阅读说明：本技术 一种头孢菌素c的发酵方法 (Fermentation method of cephalosporin C ) 是由 党建宁 王强 寇韩涛 杨丽丽 赵佳美 何彬 于 2020-07-21 设计创作，主要内容包括：本发明公开了一种头孢菌素C的发酵方法,具体步骤包括：采用顶头孢霉菌依次制备一级种子、二级种子、三级种子,然后将得到的三级种子进行发酵的同时在培养基中流加玉米浆,制得发酵液,然后将发酵液分离纯化,制得到头孢菌素C。本发明采用的玉米浆为湿磨法生产玉米淀粉的副产物,玉米浆中含有大量的亚硫酸、多肽、多糖、蛋白质及多种氨基酸如精氨酸、缬氨酸、组氨酸、苯丙氨酸、甲硫氨酸以及半胱氨酸等,这些成分均是发酵头孢菌素C的前体成分,在发酵过程中流加玉米浆能为菌体生长代谢提供全面的营养成分,从而达到提高头孢菌素C的发酵水平和头孢菌素C的发酵产量、降低发酵成本,具有优秀的产业化前景。(The invention discloses a fermentation method of cephalosporin C, which comprises the following specific steps: sequentially preparing a first-level seed, a second-level seed and a third-level seed by adopting acremonium, fermenting the obtained third-level seed, adding corn steep liquor into a culture medium at the same time to prepare fermentation liquor, and then separating and purifying the fermentation liquor to prepare the cephalosporin C. The corn steep liquor adopted by the invention is a byproduct of corn starch produced by a wet milling method, the corn steep liquor contains a large amount of sulfurous acid, polypeptide, polysaccharide, protein and a plurality of amino acids such as arginine, valine, histidine, phenylalanine, methionine, cysteine and the like, the components are precursor components of fermentation cephalosporin C, and the corn steep liquor added in the fermentation process can provide comprehensive nutrient components for the growth and metabolism of bacteria, so that the fermentation level of the cephalosporin C and the fermentation yield of the cephalosporin C are improved, the fermentation cost is reduced, and the method has excellent industrialization prospect.)

1. A fermentation method of cephalosporin C, which is characterized by comprising the following steps:

(1) preparation of first seed

Placing acremonium in an eggplant bottle inclined plane, preparing bacterial suspension with the mass-volume ratio of 20-25%, inoculating into a primary seed culture medium according to the inoculation amount of 1-2%, and culturing at 27-29 ℃ for 50h to obtain primary seeds;

(2) preparation of Secondary seeds

Inoculating the primary seeds obtained in the step (1) into a secondary seed culture medium according to the inoculation amount of 8-10%, and culturing for 45-50h under the conditions that the pH is 6.5-7.4 and the temperature is 27-29 ℃ to obtain secondary seeds;

(3) preparation of tertiary seeds

Inoculating the secondary seeds obtained in the step (2) into a tertiary seed culture medium according to the inoculation amount of 8-10%, and culturing for 45-50h under the conditions that the pH is 6.5-7.4 and the temperature is 27-29 ℃ to obtain tertiary seeds;

(4) fermentation of

Inoculating the three-stage seeds obtained in the step (3) into a fermentation culture medium according to the inoculation amount of 25-33% for culture, starting to feed corn steep liquor after fermenting and culturing for 40-100h in the fermentation process, and fermenting for 130h to obtain fermentation liquor;

(5) controlling the flow of the fermentation liquor obtained in the step (4) through ceramic membrane filtration in turn to be 15-30m³The flow rate of nanofiltration concentration is controlled to be 60-80m³The flow rate of resin adsorption is controlled to be 50-80m³The flow rate of analysis, separation and purification is controlled to be 30-60m³And h, obtaining the cephalosporin C.

2. The process for the fermentative production of cephalosporin C according to claim 1, wherein in step (1) the composition of the primary seed culture medium is: 18-32g/L of glucose syrup, 8-15g/L of soybean oil, 50-100g/L of corn steep liquor, 7.5-15g/L of dextrin, 18-36g/L of white granulated sugar, 20-40g/L of soybean cake powder, 20-40g/L of peanut cake powder and 3.6-7.2g/L of light calcium carbonate.

3. The process for the fermentative production of cephalosporin C according to claim 1, wherein in step (2) the secondary seed culture medium has the composition: 22-32g/L of soybean cake powder, 22-32g/L of white granulated sugar, 8-12g/L of dextrin, 22-32g/L of peanut cake powder, 4-6g/L of light calcium carbonate, 50-100g/L of corn steep liquor, 18-32g/L of glucose syrup and 8-15g/L of soybean oil.

4. The process for the fermentative production of cephalosporin C according to claim 1, characterised in that in step (3) the composition of the tertiary seed medium is: 9-13g/L of ammonium sulfate, 4-6g/L of anhydrous calcium sulfate, 9-13g/L, DL-methionine, 2-4g/L of wheat gluten, 8-12g/L of corn flour, 12-18g/L of dextrin, 12-16g/L of peanut cake powder, 4-7g/L of light calcium carbonate, 50-100g/L of corn steep liquor, 14-18g/L of glucose syrup and 20-30g/L of soybean oil.

5. The process for the fermentative production of cephalosporin C according to claim 1, characterised in that in step (4) the fermentation medium has a composition of: 10-26g/L of ammonium sulfate, 10-13g/L of anhydrous calcium sulfate, 20-26g/L, DL g/L of wheat gluten, 15-20g/L of corn flour, 17-22g/L of dextrin, 16-20g/L of peanut cake powder, 5-8g/L of light calcium carbonate, 50-100g/L of corn steep liquor, 10-60g/L of soybean oil and 10-60g/L of cottonseed oil.

6. The process for the fermentative production of cephalosporin C according to claim 1, wherein in step (4), the corn steep liquor is supplemented by the specific steps of:

fermenting for 40-80h to make the flow rate of supplemented corn steep liquor be 0.1-0.8L/m³H; when the fermentation is carried out for 80-100h, the flow rate of the supplemented corn steep liquor is 0.2-1.0L/m³·h。

7. The process for the fermentative production of cephalosporin C according to claim 6, characterised in that the dry matter concentration of the corn steep liquor is 100-400 g/L.

8. The process for the fermentative production of cephalosporin C according to claim 1, wherein the fermentation culture time in step (4) is 120-140 h.

9. The process for the fermentative production of cephalosporin C according to claim 1, characterised in that in step (4), the fermentation culture is carried out for 130 h; the temperature of fermentation is 28 ℃ for 0-40h, and the temperature of fermentation is 24 ℃ for 40-130 h; the fermentation pH was 5.6.

Technical Field

The invention relates to the field of microbial fermentation, in particular to a fermentation method of cephalosporin C.

Background

Cephalosporin C is produced by Cephalosporium acremonium (Cephalosporium acremonium), is a product of the continuation and development of the primary metabolism of Cephalosporium acremonium under specific conditions, and is another important beta-lactam antibiotic following penicillin. The main ways to improve the production level of cephalosporin C at present are: firstly, the original performance of the strain is changed, the high-yield strain is obtained by mutagenesis screening or genetic engineering technology, and secondly, the fermentation process is optimized.

At present, the fermentation level of cephalosporin C abroad is obviously higher than that of the cephalosporin C at home, so that the technical problem to be solved urgently by technical personnel in the field is to provide a novel fermentation method for fermenting cephalosporin C to improve the fermentation level, thereby improving the yield of cephalosporin C and reducing the fermentation cost.

Disclosure of Invention

In view of the above, the present invention provides a method for fermenting cephalosporin C;

in order to achieve the purpose, the invention adopts the following technical scheme:

a process for the fermentation of cephalosporin C comprising the steps of:

(1) preparation of first seed

Placing acremonium in an eggplant bottle inclined plane, preparing bacterial suspension with the mass-volume ratio of 20-25%, inoculating into a primary seed culture medium according to the inoculation amount of 1-2%, and culturing at 27-29 ℃ for 50h to obtain primary seeds;

(2) preparation of Secondary seeds

Inoculating the primary seeds obtained in the step (1) into a secondary seed culture medium according to the inoculation amount of 8-10%, and culturing for 45-50h under the conditions that the pH is 6.5-7.4 and the temperature is 27-29 ℃ to obtain secondary seeds;

(3) preparation of tertiary seeds

Inoculating the secondary seeds obtained in the step (2) into a tertiary seed culture medium according to the inoculation amount of 8-10%, and culturing for 45-50h under the conditions that the pH is 6.5-7.4 and the temperature is 27-29 ℃ to obtain tertiary seeds;

(4) fermentation of

Inoculating the three-stage seeds obtained in the step (3) into a fermentation culture medium according to the inoculation amount of 25-33% for culture, starting to feed corn steep liquor after fermenting and culturing for 40-100h in the fermentation process, and fermenting for 130h to obtain fermentation liquor;

(5) controlling the flow of the fermentation liquor obtained in the step (4) through ceramic membrane filtration in turn to be 15-30m³The flow rate of nanofiltration concentration is controlled to be 60-80m³The flow rate of resin adsorption is controlled to be 50-80m³The flow rate of analysis, separation and purification is controlled to be 30-60m³And h, obtaining the cephalosporin C.

Compared with the prior art, the invention has the beneficial technical effects that: the corn steep liquor adopted by the invention is a byproduct of corn starch produced by a wet milling method, the corn steep liquor contains a large amount of sulfurous acid, polypeptide, polysaccharide, protein and a plurality of amino acids such as arginine, valine, histidine, phenylalanine, methionine, cysteine and the like, the components are precursor components of fermentation cephalosporin C, and the corn steep liquor added in the fermentation process can provide comprehensive nutrient components for the growth and metabolism of bacteria, so that the fermentation level of the cephalosporin C and the fermentation yield of the cephalosporin C are improved, the fermentation cost is reduced, and the method has excellent industrialization prospect.

Further, in the step (1), the accession number of cephalosporium acremonium is ATCC 36225.

Further, in the step (1), the primary seed culture medium comprises: 18-32g/L of glucose syrup, 8-15g/L of soybean oil, 50-100g/L of corn steep liquor, 7.5-15g/L of dextrin, 18-36g/L of white granulated sugar, 20-40g/L of soybean cake powder, 20-40g/L of peanut cake powder and 3.6-7.2g/L of light calcium carbonate.

Further, in the step (2), the composition of the secondary seed culture medium is as follows: 22-32g/L of soybean cake powder, 22-32g/L of white granulated sugar, 8-12g/L of dextrin, 22-32g/L of peanut cake powder, 4-6g/L of light calcium carbonate, 50-100g/L of corn steep liquor, 18-32g/L of glucose syrup and 8-15g/L of soybean oil.

Further, in the step (3), the composition of the third-level seed culture medium is as follows: 9-13g/L of ammonium sulfate, 4-6g/L of anhydrous calcium sulfate, 9-13g/L, DL-methionine, 2-4g/L of wheat gluten, 8-12g/L of corn flour, 12-18g/L of dextrin, 12-16g/L of peanut cake powder, 4-7g/L of light calcium carbonate, 50-100g/L of corn steep liquor, 14-18g/L of glucose syrup and 20-30g/L of soybean oil.

Further, in the step (4), the fermentation medium comprises: 10-26g/L of ammonium sulfate, 10-13g/L of anhydrous calcium sulfate, 20-26g/L, DL g/L of wheat gluten, 15-20g/L of corn flour, 17-22g/L of dextrin, 16-20g/L of peanut cake powder, 5-8g/L of light calcium carbonate, 50-100g/L of corn steep liquor, 10-60g/L of soybean oil and 10-60g/L of cottonseed oil.

Further, in the step (4), the corn steep liquor supplementing step comprises:

fermenting for 40-80h to make the flow rate of supplemented corn steep liquor be 0.1-0.8L/m³H; when the fermentation is carried out for 80-100h, the flow rate of the supplemented corn steep liquor is 0.2-1.0L/m³H. (ii) a The dry matter concentration of the corn steep liquor is 100-400 g/L.

Preferably, in the step (4), the corn steep liquor is supplemented by the following specific steps:

corn steep liquor supplementing: fermenting for 40-80h to make the flow rate of supplemented corn steep liquor be 0.2-0.7L/m³H; when the fermentation is carried out for 80-100h, the flow rate of the supplemented corn steep liquor is 0.3-0.9L/m³·h。

The dry matter concentration of the corn steep liquor is 100-400 g/L.

Further, in the step (4), the conditions of the fermentation culture are as follows: the fermentation time is 130 h; the temperature of fermentation is 28 ℃ for 0-40h, and the temperature of fermentation is 24 ℃ for 40-130 h; the fermentation pH was 5.6.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.