阅读说明：本技术 一种春雷霉素发酵培养基及发酵方法 (Kasugamycin fermentation medium and fermentation method ) 是由 王晓东 潘忠成 邓钊 陈豪 翁婧 李蒲民 于 2021-11-16 设计创作，主要内容包括：本发明涉及一种春雷霉素发酵培养基及发酵方法,属于抗生素发酵生产领域,该培养基按体积百分比计算,由以下两大部分构成,发酵基础配方：黄豆饼粉2.5-6.0%,磷酸二氢钾0.05-0.1%,氯化钠0.1-0.5%,玉米油0.5-1.0%,柠檬酸钠0.15-0.25%,玉米浆干粉0.5-1.0%,GPE0.015-0.025%,pH：自然,补料配方：补料配方：葡萄糖粉0.5-1.0%,柠檬酸钠0.15-0.25%,氨水(浓度25%,用于调节pH)0.15-0.25%。本发明通过在培养基中添加柠檬酸钠,使春雷霉素效价提高15%以上,糖耗降低25%以上,缩短发酵周期36-48小时,极大的降低了生产成本,提高了生产效率。(The invention relates to a kasugamycin fermentation medium and a fermentation method, belonging to the field of antibiotic fermentation production, wherein the medium comprises the following two parts by volume percent: 2.5-6.0% of soybean cake powder, 0.05-0.1% of monopotassium phosphate, 0.1-0.5% of sodium chloride, 0.5-1.0% of corn oil, 0.15-0.25% of sodium citrate, 0.5-1.0% of corn steep liquor dry powder, 0.015-0.025% of GPE, and pH: naturally, the formula of the feed supplement is as follows: the formula of the feed supplement is as follows: 0.5-1.0% of glucose powder, 0.15-0.25% of sodium citrate and 0.15-0.25% of ammonia water (with the concentration of 25% for adjusting the pH). According to the invention, sodium citrate is added into the culture medium, so that the kasugamycin titer is improved by more than 15%, the sugar consumption is reduced by more than 25%, the fermentation period is shortened by 36-48 hours, the production cost is greatly reduced, and the production efficiency is improved.)

1. A kasugamycin fermentation medium is characterized by comprising the following components in percentage by volume; 2.5-6.0% of soybean cake powder, 0.05-0.1% of monopotassium phosphate, 0.1-0.5% of sodium chloride, 0.5-1.0% of glucose powder, 0.15-0.25% of ammonia water, 0.5-1.0% of corn oil, 0.15-0.25% of sodium citrate, 0.5-1.0% of corn steep liquor dry powder, 0.015-0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE, and pH: 6.8-7.3.

2. The kasugamycin fermentation medium according to claim 1, wherein the ammonia water has a mass concentration of 25% for adjusting the pH.

3. The kasugamycin fermentation medium according to claim 1, wherein the medium comprises 3.0% of soybean cake powder, 0.075% of monopotassium phosphate, 0.3% of sodium chloride, 0.75% of corn oil, 0.8% of corn steep liquor dry powder, 0.02% of polyoxypropylene polyoxyethylene glycerol ether GPE, 0.2% of ammonia water and 0.2% of sodium citrate.

4. The method for preparing kasugamycin fermentation medium according to any one of claims 1 to 3, comprising the following steps:

accurately weighing soybean cake powder, monopotassium phosphate, sodium chloride, corn oil, corn steep liquor dry powder and sodium citrate, adding GPE, and fixing the volume by using tap water;

preparing glucose solution, subpackaging with ammonia water, and fixing the volume with tap water;

③ the pH is natural before the elimination.

5. A fermentation method of a kasugamycin fermentation medium is characterized by comprising the following steps:

(1) preparing a fermentation basal culture medium, wherein the formula of the fermentation basal culture medium is as follows: 2.5-6.0% of soybean cake powder, 0.05-0.1% of monopotassium phosphate, 0.1-0.5% of sodium chloride, 0.5-1.0% of corn oil, 0.5-1.0% of corn steep liquor dry powder, 0.015-0.025% of GPE, and the volume of the soybean cake powder is determined by tap water, and the pH value is: naturally; preparing a supplemented medium, and preparing glucose powder into a glucose solution, wherein the mass percent of the glucose powder is 0.5-1.0%; then adding sodium citrate into the glucose solution, wherein the mass percent of the sodium citrate is 0.15-0.25%, and then adding 0.15-0.25% ammonia water to adjust the pH value;

(2) and (3) sterilization: maintaining the fermentation basal culture medium at 121-130 deg.C for 30 min; maintaining the pressure of the feed medium at 110-120 ℃ for 30 min;

(3) inoculating, cooling to 28-30 ℃ after sterilization, inoculating streptomyces aureofaciens with the inoculation amount of 5-15 vol%;

(4) fermentation conditions are as follows: temperature: the aeration ratio is 1:0.5-1:1.5 at 28-30 ℃, the stirring speed is 150-; after fermentation is finished, collecting a fermentation product to obtain a fermentation filtrate;

(5) and (3) fermentation material supplementing conditions: fermenting and culturing for 20-30 hr, when pH is reduced to 6.8-7.3, continuously adding glucose + sodium citrate mixed solution and ammonia water, and maintaining pH at 6.8-7.3.

6. The fermentation method according to claim 5, wherein in the step (1), the mass concentration of ammonia water is 25% for adjusting the pH.

7. The fermentation process of claim 5, wherein the kasugamycin is inoculated in an amount of 15%.

8. The fermentation method of claim 5, wherein the sodium citrate is added to the kasugamycin feed medium in an amount of 0.20 to 0.25%.

Technical Field

The invention belongs to the field of antibiotic fermentation production, and particularly relates to a kasugamycin fermentation medium and a fermentation method.

Background

Kasugamycin is produced by streptomyces vernalis, belongs to aminoglycoside antibiotics, is widely applied to agriculture, has particularly remarkable prevention and treatment effect on rice blast, and is also widely applied to apples, potatoes, oranges and the like. It is harmless to human and livestock, has no residue and no pollution, is one of the current green pesticides with application prospect, and is deeply welcomed by domestic and foreign users.

The kasugamycin is produced by adopting a liquid fermentation method, and the existing fermentation process has the following problems:

1) the industrial fermentation level is low, and the fermentation titer is about 12000 ug/mL;

2) the fermentation period is relatively long, and is usually about 250 hours;

3) high production energy consumption, high cost, high control requirement and lack of market competitiveness.

Chinese patent application CN201810817178 discloses a method for improving the biological value of kasugamycin.A common culture medium of streptomyces aureofaciens consists of soybean meal powder, NaCl, corn steep liquor dry powder, maltose, fish oil, KHPO and inositol, and growth factors and trace elements are added on the basis of the common culture medium. The streptomyces aureofaciens is cultured in the culture medium added with growth factors and trace elements, the kasugamycin titer is improved by 19.7 percent, and the single-pot yield is improved by 10.8 percent. The culture medium is used for culturing the streptomyces aureofaciens, so that the biological value of the kasugamycin and the yield of a single tank can be obviously improved.

Chinese patent CN201410707264 discloses a kasugamycin fermentation medium, wherein the fermentation medium contains Tween 80 and a defoaming agent, and the defoaming agent is a silicone defoaming agent and/or a polyether defoaming agent. The kasugamycin fermentation medium provided by the invention obviously reduces the generation amount of foam in the liquid fermentation process through the synergistic effect of the Tween 80, the defoaming agent and the raw materials of the medium, so that the transfer efficiency of oxygen is improved, the liquid escape and the bacterial contamination caused by a large amount of foam are better avoided, and the fermentation yield of kasugamycin can be obviously improved by utilizing the fermentation medium and the culture method provided by the invention.

Chinese patent application CN201711346595 relates to a kasugamycin fermentation medium and a fermentation method, and comprises the following steps: (1) preparing a culture medium, disinfecting the culture medium and sterilizing; the formula of the culture medium is as follows: 5.0-8.0% of soybean meal powder; NaCl: 0.3-0.5%; dry corn steep liquor powder: 0.5-1.0%; maltose: 2.0-2.5%; fish oil: 3.5-4.0%; KHPO: 0.03-0.05%; inositol: 0.01 to 0.05 percent; the pH value is 6.8-7.2; (2) inoculating a small streptomyces aureofaciens strain in a culture medium, wherein the inoculation amount is 10-15 vol%, fermenting at 28-30 ℃ for 168-170 h, filtering a fermentation product, and taking a filtrate to obtain the product. The invention improves the fermentation titer of the kasugamycin and shortens the fermentation period, thereby reducing the production cost and improving the product quality.

These studies have attempted to solve the problems associated with the prior art kasugamycin fermentation processes from different perspectives, but are still not comprehensive enough, and there is a need in the art for more effective and optimized solutions.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a kasugamycin fermentation medium and a fermentation method, and the method effectively improves the titer of kasugamycin, reduces sugar consumption, shortens the fermentation period, greatly reduces the production cost and improves the production efficiency.

In order to realize the purpose of the invention, the invention adopts the following technical scheme: a kasugamycin fermentation medium comprises the following components in percentage by volume; 2.5-6.0% of soybean cake powder, 0.05-0.1% of monopotassium phosphate, 0.1-0.5% of sodium chloride, 0.5-1.0% of glucose powder, 0.15-0.25% of ammonia water, 0.5-1.0% of corn oil, 0.15-0.25% of sodium citrate, 0.5-1.0% of corn steep liquor dry powder, 0.015-0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE, and pH: 6.8-7.3.

In a preferred embodiment of the invention, ammonia is used to adjust the pH at a mass concentration of 25%.

In a preferred embodiment of the invention, the culture medium comprises 3.0% of soybean cake powder, 0.075% of potassium dihydrogen phosphate, 0.3% of sodium chloride, 0.75% of corn oil, 0.8% of corn steep liquor dry powder, 0.02% of polyoxypropylene polyoxyethylene glycerol ether GPE, 0.2% of ammonia water and 0.2% of sodium citrate.

In a preferred embodiment of the present invention, the preparation method of the kasugamycin fermentation medium comprises:

accurately weighing soybean cake powder, monopotassium phosphate, sodium chloride, corn oil, corn steep liquor dry powder and sodium citrate, adding GPE, and fixing the volume by using tap water;

preparing glucose solution, subpackaging with ammonia water, and fixing the volume with tap water;

③ the pH is natural before the elimination.

The invention also provides a fermentation method of the kasugamycin fermentation medium, which comprises the following steps:

(1) preparing a fermentation basal culture medium, wherein the formula of the fermentation basal culture medium comprises the following components in percentage by volume: 2.5-6.0% of soybean cake powder, 0.05-0.1% of monopotassium phosphate, 0.1-0.5% of sodium chloride, 0.5-1.0% of corn oil, 0.5-1.0% of corn steep liquor dry powder, 0.015-0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE, and the volume of the soybean cake powder is determined by tap water, and the pH value is as follows: naturally; preparing a supplemented medium, and preparing glucose powder into a glucose solution, wherein the mass percent of the glucose powder is 0.5-1.0%; then adding sodium citrate into the glucose solution, wherein the mass percent of the sodium citrate is 0.15-0.25%, and then adding 0.15-0.25% ammonia water to adjust the pH value;

(2) and (3) sterilization: maintaining the fermentation basal culture medium at 121-130 deg.C for 30 min; maintaining the fed-batch culture medium at 110 deg.C for 30 min;

(3) inoculating, cooling to 30 ℃ after sterilization, inoculating streptomyces aureofaciens with the inoculation amount of 5-15 vol%;

(4) fermentation conditions are as follows: temperature: the aeration ratio is 1:0.5-1:1.5 at 28-30 ℃, the stirring speed is 150-; after fermentation is finished, collecting a fermentation product to obtain a fermentation filtrate;

(5) and (3) fermentation material supplementing conditions: fermenting and culturing for 20-30 hr, when pH is reduced to 6.8-7.3, continuously adding glucose + sodium citrate mixed solution and ammonia water, and maintaining pH at 6.8-7.3.

In a preferred embodiment of the present invention, in step (1), the ammonia water has a mass concentration of 25% for adjusting the pH.

In a preferred embodiment of the invention, the kasugamycin is inoculated in an amount of 15%.

In a preferred embodiment of the invention, sodium citrate is added to the kasugamycin feed medium in an amount of 0.20 to 0.25%.

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

in one aspect, the fermentation medium of the present invention consists of two major components, namely a fermentation base formulation (soybean cake powder 2.5-6.0%, monopotassium phosphate 0.05-0.1%, sodium chloride 0.1-0.5%, corn oil 0.5-1.0%, sodium citrate 0.15-0.25%, corn steep liquor dry powder 0.5-1.0%, polyoxypropylene polyoxyethylene glycerol ether GPE0.015-0.025%, pH: natural) and a supplement formulation (glucose powder 0.5-1.0%, sodium citrate 0.15-0.25%, ammonia 0.15-0.25%). According to the invention, the fermentation titer of kasugamycin is effectively improved by adding the sodium citrate into the kasugamycin feed supplement culture medium.

On the other hand, the fermentation method and the fermentation parameters are optimized aiming at the specific fermentation culture medium composition, the auxiliary materials are added at one time, the actual operation and the trouble are reduced, the culture medium and the fermentation method are adopted to produce the kasugamycin, the titer of the kasugamycin is improved by more than 20 percent, the sugar consumption is reduced by more than 25 percent, the fermentation period is shortened by 36-48 hours, and the production cost is greatly saved.

Detailed Description

The present invention will be described in further detail with reference to examples, but the present invention is not limited to the following examples.

Example 1

(1) Preparing a culture medium, preparing 35L of a basic culture medium according to the formula ratio of the kasugamycin liquid fermentation culture medium, and putting the basic culture medium into a 50L fermentation tank for later use. The formula of the culture medium is as follows: the culture medium comprises a basic culture medium, 2.5% of bean cake powder, 0.05% of potassium dihydrogen phosphate, 0.1% of sodium chloride, 0.5% of corn oil, 0.5% of corn steep liquor dry powder, 0.015% of polyoxypropylene polyoxyethylene glycerol ether GPE, natural pH, a supplemented culture medium, 0.5% of glucose powder, 0.15% of sodium citrate, tap water, 5L of mixed constant volume for later use, and 1L of ammonia water (25% concentration) for later use.

(2) And (3) sterilization: the basic culture medium is sterilized for 30min at 121 ℃, the supplementary culture medium is mixed with the glucose and sodium citrate culture medium for 30min at 110 ℃, and ammonia water is not sterilized.

(3) Inoculating, cooling the temperature of the fermentation tank to 30 ℃, inoculating 15vol% of streptomyces aureofaciens seed liquid, and performing fermentation culture.

(4) Fermentation culture: temperature 28 ℃, aeration ratio 1:1, stirring at the rotation speed of 150rpm, tank pressure of 0.05MPa, fermenting and culturing for 24 hours, feeding a glucose and sodium citrate mixed solution when the pH is 7.0, naturally feeding ammonia water, controlling the pH to be 6.8-7.3, fermenting for 160 hours, collecting a fermentation product, filtering to obtain a filtrate, and measuring 13800u/mL by adopting a liquid chromatography, the sugar consumption is 0.5 percent and the ammonia water is 205 mL.

For comparison, under the above operating conditions, when glucose and sodium citrate are not added when the pH is 7.0, ammonia water is naturally fed to control the pH to be 6.8-7.3, the fermentation period is 160h, the fermentation product is collected and filtered to obtain filtrate, and the filtrate is measured by liquid chromatography to obtain 12900u/mL which is far lower than the titer of the application.

Example 2

(1) Preparing a culture medium, preparing 35L of the culture medium according to the formula ratio of the kasugamycin liquid fermentation culture medium, and putting the culture medium into a 50L fermentation tank for later use. The formula of the culture medium is as follows: the culture medium comprises a basic culture medium, 3.5% of bean cake powder, 0.05% of potassium dihydrogen phosphate, 0.1% of sodium chloride, 0.5% of corn oil, 0.5% of corn steep liquor dry powder, 0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE, natural pH, a feed supplement culture medium, 0.8% of glucose powder and 0.20% of sodium citrate, wherein the glucose powder and the sodium citrate are uniformly mixed by tap water to a constant volume of 5L for later use, and 1L of ammonia water (the concentration is 25%) for later use.

(2) And (3) sterilization: the basic culture medium is sterilized for 30min at 121 ℃, the supplementary culture medium is mixed with the glucose and sodium citrate culture medium for 30min at 110 ℃, and ammonia water is not sterilized.

(3) Inoculating, cooling the temperature of the fermentation tank to 30 ℃, inoculating 15vol% of streptomyces aureofaciens seed liquid, and performing fermentation culture.

(4) Fermentation culture: temperature 28 ℃, aeration ratio 1:1, stirring at the rotation speed of 150rpm, tank pressure of 0.05MPa, fermentation culture for 24 hours, feeding a glucose and sodium citrate mixed solution when the pH is 7.1, naturally feeding ammonia water at the controlled pH of 6.8-7.3 for a fermentation period of 160 hours, collecting a fermentation product, filtering to obtain a filtrate, and measuring 13904u/mL, sugar consumption of 0.8% and ammonia water of 217mL by liquid chromatography.

For comparison, under the above operating conditions, when the pH is 7.1, the glucose + sodium citrate group is not added, ammonia water is naturally fed and pH is controlled to be 6.8-7.3, the fermentation period is 160h, the fermentation product is collected and filtered to obtain filtrate, and 12560u/mL is measured by liquid chromatography and is far lower than the titer of the application.

Example 3

(1) Preparing a culture medium, preparing 35L of the culture medium according to the formula ratio of the kasugamycin liquid fermentation culture medium, and putting the culture medium into a 50L fermentation tank for later use. The formula of the culture medium is as follows: the culture medium comprises a basic culture medium, 4.5% of bean cake powder, 0.05% of potassium dihydrogen phosphate, 0.1% of sodium chloride, 0.5% of corn oil, 0.8% of corn steep liquor dry powder, 0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE and natural pH, wherein 1.0% of glucose powder and 0.25% of sodium citrate are weighed in a supplemented culture medium, tap water is used for uniformly mixing and metering the volume to 5L for later use, and 1L of ammonia water (the concentration is 25%) is used for later use.

(2) And (3) sterilization: the basic culture medium is sterilized for 30min at 121 ℃, the supplementary culture medium is mixed with the glucose and sodium citrate culture medium for 30min at 110 ℃, and ammonia water is not sterilized.

(3) Inoculating, cooling the temperature of the fermentation tank to 30 ℃, inoculating 15vol% of streptomyces aureofaciens seed liquid, and performing fermentation culture.

(4) Fermentation culture: temperature 28 ℃, aeration ratio 1:1, stirring at the rotation speed of 150rpm, tank pressure of 0.05MPa, fermenting and culturing for 24 hours, feeding a mixed solution of glucose and sodium citrate when the pH is 7.2, naturally feeding ammonia water at the controlled pH of 6.8-7.3 for a fermentation period of 160 hours, collecting a fermentation product, filtering to obtain a filtrate, and measuring 13900u/mL, sugar consumption of 1.0% and ammonia water of 225mL by adopting a liquid chromatography.

Example 4

(1) Preparing a culture medium, preparing 35L of the culture medium according to the formula ratio of the kasugamycin liquid fermentation culture medium, and putting the culture medium into a 50L fermentation tank for later use. The formula of the culture medium is as follows: the culture medium comprises a basic culture medium, 5.5% of bean cake powder, 0.05% of potassium dihydrogen phosphate, 0.1% of sodium chloride, 1.0% of corn oil, 1.0% of corn steep liquor dry powder, 0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE and natural pH, wherein 1.0% of glucose powder and 0.25% of sodium citrate are weighed in a supplemented culture medium, tap water is used for uniformly mixing and metering the volume to 5L for later use, and 1L of ammonia water (the concentration is 25%) is used for later use.

(2) And (3) sterilization: the basic culture medium is sterilized for 30min at 121 ℃, the supplementary culture medium is mixed with the glucose and sodium citrate culture medium for 30min at 110 ℃, and ammonia water is not sterilized.

(3) Inoculating, cooling the temperature of the fermentation tank to 30 ℃, inoculating 15vol% of streptomyces aureofaciens seed liquid, and performing fermentation culture.

(4) Fermentation culture: temperature 28 ℃, aeration ratio 1:1, stirring at the rotation speed of 150rpm, tank pressure of 0.03MPa, fermentation culture for 24 hours, feeding a glucose and sodium citrate mixed solution when the pH is 6.9, naturally feeding ammonia water at the controlled pH of 6.8-7.3, fermenting for 160 hours, collecting a fermentation product, filtering to obtain a filtrate, and measuring 14007u/mL, sugar consumption of 1.0% and 253mL by adopting a liquid chromatography.

Example 5

(1) Preparing a culture medium, preparing 35L of the culture medium according to the formula ratio of the kasugamycin liquid fermentation culture medium, and putting the culture medium into a 50L fermentation tank for later use. The formula of the culture medium is as follows: the culture medium comprises a basic culture medium, 5.5% of bean cake powder, 0.1% of potassium dihydrogen phosphate, 0.1% of sodium chloride, 1.0% of corn oil, 1.0% of corn steep liquor dry powder, 0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE and natural pH, wherein 1.0% of glucose powder and 0.25% of sodium citrate are weighed in a supplemented culture medium, tap water is used for uniformly mixing and metering the volume to 5L for later use, and 1L of ammonia water (the concentration is 25%) is used for later use.

(2) And (3) sterilization: the basic culture medium is sterilized for 30min at 121 ℃, the supplementary culture medium is mixed with the glucose and sodium citrate culture medium for 30min at 110 ℃, and ammonia water is not sterilized.

(3) Inoculating, cooling the temperature of the fermentation tank to 30 ℃, inoculating 15vol% of streptomyces aureofaciens seed liquid, and performing fermentation culture.

(4) Fermentation culture: temperature 28 ℃, aeration ratio 1:1, stirring at the rotation speed of 150rpm, tank pressure of 0.05MPa, fermenting and culturing for 24 hours, feeding a mixed solution of glucose and sodium citrate when the pH is 6.8, naturally feeding ammonia water at the controlled pH of 6.8-7.3, fermenting for 160 hours, collecting a fermentation product, filtering to obtain a filtrate, and measuring 14507u/mL, sugar consumption of 1.0% and ammonia water of 230mL by adopting a liquid chromatography.

Example 6

(1) Preparing a culture medium, preparing 35L of the culture medium according to the formula ratio of the kasugamycin liquid fermentation culture medium, and putting the culture medium into a 50L fermentation tank for later use. The formula of the culture medium is as follows: the culture medium comprises a basic culture medium, 5.5% of bean cake powder, 0.1% of potassium dihydrogen phosphate, 0.1% of sodium chloride, 1.0% of corn oil, 1.0% of corn steep liquor dry powder, 0.025% of polyoxypropylene polyoxyethylene glycerol ether GPE and natural pH, wherein 1.0% of glucose powder and 0.25% of sodium citrate are weighed in a supplemented culture medium, tap water is used for uniformly mixing and metering the volume to 5L for later use, and 1L of ammonia water (the concentration is 25%) is used for later use.

(2) And (3) sterilization: the basic culture medium is sterilized for 30min at 121 ℃, the supplementary culture medium is mixed with the glucose and sodium citrate culture medium for 30min at 110 ℃, and ammonia water is not sterilized.

(3) Inoculating, cooling the temperature of the fermentation tank to 30 ℃, inoculating 15vol% of streptomyces aureofaciens seed liquid, and performing fermentation culture.

(4) Fermentation culture: temperature 28 ℃, aeration ratio 1: 0.8, stirring speed of 150rpm, tank pressure of 0.05MPa, fermentation culture for 24 hours, feeding glucose and sodium citrate mixed solution when pH is reduced to 6.8, naturally feeding ammonia water, controlling pH to 6.8-7.3, fermenting for 160 hours, collecting fermentation product, filtering to obtain filtrate, and measuring 14808u/mL, sugar consumption of 1.0% and ammonia water of 245mL by liquid chromatography.

Although the invention has been described in detail in the foregoing with reference to general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto based on the invention. Therefore, it is intended that the present invention cover the modifications and improvements made without departing from the spirit and scope of the present invention.