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,包括以下步骤：步骤(1)取顶头孢霉活化,取质量体积浓度为25％的菌液,按照1％的接种量接种至种子培养基中培养,在pH为7.0、29℃培养90h,获得一级种子液；步骤(2)取步骤(1)中制备的一级种子液,按照10％的接种量接种至种子培养基,在pH为6.5、28℃培养50h,获得二级种子液；步骤(3)取步骤(2)中制备的二级种子液,按照20％的接种量接种至发酵培养基中发酵,在发酵30-140h内流加全料,发酵140h后停止补全料,过滤获得发酵液；步骤(4)取步骤(3)中头孢菌素C液吸附,干燥,结晶,洗涤,干燥获得头孢菌素C；采用上述制备方法能够提高头孢菌素C的产率及效价。(The invention discloses a fermentation method of cephalosporin C, which comprises the following steps: activating cephalosporium acremonium, inoculating a bacterial liquid with the mass volume concentration of 25% into a seed culture medium according to the inoculation amount of 1% for culture, and culturing at the temperature of 29 ℃ and the pH value of 7.0 for 90 hours to obtain a primary seed liquid; step (2) taking the primary seed solution prepared in the step (1), inoculating the primary seed solution to a seed culture medium according to the inoculation amount of 10%, and culturing at the temperature of 28 ℃ for 50h at the pH value of 6.5 to obtain a secondary seed solution; step (3) inoculating the secondary seed liquid prepared in the step (2) into a fermentation culture medium according to the inoculation amount of 20% for fermentation, adding a complete material within 30-140h of fermentation, stopping supplementing the complete material after 140h of fermentation, and filtering to obtain a fermentation liquid; step (4) taking the cephalosporin C liquid obtained in the step (3) for adsorption, drying, crystallizing, washing and drying to obtain cephalosporin C; the preparation method can improve the yield and the titer of the cephalosporin C.)

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

preparing a first-stage seed solution, namely inoculating cephalosporium acremonium with the mass volume concentration of 20-25% into a seed culture medium according to the inoculation amount of 1-2% for culture, and culturing for 80-90h under the conditions that the pH is 7.0-8.0 and the temperature is 27-29 ℃ to obtain the first-stage seed solution;

preparing a secondary seed solution in the step (2), inoculating the primary seed solution prepared in the step (1) to a seed culture medium according to the inoculation amount of 8-10%, and culturing for 40-50h under the conditions that the pH is 6.5-7.5 and the temperature is 28-30 ℃ to obtain the secondary seed solution;

step (3) inoculating the secondary seed liquid prepared in the step (2) into a fermentation culture medium according to the inoculation amount of 20-30% for fermentation, adding complete materials within 30-140h of fermentation, stopping supplementing complete materials after 140h of fermentation, and filtering to obtain fermentation liquor;

and (4) separating and purifying the cephalosporin C liquid obtained in the step (3), adsorbing, drying, crystallizing, washing and drying to obtain the cephalosporin C.

2. The process for fermentation of cephalosporin C as claimed in claim 1, wherein the cephalospora acremonium of step (1) is cephalospora acremonium deposited under accession number ATCC.36225.

3. The method for fermenting cephalosporin C in claim 1, wherein the culture medium in step (1) comprises the following raw materials in parts by weight: 15-20 parts of corn steep liquor, 13-15 parts of glucose, 8-10 parts of white granulated sugar, 10-15 parts of soybean oil, 0.7-0.8 part of calcium carbonate and 0.5 part of defoaming agent.

4. The method for fermenting cephalosporin C in claim 1, wherein the culture medium in step (2) comprises the following raw materials in parts by weight: 20-30 parts of corn steep liquor, 4-6 parts of peanut powder, 4-6 parts of soybean meal, 3-6 parts of white granulated sugar, 5-7 parts of calcium sulfate, 8-20 parts of glucose, 30-46 parts of soybean oil, 4-5 parts of calcium carbonate and 0.5-0.8 part of defoaming agent.

5. The method for fermenting cephalosporin C in claim 1, wherein the fermentation medium in step (3) comprises the following raw materials in parts by weight: 25-50 parts of corn steep liquor, 5-13 parts of peanut powder, 5-12 parts of glucose, 15-25 parts of dextrin, 3-8 parts of methionine, 40-55 parts of soybean oil, 1.5-3 parts of defoaming agent and CaSO₄2-5 parts of (NH)₄)₂SO₄5-10 parts of FeSO₄0.05 to 0.1 portion of MnSO₄0.01 to 0.04 portion of ZnSO₄0.01-0.04 part of CuSO₄0.01 to 0.04 portion of CaCO₃5-10 parts.

6. The method for fermenting cephalosporin C of claim 1, wherein the fermentation conditions in step (3) are: the inoculation amount is 18-22% (v/v); the fermentation temperature is 28 ℃ in 0-40h, and the fermentation temperature is 24-25 ℃ in 40-140 h; the fermentation pH is 5.5-5.8; the fermentation time was 140 h.

7. The method for fermenting cephalosporin C in claim 1, wherein the process of supplementing the material in step (3) is: feeding the whole materials within 30-50h of fermentation according to the feeding amount of (6-8) g/L.h; feeding the whole materials within 50-70h of fermentation according to the feeding amount of (12-14) g/L.h; feeding the whole materials within 70-110h of fermentation according to the feeding amount of (8-10) g/L.h; and (4) feeding the whole materials according to the feeding amount of (8-10) g/L.h within the fermentation period of 110-140 h.

8. The method for fermenting cephalosporin C in claim 1, wherein the complete material in step (3) comprises the following raw materials in parts by weight: 0.5-1.0 part of corn steep liquor, 0.5-1.0 part of peanut powder, 1.0-3.0 parts of glucose, 0.5-1.0 part of dextrin, 0.5-1.0 part of methionine, 1.0-3.0 parts of soybean oil, CaSO₄0.5-1.0 part of (NH)₄)₂SO₄8-15 parts.

9. The method for fermenting cephalosporin C of any of claims 1-8, characterized in that the ratio of mass of the whole material solution to volume of fermentation broth in step (3) is 600-700 g/L.

10. The method for fermenting cephalosporin C in any of claims 1-8, characterized in that the separation and purification in step (4) comprises the steps of: adjusting the pH value of the fermentation liquor obtained in the step (3) to 2.5-3.0, adjusting the temperature to be below 10 ℃, adsorbing by using adsorbent macroporous resin, desorbing the saturated macroporous resin by using ethanol or acetone, adsorbing the obtained ethyl acetate or acetone solution by using anion resin, desorbing the saturated anion resin by using sodium acetate, drying the obtained sodium acetate solution at the temperature of 55-60 ℃ for 35-40min, then carrying out crystallization reaction, washing by using ethanol, and drying to obtain cephalosporin C.

Technical Field

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

Background

Cephalosporin C is produced by Cephalosporium acremonium and is another important beta-lactam antibiotic following penicillin, and has a structure similar to that of penicillin, has a monoacyl side chain connected to an amino group on a bicyclic nucleus, and has a quaternary lactam ring as with penicillin, but cephalosporin C has a six-membered dihydrothiazine ring instead of the five-membered thiazole ring characteristic of penicillin. Cephalosporin C formula C ₁₆H₂₁O₈N₃S, molecular weight 415.4. The cephalosporin antibiotics are anti-inflammatory and antibacterial drugs widely used clinically due to the characteristics of wide antibacterial spectrum and weak anaphylaxis. Cephalosporin C is a basic raw material for producing various semi-synthetic cephalosporins for injection.

In the process of preparing cephalosporin C by fermentation, a fermentation medium is an important factor influencing the yield of cephalosporin C, and the culture medium is rich in nutrition, so that thallus metabolism is vigorous in the early stage of fermentation, and the hypha growth speed is too fast, thus being not beneficial to the synthesis of cephalosporin C. The supplement of proper carbon source, nitrogen source, sulfur source and growth factor is the key factor for determining the yield of cephalosporin C. Segment soldiers and the like 'optimization of a process for producing cephalosporin C by fermentation of a mixed carbon source', industrial microorganisms 2014 and 2, and when a strategy of adding the mixed carbon source (glucose and soybean oil) is adopted, the final concentration of CPC is the highest and reaches 36.99g/L, so that the production requirement is met.

At present, the cephalosporin C prepared by the fermentation method reported in the literature has low titer and is difficult to meet the requirement of industrial application. Therefore, how to provide a fermentation method capable of improving the yield and titer of cephalosporin C is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the present invention provides a fermentation method of cephalosporin C capable of increasing cephalosporin C and titer.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for fermentation of cephalosporin C comprising the steps of:

preparing a first-stage seed solution, placing cephalosporium acremonium on an inclined plane of an eggplant bottle, inoculating a cephalosporium acremonium bacterium solution with the mass volume concentration of 25% into a seed culture medium according to the inoculation amount of 1% -2% for culture, and culturing for 90 hours under the conditions that the pH is 7.0-8.0 and the temperature is 27-29 ℃ to obtain the first-stage seed solution.

Preparing the secondary seed solution in the step (2), inoculating the primary seed solution prepared in the step (1) to a seed culture medium according to the inoculation amount of 8-10%, and culturing for 40-50h under the conditions that the pH is 6.5-7.5 and the temperature is 28-30 ℃ to obtain the secondary seed solution.

Step (3) inoculating the secondary seed liquid prepared in the step (2) into a fermentation culture medium according to the inoculation amount of 20-30% for fermentation, supplementing materials in the fermentation for 30-140h, stopping supplementing materials after the fermentation for 140h, and filtering to obtain fermentation liquor;

and (4) separating and purifying the cephalosporin C liquid obtained in the step (3), adsorbing, drying, crystallizing, washing and drying to obtain the cephalosporin C.

The invention has the beneficial effects that: the fermentation method adopted in the invention improves the titer of the cephalosporin C and greatly reduces the production cost.

Preferably, the cephalospora acremonium of step (1) is a cephalospora acremonium deposited under accession number atcc.36225.

Preferably, the culture medium in the step (1) comprises the following raw materials in parts by weight: 15-20 parts of corn steep liquor, 13-15 parts of glucose, 8-10 parts of white granulated sugar, 10-15 parts of soybean oil, 0.7-0.8 part of calcium carbonate and 0.5 part of defoaming agent.

Adopt above-mentioned further beneficial effect to lie in: the culture medium contains nutrient substances such as carbon sources, nitrogen sources and the like, and is favorable for the rapid growth of thalli.

Preferably, the culture medium in the step (2) comprises the following raw materials in parts by weight: 20-30 parts of corn steep liquor, 4-6 parts of peanut powder, 4-6 parts of soybean meal, 3-6 parts of white granulated sugar, 5-7 parts of calcium sulfate, 8-20 parts of glucose, 30-46 parts of soybean oil, 4-5 parts of calcium carbonate and 0.5-0.8 part of defoaming agent.

Adopt above-mentioned further beneficial effect to lie in: the culture medium contains nutrient substances such as carbon sources, nitrogen sources and the like, which is beneficial to the rapid growth of thalli, so that the seeds can be quickly adapted when entering the fermentation culture medium.

Preferably, the fermentation medium in the step (3) comprises the following raw materials in parts by weight: 25-50 parts of corn steep liquor, 5-13 parts of peanut powder, 5-12 parts of glucose, 15-25 parts of dextrin, 3-8 parts of methionine, 40-55 parts of soybean oil, 1.5-3 parts of defoaming agent and CaSO ₄2-5 parts of (NH)₄)₂SO₄5-10 parts of FeSO₄0.05 to 0.1 portion of MnSO₄0.01 to 0.04 portion of ZnSO₄0.01-0.04 part of CuSO₄0.01 to 0.04 portion of CaCO₃5-10 parts.

Adopt above-mentioned further beneficial effect to lie in: the culture medium contains specific elements, precursors, promoters and the like required for the product, in addition to elements and compounds necessary for the growth of the cells.

Preferably, the fermentation conditions in step (3) are: the inoculation amount is 18-22% (v/v); the fermentation temperature is 28 ℃ in 0-40h, and the fermentation temperature is 24-25 ℃ in 40-140 h; the fermentation pH is 5.5-5.8; the fermentation time was 140 h.

Adopt above-mentioned further beneficial effect to lie in: provides a proper environment for the growth and metabolism of the thalli so as to maximize the product.

Preferably, the process of the supplement in the step (3) is as follows: supplementing the whole materials within 30-50h of fermentation according to the flow addition of (6-8) g/L.h; supplementing the whole materials within 50-70h of fermentation according to the flow addition of (12-14) g/L.h; supplementing the whole materials within 70-110h of fermentation according to the flow addition of (8-10) g/L.h; in the fermentation period of 110-140h, the whole materials are supplemented according to the flow addition of (8-10) g/L.h.

Adopt above-mentioned further beneficial effect to lie in: continuously providing nutrient substances for the growth and metabolism of the thalli in the fermentation process.

Preferably, the complete material in the step (3) comprises the following raw materials in parts by weight: 0.5 to 1.0 portion of corn steep liquor, 0.5 to 1.0 portion of peanut powder, 1.0 to 3.0 portions of glucose, Dextrin 0.5-1.0 part, methionine 0.5-1.0 part, soybean oil 1.0-3.0 parts, CaSO₄0.5-1.0 part of (NH)₄)₂SO₄8-15 parts. Preferably, the ratio of the mass of the whole material solution to the volume of the fermentation liquid in the step (3) is 600-700 g/L.

Preferably, in step (4), the separation and purification steps are: adsorbing the fermentation liquor obtained in the step (3) by using adsorbent type macroporous resin, desorbing the saturated macroporous resin by using ethanol or acetone, adsorbing the obtained ethyl acetate or acetone solution by using anion resin, desorbing the saturated anion resin by using sodium acetate, drying the obtained sodium acetate solution at the temperature of 55-60 ℃ for 35-40min, then carrying out crystallization reaction, washing by using ethanol, and drying to finally obtain cephalosporin C.

According to the technical scheme, compared with the prior art, the invention discloses the fermentation method of the cephalosporin C, the complete material obtained by compounding the raw materials in the invention is supplemented in the fermentation process, the requirements of the growth of thalli at the early stage of fermentation and CPC synthesis on a nitrogen source and a sulfur source are met, the differentiation of the mycelia of the cephalosporium acremonium is promoted, and the synthesis of the cephalosporin C can be promoted; thereby improving the yield and the titer of the cephalosporin C and being more suitable for industrial production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the titer and the feed amount in examples 1 to 8 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.