阅读说明：本技术 一种发酵制备头孢菌素c的方法 (Method for preparing cephalosporin C through fermentation ) 是由 周如国 宋更申 高彬洁 顾洹 于 2019-12-09 设计创作，主要内容包括：本发明涉及一种发酵制备头孢菌素C的方法,包括如下步骤：(1)取顶头孢霉菌,制备一级种子、二级种子、三级种子；(2)取步骤(1)制备的三级种子,接种至发酵培养基中发酵,发酵过程中及时补加碳源、豆油、硫酸铵；其中,碳源为液体葡萄糖和秸秆液化糖的混合物；(3)将步骤(2)所得发酵液经过板框过滤后得到头孢菌素C溶液。本发明通过发酵过程中及时补加碳源、豆油、硫酸铵,使得头孢菌素C的效价达到了29951-35441U/mL,显著高于现有技术制备得到的头孢菌素C,同时,本发明发酵过程中补加的碳源为液体葡萄糖和秸秆液化糖的混合物代替了现有技术中的液体葡萄糖,不仅大大降低了生产成本,同时,也不会降低发酵液质量。(The invention relates to a method for preparing cephalosporin C by fermentation, which comprises the following steps: (1) taking cephalosporium acremonium, and preparing first-level seeds, second-level seeds and third-level seeds; (2) inoculating the three-stage seeds prepared in the step (1) into a fermentation culture medium for fermentation, and supplementing a carbon source, soybean oil and ammonium sulfate in time in the fermentation process; wherein the carbon source is a mixture of liquid glucose and straw liquefied sugar; (3) and (3) filtering the fermentation liquor obtained in the step (2) by using a plate frame to obtain a cephalosporin C solution. According to the invention, the carbon source, the soybean oil and the ammonium sulfate are timely supplemented in the fermentation process, so that the titer of the cephalosporin C reaches 29951-.)

1. A process for the fermentative preparation of cephalosporin C, characterized in that: it comprises the following steps:

(1) taking cephalosporium acremonium, and preparing first-level seeds, second-level seeds and third-level seeds;

(2) inoculating the three-stage seeds prepared in the step (1) into a fermentation culture medium for fermentation, and supplementing a carbon source, soybean oil and ammonium sulfate in time in the fermentation process;

the carbon source is a mixture of liquid glucose and straw liquefied sugar;

wherein the fermentation medium comprises the following components: using drinking water as medium, peanut cake powder 1.5-1.7g/L, glucose 0.17-0.19g/L, corn steep liquor 4.9-5.1g/L, dextrin 3.1-3.3g/L, soybean oil 5.0-5.2g/L, ammonium sulfate 0.63-0.65g/L, potassium dihydrogen phosphate 0.22-0.24g/L, magnesium sulfate heptahydrate 0.18-0.20g/L, light calcium carbonate 0.37-0.39g/L, copper sulfate pentahydrate 0.0019-0.0021g/L, zinc sulfate heptahydrate 0.0019-0.0021g/L, manganese sulfate monohydrate 0.0019-0.0021g/L, ferrous sulfate heptahydrate 0.0059-0.0061g/L, calcium sulfate 0.67-0.69g/L, DL-methionine 0.45-0.47g/L, wheat gluten 1.12-1.91 g/L, prion powder 1.93-1.93 g/L, wheat gluten powder 0.9-0.0021 g/L, 0.02-0.04g/L of antifoaming agent;

(3) and (3) filtering the fermentation liquor obtained in the step (2) by using a plate frame to obtain a cephalosporin C solution.

2. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that:

the fermentation medium in the step (2) comprises the following components: 1.69g/L of peanut cake powder, 0.18g/L of glucose, 5.00g/L of corn steep liquor, 3.21g/L of dextrin, 5.10g/L of soybean oil, 0.64g/L of ammonium sulfate, 0.23g/L of monopotassium phosphate, 0.19g/L of magnesium sulfate heptahydrate, 0.38g/L of light calcium carbonate, 0.002g/L of copper sulfate pentahydrate, 0.002g/L of zinc sulfate heptahydrate, 0.002g/L of manganese sulfate monohydrate, 0.006g/L of ferrous sulfate heptahydrate, 0.68g/L, DL-methionine, 1.13g/L of corn flour, 1.92g/L of wheat gluten and 0.03g/L of defoaming agent by taking domestic drinking water as a medium.

3. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that:

the primary seed culture medium in the step (1) comprises the following components: taking drinking water as a medium, 2.55g/L of peanut cake powder, 0.89g/L of glucose, 2.55g/L of soybean cake powder, 4.48g/L of corn steep liquor, 2.23g/L of cane sugar, 0.89g/L of dextrin, 0.95g/L of soybean oil, 0.45g/L of light calcium carbonate and 0.04g/L of defoaming agent;

the first-order seed preparation conditions were as follows: temperature 26-30 ℃, seed age: 80-100h, PH 6.8-7.8.

4. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that:

the secondary seed culture medium in the step (1) comprises the following components: taking drinking water as a medium, 2.77g/L of peanut cake powder, 0.97g/L of glucose, 2.78g/L of soybean cake powder, 4.86g/L of corn steep liquor, 2.43g/L of sucrose, 0.97g/L of dextrin, 0.10g/L of soybean oil, 0.49g/L of light calcium carbonate and 0.04g/L of defoaming agent;

preparation conditions of the second-level seeds: temperature 26-30 ℃, seed age: 45-55h and pH 6.8-7.8.

5. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that:

the tertiary seed culture medium in the step (1) comprises the following components: 2.52g/L of peanut cake powder, 0.88g/L of glucose, 2.52g/L of soybean cake powder, 4.77g/L of corn steep liquor, 2.23g/L of cane sugar, 0.44g/L of dextrin, 1.35g/L of soybean oil, 0.45g/L of light calcium carbonate and 0.03g/L of defoaming agent by taking domestic drinking water as a medium;

preparation conditions of the third-level seeds: temperature 26-30 ℃, seed age: 30-40h and pH 6.8-7.8.

6. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that: the fermentation conditions in the step (2) are as follows: fermentation time: 130-150 h; fermentation temperature: 27-29 ℃ before 40-50h and 24-26 ℃ after 40-50 h; the fermentation pH is 5.55-5.65.

7. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that: the mass ratio of the liquid glucose to the straw liquefied sugar in the carbon source in the step (2) is 1: 0.8-1.2.

8. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that: the process for supplementing the carbon source in the step (2) is as follows:

the carbon source is fed in at the following specific rate from 50 h:

50-54 h: 1.2 g/L.h, 55-59 h: 1.4 g/L.h, 60-64 h: 1.8g/L · h, 65-69 h: 2.3g/L · h, 70-110 h: 2.5 g/L.h, 111-115 h: 2.3 g/L.h, 116-120 h: 2.2 g/L.h, 121 h-end of fermentation: 2.0 g/L.multidot.h.

9. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that: the oil supplementing process in the step (2) is as follows: and when the fermentation is carried out for 65-75 hours, the soybean oil is started to be supplemented, the oil layer content is controlled to be 2-4%, and the soybean oil is stopped to be supplemented when the fermentation is stopped for 5-7 hours.

10. A process for the fermentative preparation of cephalosporin C according to claim 1, characterized in that: the ammonium sulfate supplementing process in the step (2) is as follows: when the ammonia nitrogen value is 1.5g/L, beginning to supplement ammonium sulfate, controlling the content of the ammonium sulfate to be 1.2-1.5g/L, and stopping supplementing the ammonium sulfate when fermenting for 85-95 h.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a method for preparing cephalosporin C by fermentation.

Background

Cephalosporin C (CPC) is β -lactam antibiotic, has the characteristics of wide antibacterial spectrum, high activity, good curative effect and low toxicity, is an antibiotic variety with the widest application at present, and a fermentation production strain of the cephalosporin C is Cephalosporium acremonium (CPC) which is a secondary metabolite of Cephalosporium acremonium.

The cephalosporin C is used as a semi-synthetic precursor of the cephalosporin antibiotics, the production level of the cephalosporin C determines the market supply amount of the cephalosporin antibiotics, and the cephalosporin C has important social and economic significance for the deep research and process optimization of a fermentation method for preparing the cephalosporin C. How to optimize the fermentation process to improve the fermentation titer, further improve the production efficiency and reduce the production cost becomes very important.

Disclosure of Invention

The invention aims to: a process for the fermentative preparation of cephalosporin C is provided.

In order to achieve the above purpose, the invention provides the following technical scheme:

a process for the fermentative preparation of cephalosporin C, which comprises the following steps:

(1) taking cephalosporium acremonium, and preparing first-level seeds, second-level seeds and third-level seeds;

(2) inoculating the three-stage seeds prepared in the step (1) into a fermentation culture medium for fermentation, and supplementing a carbon source, soybean oil and ammonium sulfate in time in the fermentation process;

the carbon source is a mixture of liquid glucose and straw liquefied sugar;

wherein the fermentation medium comprises the following components: using drinking water as medium, peanut cake powder 1.5-1.7g/L, glucose 0.17-0.19g/L, corn steep liquor 4.9-5.1g/L, dextrin 3.1-3.3g/L, soybean oil 5.0-5.2g/L, ammonium sulfate 0.63-0.65g/L, potassium dihydrogen phosphate 0.22-0.24g/L, magnesium sulfate heptahydrate 0.18-0.20g/L, light calcium carbonate 0.37-0.39g/L, copper sulfate pentahydrate 0.0019-0.0021g/L, zinc sulfate heptahydrate 0.0019-0.0021g/L, manganese sulfate monohydrate 0.0019-0.0021g/L, ferrous sulfate heptahydrate 0.0059-0.0061g/L, calcium sulfate 0.67-0.69g/L, DL-methionine 0.45-0.47g/L, wheat gluten 1.12-1.91 g/L, prion powder 1.93-1.93 g/L, wheat gluten powder 0.9-0.0021 g/L, 0.02-0.04g/L of antifoaming agent;

(3) and (3) filtering the fermentation liquor obtained in the step (2) by using a plate frame to obtain a cephalosporin C solution.

Preferably, the fermentation medium in step (2) comprises the following components: 1.69g/L of peanut cake powder, 0.18g/L of glucose, 5.00g/L of corn steep liquor, 3.21g/L of dextrin, 5.10g/L of soybean oil, 0.64g/L of ammonium sulfate, 0.23g/L of monopotassium phosphate, 0.19g/L of magnesium sulfate heptahydrate, 0.38g/L of light calcium carbonate, 0.002g/L of copper sulfate pentahydrate, 0.002g/L of zinc sulfate heptahydrate, 0.002g/L of manganese sulfate monohydrate, 0.006g/L of ferrous sulfate heptahydrate, 0.68g/L, DL-methionine, 1.13g/L of corn flour, 1.92g/L of wheat gluten and 0.03g/L of defoaming agent by taking domestic drinking water as a medium.

Preferably, the primary seed culture medium in step (1) comprises the following components: taking drinking water as a medium, 2.55g/L of peanut cake powder, 0.89g/L of glucose, 2.55g/L of soybean cake powder, 4.48g/L of corn steep liquor, 2.23g/L of cane sugar, 0.89g/L of dextrin, 0.95g/L of soybean oil, 0.45g/L of light calcium carbonate and 0.04g/L of defoaming agent;

the first-order seed preparation conditions were as follows: temperature 26-30 ℃, seed age: 80-100h, PH 6.8-7.8.

Preferably, the secondary seed culture medium in step (1) comprises the following components: taking drinking water as a medium, 2.77g/L of peanut cake powder, 0.97g/L of glucose, 2.78g/L of soybean cake powder, 4.86g/L of corn steep liquor, 2.43g/L of sucrose, 0.97g/L of dextrin, 0.10g/L of soybean oil, 0.49g/L of light calcium carbonate and 0.04g/L of defoaming agent;

preparation conditions of the second-level seeds: temperature 26-30 ℃, seed age: 45-55h and pH 6.8-7.8.

Preferably, the tertiary seed medium in step (1) comprises the following components: 2.52g/L of peanut cake powder, 0.88g/L of glucose, 2.52g/L of soybean cake powder, 4.77g/L of corn steep liquor, 2.23g/L of cane sugar, 0.44g/L of dextrin, 1.35g/L of soybean oil, 0.45g/L of light calcium carbonate and 0.03g/L of defoaming agent by taking domestic drinking water as a medium;

preparation conditions of the third-level seeds: temperature 26-30 ℃, seed age: 30-40h and pH 6.8-7.8.

Preferably, the fermentation conditions in step (2) are as follows: fermentation time: 130-150 h; fermentation temperature: 27-29 ℃ before 40-50h and 24-26 ℃ after 40-50 h; the fermentation pH is 5.55-5.65.

Preferably, the mass ratio of the liquid glucose to the straw liquefied sugar in the carbon source in the step (2) is 1: 0.8-1.2.

Preferably, the process for supplementing the carbon source in step (2) is as follows:

the carbon source is fed in at the following specific rate from 50 h:

50-54 h: 1.2 g/L.h, 55-59 h: 1.4 g/L.h, 60-64 h: 1.8g/L · h, 65-69 h: 2.3g/L · h, 70-110 h: 2.5 g/L.h, 111-115 h: 2.3 g/L.h, 116-120 h: 2.2 g/L.h, 121 h-end of fermentation: 2.0 g/L.multidot.h.

Preferably, the oil supplementing process in the step (2) is as follows: and when the fermentation is carried out for 65-75 hours, the soybean oil is started to be supplemented, the oil layer content is controlled to be 2-4%, and the soybean oil is stopped to be supplemented when the fermentation is stopped for 5-7 hours.

Preferably, the ammonium sulfate supplementing process in the step (2) is as follows: when the ammonia nitrogen value is 1.5g/L, beginning to supplement ammonium sulfate, controlling the content of the ammonium sulfate to be 1.2-1.5g/L, and stopping supplementing the ammonium sulfate when fermenting for 85-95 h.

The invention has the beneficial effects that:

according to the invention, the carbon source, the soybean oil and the ammonium sulfate are timely supplemented in the fermentation process, so that the titer of the prepared cephalosporin C reaches 29951-.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

A method for preparing cephalosporin C by fermentation, comprising the following steps:

(1) preparation of first seed

Primary seed culture medium: taking drinking water as a medium, 2.55g/L of peanut cake powder, 0.89g/L of glucose, 2.55g/L of soybean cake powder, 4.48g/L of corn steep liquor, 2.23g/L of cane sugar, 0.89g/L of dextrin, 0.95g/L of soybean oil, 0.45g/L of light calcium carbonate and 0.04g/L of defoaming agent;

taking Acremonium acremonium, activating, taking a bacterial liquid with the concentration of 120(mg/ml), inoculating the bacterial liquid to a seed culture medium according to the inoculation amount of 1% (v/v), and culturing for 90 hours at the temperature of 28 ℃ and the pH of 6.8-7.8.

(2) Preparation of Secondary seeds

Secondary seed culture medium: taking drinking water as a medium, 2.77g/L of peanut cake powder, 0.97g/L of glucose, 2.78g/L of soybean cake powder, 4.86g/L of corn steep liquor, 2.43g/L of sucrose, 0.97g/L of dextrin, 0.10g/L of soybean oil, 0.49g/L of light calcium carbonate and 0.04g/L of defoaming agent;

inoculating the first-class seed into seed culture medium at 5% (v/v) inoculum size, and culturing at 28 deg.C and pH of 6.8-7.8 for 50 h.

(3) Preparation of tertiary seeds

Third-level seed culture medium: 2.52g/L of peanut cake powder, 0.88g/L of glucose, 2.52g/L of soybean cake powder, 4.77g/L of corn steep liquor, 2.23g/L of cane sugar, 0.44g/L of dextrin, 1.35g/L of soybean oil, 0.45g/L of light calcium carbonate and 0.03g/L of defoaming agent by taking domestic drinking water as a medium;

inoculating the second-level seed into seed culture medium according to 1.5% (v/v) inoculum size, and culturing at 28 deg.C and pH of 6.8-7.8 for 35 h.

(4) Fermentation of

Fermentation medium: 1.69g/L of peanut cake powder, 0.18g/L of glucose, 5.00g/L of corn steep liquor, 3.21g/L of dextrin, 5.10g/L of soybean oil, 0.64g/L of ammonium sulfate, 0.23g/L of monopotassium phosphate, 0.19g/L of magnesium sulfate heptahydrate, 0.38g/L of light calcium carbonate, 0.002g/L of copper sulfate pentahydrate, 0.002g/L of zinc sulfate heptahydrate, 0.002g/L of manganese sulfate monohydrate, 0.006g/L of ferrous sulfate heptahydrate, 0.68g/L, DL-methionine, 1.13g/L of corn flour, 1.92g/L of wheat gluten and 0.03g/L of defoaming agent by taking domestic drinking water as a medium.

And (3) inoculating the third-level seeds prepared in the step (3) into a fermentation culture medium according to the inoculation amount of 18% (v/v) for fermentation for 140h, wherein the fermentation time is 28 ℃ for 0-45h, the fermentation time is 25 ℃ for 46-140h, and the pH value is 5.55-5.65.

Timely supplementing a carbon source, soybean oil and ammonium sulfate in the fermentation process;

wherein the carbon source is a mixture of liquid glucose and straw liquefied sugar in a mass ratio of 1: 1;

the process for supplementing the carbon source comprises the following steps:

the carbon source is fed in at the following specific rate from 50 h:

50-54h：1.2g/L·h，55-59h：1.4g/L·h，60-64h：1.8g/L·h，65-69h：2.3g/L·h，70-110h：2.5g/L·h，111-115h：2.3g/L·h，116-120h：2.2g/L·h，121h-140h：2.0g/L·h。

the oil supplementing process comprises the following steps: and (3) when the fermentation is carried out for 70 hours, the soybean oil is started to be supplemented, the oil layer content is controlled to be 2-4%, and the soybean oil is stopped to be supplemented when the fermentation is stopped for 6 hours.

The ammonium sulfate supplementing process comprises the following steps: when the ammonia nitrogen value is 1.5g/L, ammonium sulfate is started to be supplemented, the content of the ammonium sulfate is controlled to be 1.2-1.5g/L, and the ammonium sulfate is stopped to be supplemented when the fermentation is carried out for 90 hours.

(5) And (3) filtering: and filtering the fermentation liquor by using a plate frame to obtain filtrate. And (3) potency detection: adding 1mL of filtrate into a 50mL volumetric flask, adding water to the scale, shaking up to prepare a cephalosporin C mixed solution, and taking the prepared cephalosporin C, wherein the titer is detected to be 35441U/mL, and the concentration is 58%.