplementing and volume fixing.
3) The formula of the fermentation medium 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 plant peptone, 30.0g/L of soybean oil, 0.5g/L, GPE of potassium dihydrogen phosphate and 0.03g/L of defoamer, and the pH value after water supplementing and volume fixing is 5.9-6.2.
4) After the primary seed culture medium is sterilized, inoculating mature streptomyces parvulus seeds as shake flask seeds for microscopic examination of hyphae stretching and branching into a net. Culture conditions: the air quantity 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 hours. Monitoring various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like, and qualified indexes of first-class seeds: the pH value is between 7.2 and 7.3, the microscopic hyphae are spread and branched to form a sparse net shape, and the sparse net shape is transferred into a secondary seed tank according to the proportion of 10 percent.
5) Various culture conditions of the secondary seeds: 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 hours. Monitoring various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like, and qualified indexes of the secondary seeds: the pH value is between 6.8 and 7.0, and the hypha branches are microscopic examination and broken to form a firm and dense net shape, and the hypha branches are 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%, the rotating speed is 80-120rmp, and the pH is natural in the early stage. The pH value is reduced to about 6.7 in the earlier fermentation period of 24-36h, and the constant rate is 0.15-0.20kg/t.h -1 Ammonia water in the flow processing industry, stable fermentation pH of 6.5-6.7, starting to flow 45-50% liquid sugar according to the ammonia supplementing condition within 3-5h, and the highest flow adding rate is not more than 1.8kg/t.h -1 The dissolved oxygen is controlled between 55 and 75 percent in the middle fermentation period of 90 to 120 hours, and between 75 and 85 percent after 140 hours in the later fermentation period. The fermentation yield fluctuation is more than 0.45kg/t every 8 hours in the later fermentation period, the fermentation period is prolonged and is lower than 0.3kg/t, the fermentation titer and the yield curve inflection point are selected, the fermentation period is between 210 and 230 hours, the lower tank is selected at a proper time, and the lower tank titer is between 19500 and 23500 mu g/L.
Example 2
1) The formula of the primary seed culture medium comprises: 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 of potassium dihydrogen phosphate and 0.15g/L of defoamer, and after water supplementing and volume fixing, the pH value is 6.2-6.4.
2) The formula of the secondary seed culture medium comprises: 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, 2.0g/L, GPE of potassium dihydrogen phosphate and 0.03g/L of defoamer, and after water supplementing and volume fixing, the pH value is 6.2-6.4.
3) The formula of the fermentation medium 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 plant peptone, 20.0g/L of soybean oil, 1.0g/L, GPE of potassium dihydrogen phosphate and 0.03g/L of defoamer, and after water supplementing and volume fixing, the pH value is 5.9-6.2.
4) After the primary seed culture medium is sterilized, inoculating mature streptomyces parvulus seeds to be a test tube inclined plane for microscopic spore maturation, and washing the bacteria body by using sterile saline-free water or normal saline to form a suspension. Culture conditions: the air volume coefficient is 0.8, the temperature is 29.0-30.0 ℃, the rotating speed is 300-350rmp, dissolved oxygen is natural, the culture period is 40-48 hours, various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like are monitored, and the first-level seed qualification index is obtained: the pH value is between 7.3 and 7.5, the microscopic hyphae are spread and branched to form a sparse net shape, and the sparse net shape is transferred into a secondary seed tank according to the proportion of 13 percent.
5) Various culture conditions of the secondary seeds: 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%, and the culture period is 30-36h. Monitoring various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like, and qualified indexes of the secondary seeds: the pH value is between 7.0 and 7.2, the branches of the hyphae are broken and are in a firm and dense net shape after microscopic examination, and the hyphae are transferred into a fermentation tank according to the proportion of 10 percent.
6) Fermenting all culture conditions: the air volume coefficient is 0.8, the temperature is 26.0 ℃, the dissolved oxygen is more than 65%, the rotating speed is 120-150rpm, and the pH is natural in the earlier stage. The pH value is reduced to between 6.8 and 6.9 in the early fermentation period of 24 to 36 hours, the downward trend has an inflection point, 45 to 50 percent of liquid sugar starts to be fed, and the initial feeding rate is 0.2 to 0.3kg/t.h -1 At the same time at a constant rate of 0.15-0.20kg/t.h -1 Ammonia water in the fluid processing industry is used for stabilizing fermentation pH between 6.8 and 6.9, the fluid sugar feeding amount is gradually increased according to the condition of ammonia feeding amount, and the highest feeding rate is not more than 1.8kg/t.h -1 The dissolved oxygen is controlled between 65 and 75 percent in the middle fermentation period of 90 to 120 hours, and 80 to 85 percent after 140 hours in the later fermentation period. The fermentation yield fluctuation is more than 0.45kg/t every 8 hours in the later fermentation period, the fermentation period is prolonged and is lower than 0.3kg/t, the fermentation titer and the yield curve inflection point are selected, the fermentation period is between 210 and 230 hours, the lower tank is selected at a proper time, and the lower tank titer is between 17500 and 19500 mug/L.
Example 3
1) The formula of the primary seed culture medium comprises: 20.0g/L of soybean cake powder, 1.0g/L of yeast powder, 1.0g/L of fish meal, 1.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 15.0g/L of soybean oil, 2.0g/L, GPE of potassium dihydrogen phosphate and 0.03g/L of defoamer, and after water supplementing and volume fixing, the pH value is 6.2-6.4.
2) The formula of the secondary seed culture medium comprises: 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 of monopotassium phosphate and 0.01-0.03g/L of defoamer, and after water supplementing and volume fixing, the pH value is 6.2-6.4.
3) The formula of the fermentation medium 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 plant peptone, 15.0g/L of soybean oil, 1.0g/L, GPE of potassium dihydrogen phosphate and 0.05g/L of defoamer, and after water supplementing and volume fixing, the pH value is 5.9-6.2.
4) After the primary seed culture medium is sterilized, inoculating mature streptomyces parvulus seeds to obtain a seed inclined plane of an eggplant-shaped bottle with mature microscopic spores, and washing the bacteria body to form a suspension by using sterile saline-free water or normal saline. Culture conditions: the air volume coefficient is 1.0, the temperature is 28.0-29.0 ℃, the rotating speed is 400-450rmp, dissolved oxygen is natural, the culture period is 30-36h, and various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like are monitored, so that the first-level seed qualification index is obtained: the pH value is between 7.5 and 7.7, and the microscopic hyphae are spread and branched to form a sparse net shape and are transferred into a secondary seed tank according to the proportion of 10 percent.
5) Various culture conditions of the secondary seeds: 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 hours. Monitoring various physicochemical indexes such as mycelium morphological change, pH, fungus concentration and the like, and qualified indexes of the secondary seeds: the pH value is between 7.0 and 7.2, the branches of the 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 all 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%, and the pH is natural in the earlier stage. 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 to 6.9-7.0, the inflection point appears in the descending trend, the temperature is reduced to 28 ℃, 45-50% of liquid sugar starts to be fed, and the initial feeding rate is 0.2-0.3kg/t.h -1 After the pH is reduced to 6.8-6.9, the mixture is pressedAt a constant rate of 0.15-0.20kg/t.h -1 Ammonia water in the fluid processing industry is stable and the fermentation pH is constant, the fluid sugar feeding amount is gradually increased according to the condition of the ammonia feeding amount, and the highest feeding rate is not more than 1.8kg/t.h -1 The dissolved oxygen is controlled to be between 70 and 80 percent in the middle fermentation period of 90 to 120 hours, the dissolved oxygen is excessively raised after 140 hours in the later fermentation period, the rotating speed and the air quantity are reduced, and the dissolved oxygen is controlled to be about 80 percent. The fermentation yield fluctuation is more than 0.45kg/t every 8 hours in the later fermentation period, the fermentation period is prolonged and is lower than 0.3kg/t, the fermentation titer and the yield curve inflection point are selected, the fermentation period is before 210 hours, the lower tank is selected at a proper time, and the lower tank titer is between 17500 and 18500 mug/L.
Example 4 (production control Process)
1) The formula of the primary seed culture medium comprises: 20.0g/L of soybean cake powder, 1.0g/L of yeast powder, 1.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 15.0g/L, GPE of soybean oil and 0.01g/L of defoamer, and after water supplementing and volume fixing, the pH value is 6.2-6.4.
2) The formula of the secondary seed culture medium comprises: 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, 10.0g/L, GPE of soybean oil and 0.03g/L of defoamer, and after water supplementing and volume fixing, the pH value is 6.2-6.4.
3) The formula of the fermentation medium comprises: 30.0g/L of soybean cake powder, 1.0g/L of sodium chloride, 25.0g/L of soybean oil, 0.5g/L, GPE of monopotassium phosphate and 0.03g/L of defoamer, and after water supplementing and volume fixing, the pH value is 5.9-6.2.
4) After the primary seed culture medium is sterilized, inoculating mature streptomyces parvulus seeds to obtain an eggplant-shaped bottle seed inclined plane with mature microscopic spores, and washing the bacteria body with sterilized normal saline to form a suspension. Culture conditions: the air quantity coefficient is 1.0, the temperature is 28.0 ℃, the rotating speed is 450rmp, the dissolved oxygen is natural, the culture period is 33 hours, the microscopic hypha is stretched and branched into a dispersed net shape, the pH is natural, and the hypha is transferred into a secondary seed tank according to the proportion of 10 percent.
5) Various culture conditions of the secondary seeds: the air volume coefficient is 0.8, the temperature is 28.0 ℃, the rotating speed is 350rpm, dissolved oxygen is natural, the culture period is 24 hours, the microscopic examination hypha branches are broken, the hypha branches are solid and compact, the pH is natural, and the hypha branches are transferred into a fermentation tank according to the proportion of 15%.
6) Fermenting all culture conditions: the air quantity coefficient is 0.7, the temperature is 28.0 ℃, and the rotating speed is constant at 120-150rpmThe pH is natural in the early stage. After 24h fermentation, the pH is reduced to 6.5-6.8, a suitable control point is selected, and the pH is initially controlled at a constant rate of 0.10kg/t.h -1 Ammonia water in the fluid processing industry is increased to 0.15kg/t.h in the middle period -1 The fermentation pH is constant. According to the actual ammonia supplementing amount, 45-50% of liquid sugar is fed in the middle of 24-30h, fermentation is carried out for 60-85h, and the highest feeding of liquid sugar is increased to 1.5kg/t.h -1 . The fermentation period is 185-195h, the fermentation titer is 10500-12500 mug/L, and the tank is ready to be put.
The foregoing is a further detailed description of the invention in connection with specific/preferred embodiments, and is not intended to limit the practice of the invention to such description. Several alternatives or modifications to these described embodiments should be considered to fall within the scope of the invention without departing from the inventive concept.

Claims (1)

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) Proportioning according to basic formulas of kasugamycin seeds and a fermentation medium, supplementing water, fixing the volume, and naturally regulating the pH;
2) Inoculating mature Streptomyces parvus seeds after the primary seed culture medium is sterilized, sampling and microscopic examination are carried out in a culture period according to various culture conditions of a primary seed tank, monitoring various physicochemical indexes of mycelium morphological change, pH and fungus concentration, and transferring the seeds into a secondary seed tank according to a proportion when the seed indexes are qualified;
3) Sampling and microscopic examination are carried out in a culture period according to each culture condition of the secondary seeds, each physicochemical index of the mycelium form change, pH and fungus concentration is monitored, and when the seed index is qualified, the seeds are transferred into a fermentation tank according to a proportion;
4) According to various culture conditions of fermentation, according to constant or non-constant speed, glucose is extracted and ammonia is controlled according to process requirements, stable pH and dissolved oxygen are constantly or regularly fluctuated within a set range, the dissolved oxygen is controlled to be relatively stable when fermentation enters a production stability period, fermentation titer and yield curve inflection points are monitored in the later period of fermentation, and a lower tank is selected for proper time;
the formula of the seed culture medium is as follows: 10.0 to 20.0g/L of soybean cake powder, 1.0 to 5.0g/L of yeast powder, 0.5 to 1.0g/L of fish meal, 1.0 to 3.0g/L of sodium chloride, 3.0g/L of ammonium sulfate, 10.0 to 30.0g/L of soybean oil, 2.0g/L, GPE of monopotassium phosphate and 0.01 to 0.03g/L of defoamer;
in the step 1), the material proportion of the fermentation medium is as follows: 20.0 to 40.0g/L of soybean cake powder, 10.0 to 30.0g/L of corn steep liquor or corn steep liquor dry powder, 1.0 to 5.0g/L of sodium chloride, 5.0 to 1.0g/L of plant peptone, 10.0 to 30.0g/L of soybean oil, 0.5 to 1.0g/L, GPE of potassium dihydrogen phosphate and 0.03 to 0.05g/L of defoamer are also fixedly contained;
in the step 2), inoculating mature streptomycete parvulus seeds as microscopic hyphae stretching and branching net-forming shake flask seeds, or using microscopic spore mature test tube inclined planes or eggplant-shaped bottles; in step 2), the culture conditions of the primary seed tank: the air volume coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 60%, the rotating speed is 200-450rmp, and the culture period is 24-48 hours; first-level seed qualification index: the pH value is between 7.2 and 7.7, and the microscopic hyphae are stretched and branched to form a sparse net shape;
in the step 3), each culture condition of the secondary seeds: the air volume coefficient is 0.6-1.0, the temperature is 29.0-31.0 ℃, the dissolved oxygen is more than 10%, the rotating speed is 200-350rmp, and the culture period is 18-36h; second grade seed qualification index: the pH value is between 6.8 and 7.5, and the hypha branches are microscopic to be broken and are in a firm and dense net shape;
in step 4), fermenting each culture condition: the air quantity coefficient is 0.5-0.8, the temperature is 26.0-30.0 ℃, the dissolved oxygen is more than 60%, the rotating speed is 80-150 rpm, and the pH value is controlled to be 6.5-7.0 after ammonia is supplemented; sugar and ammonia are added in the early fermentation period for 24-36h, and the whole process of the industrial ammonia water flow adding rate is controlled to be 0.15-0.3kg/t.h -1 45% -55% liquid sugar maximum feeding rate 1.4-1.8kg/t.h -1 The method comprises the steps of carrying out a first treatment on the surface of the The dissolved oxygen is controlled between 55% and 75% by using the conditions of the feeding rate, the air quantity and the rotating speed in the middle fermentation period of 90-120 hours, and the dissolved oxygen is controlled between 75% and 85% after 140 hours in the later fermentation period; the fermentation yield fluctuation is more than 0.45kg/t every 8 hours in the later fermentation period, the fermentation period is prolonged and is lower than 0.3kg/t, the fermentation period is controlled between 210 and 230 hours, and the preparation is carried out.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105624240A (en) * 2014-11-28 2016-06-01 牡丹江佰佳信生物科技有限公司 Kasugamycin fermentation medium and fermentation method thereof
WO2016101720A1 (en) * 2014-12-24 2016-06-30 浙江海正药业股份有限公司 Method for preparing stallimycin
CN106011201A (en) * 2016-07-01 2016-10-12 宁夏泰瑞制药股份有限公司 Culture medium for producing kasugamycin by fermenting actinomyces microaureus and culture method
CN107815478A (en) * 2016-09-12 2018-03-20 牡丹江佰佳信生物科技有限公司 Utilize the method for streptomyces microaureus BJX007 fermenting and producing kasugarnycin
CN107828702A (en) * 2017-12-15 2018-03-23 陕西麦可罗生物科技有限公司 A kind of kasugarnycin fermentation medium and fermentation process
CN111184030A (en) * 2020-01-09 2020-05-22 枣庄市杰诺生物酶有限公司 Kasugamycin raw powder and preparation method and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105624240A (en) * 2014-11-28 2016-06-01 牡丹江佰佳信生物科技有限公司 Kasugamycin fermentation medium and fermentation method thereof
WO2016101720A1 (en) * 2014-12-24 2016-06-30 浙江海正药业股份有限公司 Method for preparing stallimycin
CN106011201A (en) * 2016-07-01 2016-10-12 宁夏泰瑞制药股份有限公司 Culture medium for producing kasugamycin by fermenting actinomyces microaureus and culture method
CN107815478A (en) * 2016-09-12 2018-03-20 牡丹江佰佳信生物科技有限公司 Utilize the method for streptomyces microaureus BJX007 fermenting and producing kasugarnycin
CN107828702A (en) * 2017-12-15 2018-03-23 陕西麦可罗生物科技有限公司 A kind of kasugarnycin fermentation medium and fermentation process
CN111184030A (en) * 2020-01-09 2020-05-22 枣庄市杰诺生物酶有限公司 Kasugamycin raw powder and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
春雷霉素生产发酵工艺优化的研究;任朝君 等;中小企业管理与科技(中旬刊);241 *

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