阅读说明：本技术 一种发酵生产去乙酰氧基脱头孢烷酸的方法 (Method for producing desacetoxy cephalosporanic acid by fermentation ) 是由 党建宁 张宝新 王强 马俊 寇韩涛 于 2021-07-23 设计创作，主要内容包括：本发明提供了一种发酵生产去乙酰氧基脱头孢烷酸的方法。以诱变的顶头孢霉菌通过三级种子培养,发酵生产而得,本发明方法能够以特定的诱变菌株成功发酵制备乙酰氧基脱头孢烷酸,简称DAOC,产物效价高达29300μg/mL,能够实现工业化生产,有利于进一步制备合成7-ADCA,具备极大的应用价值。(The invention provides a method for producing desacetoxy cephalosporanic acid by fermentation. The method can successfully prepare the acetoxy-desocerotic acid, called DAOC for short, by fermenting specific mutagenic strains, has the product titer as high as 29300 mu g/mL, can realize industrial production, is favorable for further preparing and synthesizing 7-ADCA, and has great application value.)

1. A method for producing desacetoxycephalosporanic acid by fermentation is characterized by comprising the following steps:

(1) preparing Cephalosporium acremonium D-G3-B-2001 with preservation number of CGMCC NO:20270 into bacterial suspension;

(2) inoculating the bacterial suspension obtained in the step (1) into a primary seed culture medium, and culturing for 80-90h at 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.1 to obtain a primary seed solution with the centrifugal bacterial concentration of 20-30%;

(3) inoculating the primary seed liquid obtained in the step (2) into a secondary seed culture medium, and culturing for 40-60h at 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.2 to obtain a secondary seed liquid with the centrifugal bacterium concentration of 20-30%;

(4) inoculating the secondary seed liquid obtained in the step (3) into a tertiary seed culture medium, and culturing for 40-60h at 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.3 to obtain a tertiary seed liquid with the centrifugal bacterium concentration of 20-30%;

(5) inoculating the third-level seed liquid obtained in the step (4) into a fermentation culture medium, culturing for 120-128h at the temperature of 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.5 to obtain DAOC fermentation liquid, and supplementing vegetable oil, ammonium sulfate, liquid sugar and ammonia water in the fermentation process.

2. The method according to claim 1, wherein the concentration of the bacterial suspension in step (1) is 20-30% w/v, preferably 25% w/v; and/or the inoculation in the step (2) is as follows: the inoculation amount of (1) and the concentration of the centrifugal bacteria is 26%; and/or the inoculation amount of the inoculation in the step (3) is 5 percent, and the centrifugal bacterium concentration is 26 percent; and/or the inoculation amount of the inoculation in the step (4) is 10 percent, and the centrifugal bacterium concentration is 26 percent; and/or the inoculation amount of the inoculation in the step (5) is 25 percent.

3. The method of claim 1, wherein the primary seed medium of step (2) comprises the following components: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin;

and/or the secondary seed culture medium in the step (3) comprises the following components: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate;

and/or the tertiary seed culture medium in the step (4) comprises the following components: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

4. The method of claim 1, wherein: the fermentation medium in the step (5) comprises the following components: 10-33 parts of corn steep liquor, 9-21 parts of peanut powder, 4-13 parts of glucose, 3-10 parts of dextrin, 1-5 parts of methionine, 4-20 parts of vegetable oil, 0.01-0.06 part of defoaming agent, 2-7 parts of ammonium sulfate, 0.01-0.04 part of ferrous sulfate, 0.01-0.05 part of manganese sulfate, 0.01-0.06 part of zinc sulfate, 0.01-0.06 part of copper sulfate, 2-7 parts of calcium carbonate and 3-10 parts of gluten powder.

5. The method of claim 4, wherein: 11-32 parts of corn steep liquor, 10-20 parts of peanut powder, 5-12 parts of glucose, 4-9 parts of dextrin, 2-4 parts of methionine, 5-19 parts of vegetable oil, 0.02-0.05 part of defoaming agent, 3-6 parts of ammonium sulfate, 0.02-0.03 part of ferrous sulfate, 0.02-0.04 part of manganese sulfate, 0.02-0.05 part of zinc sulfate, 0.02-0.05 part of copper sulfate, 3-6 parts of calcium carbonate and 4-9 parts of gluten powder;

preferably: 12-15 parts of corn steep liquor, 12-15 parts of peanut powder, 8-11 parts of glucose, 5-9 parts of dextrin, 1-3 parts of methionine, 7-8 parts of vegetable oil, 0.01-0.02 part of defoaming agent, 4-5 parts of ammonium sulfate, 0.01-0.02 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 4 parts of gluten powder.

6. The method according to claim 1, wherein in the step (5), the vegetable oil is one or more of soybean oil, cottonseed oil, corn oil and rice bran oil, and the oil supplementing time is any time from 20 h to 128h after the fermentation is started; oil supplement 0.05-0.5L/h³。

7. The method according to claim 1, wherein in step (5), the liquid sugar used is 64% -70% (w/v) concentration liquid sugar, preferably 65% -69% (w/v) concentration liquid sugar; the sugar supplementing time is any time from 20 h to 127h after fermentation is started; sugar supplement 1-7.5L/h.m³。

8. The method as claimed in claim 1, wherein in the step (5), the concentration of ammonium sulfate used is 20-30% (w/v), and the time for supplementing ammonium sulfate is any time from 20 h to 80h after the start of fermentation; ammonium sulfate supplement with 0.05-0.4L/h³。

9. The method according to claim 1, wherein in step (5), the concentration of liquid ammonia used is 20-30% (w/v), and the pH of the fermentation broth is controlled to 5.6 ± 0.2 throughout the fermentation.

10. A fermentation medium for producing desacetoxycephalosporanic acid by fermentation is characterized by comprising the following components: 10-33 parts of corn steep liquor, 9-21 parts of peanut powder, 4-13 parts of glucose, 3-10 parts of dextrin, 1-5 parts of methionine, 4-20 parts of vegetable oil, 0.01-0.06 part of defoaming agent, 2-7 parts of ammonium sulfate, 0.01-0.04 part of ferrous sulfate, 0.01-0.05 part of manganese sulfate, 0.01-0.06 part of zinc sulfate, 0.01-0.06 part of copper sulfate, 2-7 parts of calcium carbonate and 3-10 parts of gluten powder;

preferably: 11-32 parts of corn steep liquor, 10-20 parts of peanut powder, 5-12 parts of glucose, 4-9 parts of dextrin, 2-4 parts of methionine, 5-19 parts of vegetable oil, 0.02-0.05 part of defoaming agent, 3-6 parts of ammonium sulfate, 0.02-0.03 part of ferrous sulfate, 0.02-0.04 part of manganese sulfate, 0.02-0.05 part of zinc sulfate, 0.02-0.05 part of copper sulfate, 3-6 parts of calcium carbonate and 4-9 parts of gluten powder;

more preferably: 12-15 parts of corn steep liquor, 12-15 parts of peanut powder, 8-11 parts of glucose, 5-9 parts of dextrin, 1-3 parts of methionine, 7-8 parts of vegetable oil, 0.01-0.02 part of defoaming agent, 4-5 parts of ammonium sulfate, 0.01-0.02 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 4 parts of gluten powder.

Technical Field

The invention belongs to the field of biological fermentation, and particularly relates to a method for preparing desacetoxycephalosporanic acid.

Background

7-aminodeacetyl cephalosporanic acid (7-ADCA) is a synthetic cephalosporin antibiotic, which is a main starting material of cefadroxil, cefalexin, cephradine and the like, the existing 7-ADCA production process is to synthesize the cephalosporin antibiotic by using penicillin G potassium salt as a raw material through the steps of oxidation, ring expansion, cracking and the like, however, the traditional process for producing the raw material penicillin G potassium salt needs to use penicillium chrysogenum for fermentation and then cracking to obtain 7-ADCA, a large amount of peracetic acid and butyl acetate are used in the process, a large amount of waste acid water is generated, and environmental pollution is easily caused; furthermore, 7-ADCA crystals produced by taking penicillin G potassium salt as a raw material are not easy to crystallize, and have the problems of unstable 7-ADCA, low purity and the like.

The method for producing 7-ADCA by using the deacetoxy cephalosporanic acid (DAOC) as the raw material through the enzymolysis method has the advantages of no need of solvent extraction and full-water phase treatment, and has low cost, obvious advantages compared with the chemical method for producing 7-ADCA and very excellent industrial application value.

Patent publication No. CN1357051A discloses a new strain obtained by inactivating cefEF gene of a. chrysogenum strain and expressing cefE gene, which can improve the yield of DAOC, but the strain engineered by genetic engineering method has great instability.

Compared with a genetic engineering method, the fermentation method for producing the DAOC has the unique advantages that the DAOC can be obtained from a cephalosporin C (CPC) fermentation process, however, products of the CPC fermentation process also comprise CPC, desacetyl cephalosporin C (DCPC) and other substances, the DAOC is a byproduct of the CPC fermentation process and a structural analogue of the CPC, and therefore a pure DAOC product cannot be obtained in the fermentation process. Microbial fermentation is a complex process, and factors influencing the quality of fermentation products are more, such as the influence of process parameters such as inoculation amount, fermentation pH, temperature, time, dissolved oxygen and the like on the fermentation products is particularly obvious.

Therefore, there is still a need for a method for preparing high-titer DAOC products by microbial fermentation, which has important significance and high industrial application value.

Disclosure of Invention

The invention aims to provide a method for producing high-titer desacetoxy cephalosporanic acid by fermentation through a specific strain.

The invention provides a method for producing desacetoxycephalosporanic acid by fermentation, which comprises the following steps:

(1) preparing Cephalosporium acremonium D-G3-B-2001 with preservation number of CGMCC NO:20270 into bacterial suspension;

(2) inoculating the bacterial suspension obtained in the step (1) into a primary seed culture medium, and culturing for 80-90h at 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.1 to obtain a primary seed solution with the centrifugal bacterial concentration of 20-30%;

(3) inoculating the primary seed liquid obtained in the step (2) into a secondary seed culture medium, and culturing for 40-60h at 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.2 to obtain a secondary seed liquid with the centrifugal bacterium concentration of 20-30%;

(4) inoculating the secondary seed liquid obtained in the step (3) into a tertiary seed culture medium, and culturing for 40-60h at 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.3 to obtain a tertiary seed liquid with the centrifugal bacterium concentration of 20-30%;

(5) inoculating the third-level seed liquid obtained in the step (4) into a fermentation culture medium, culturing for 120-128h at the temperature of 28 +/-0.5 ℃ and the aeration ratio of 0.8-1.5 to obtain DAOC fermentation liquid, and supplementing vegetable oil, ammonium sulfate, liquid sugar and ammonia water in the fermentation process.

Further, the inoculation amount of the inoculation in the step (2) is 1%; and/or the inoculation amount of the inoculation in the step (3) is 5 percent; and/or the inoculation amount of the inoculation in the step (4) is 10 percent; and/or the inoculation amount of the inoculation in the step (5) is 25 percent.

Further, the primary seed culture medium in the step (2) comprises the following components: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin;

and/or the secondary seed culture medium in the step (3) comprises the following components: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate;

and/or the tertiary seed culture medium in the step (4) comprises the following components: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

Further, the fermentation medium in the step (5) comprises the following components: 10-33 parts of corn steep liquor, 9-21 parts of peanut powder, 4-13 parts of glucose, 3-10 parts of dextrin, 1-5 parts of methionine, 4-20 parts of vegetable oil, 0.01-0.06 part of defoaming agent, 2-7 parts of ammonium sulfate, 0.01-0.04 part of ferrous sulfate, 0.01-0.05 part of manganese sulfate, 0.01-0.06 part of zinc sulfate, 0.01-0.06 part of copper sulfate, 2-7 parts of calcium carbonate and 3-10 parts of gluten powder.

Further, the fermentation medium comprises the following components: 11-32 parts of corn steep liquor, 10-20 parts of peanut powder, 5-12 parts of glucose, 4-9 parts of dextrin, 2-4 parts of methionine, 5-19 parts of vegetable oil, 0.02-0.05 part of defoaming agent, 3-6 parts of ammonium sulfate, 0.02-0.03 part of ferrous sulfate, 0.02-0.04 part of manganese sulfate, 0.02-0.05 part of zinc sulfate, 0.02-0.05 part of copper sulfate, 3-6 parts of calcium carbonate and 4-9 parts of gluten powder.

Further, the fermentation medium comprises the following components: 12-15 parts of corn steep liquor, 12-15 parts of peanut powder, 8-11 parts of glucose, 5-9 parts of dextrin, 1-3 parts of methionine, 7-8 parts of vegetable oil, 0.01-0.02 part of defoaming agent, 4-5 parts of ammonium sulfate, 0.01-0.02 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 4 parts of gluten powder.

Further, in the step (5), the vegetable oil is one or more of soybean oil, cottonseed oil, corn oil and rice bran oil, and the oil supplementing time is any time from 20 h to 128h after the fermentation is startedAmount of 0.05-0.5L/h. m³。

Further, in the step (5), the liquid sugar used is 64% to 70% (w/v) concentration liquid sugar, preferably 65% to 69% concentration liquid sugar; the sugar supplementing time is any time from 20 h to 127h after fermentation is started; sugar supplement 1-7.5L/h.m³。

Further, in the step (5), the concentration of the ammonium sulfate used is 20-30% (w/v), and the time for supplementing the ammonium sulfate is any time from 20 h to 80h after the start of fermentation; ammonium sulfate supplement with 0.05-0.4L/h³。

Further, in the step (5), the concentration of the liquid ammonia is 20-30% (w/v), and the pH of the fermentation liquid in the whole fermentation process is controlled to be 5.6 +/-0.2.

Experimental results show that the invention realizes that DAOC is prepared by taking the specific strain of the invention as a fermentation strain under the fermentation condition of the invention, can realize industrial production, has product titer as high as 29300 mug/mL, is beneficial to further preparing and synthesizing 7-ADCA, and has great application value.

Description of terms: the liquid sugar is prepared from corn starch.

Biological material preservation:

the Cephalosporium acremonium D-G3-B-2001 has been preserved in China general microbiological culture Collection center (CGMCC, China, Beijing, China academy of sciences institute of sciences) in 9, 16 days 2020, and the preservation number is CGMCC NO: 20270.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: IR spectrum of DAOC.

FIG. 2: H-NMR spectrum of DAOC.

FIG. 3: C-NMR spectrum of DAOC.

FIG. 4: gcosyl spectrum of DAOC.

FIG. 5: gHSQC spectrum of DAOC.

FIG. 6: the gHMBC spectrum of DAOC.

FIG. 7: mass spectrum of DAOC.

Detailed Description

The strains used in the invention: the strain produced by mutagenesis of cephalospora acremonium is obtained by a mutagenesis method. Cephalosporium acremonium D-G3-B-2001 (Cephalosporium acremonium) was deposited in China general microbiological culture Collection center (CGMCC, China, Beijing, institute of microbiology, China) at 16/9 in 2020 with the collection number of CGMCC NO: 20270.

The starting materials and equipment used in the present invention are, unless otherwise stated, known products obtained by purchasing commercially available products.

Example 1 Process for fermentative production of Deacetoxy-Decephalosporanic acid according to the invention

(1) Preparation of first seed

Taking the Acremonium terricola strain eggplant bottle inclined plane verified by a shake flask, scraping off the inclined plane strain by using sterile water, and preparing into a strain suspension of 25% (w/v). Inoculating to seed culture medium according to 1% inoculum size, culturing at pH of 7.0, 28 + -0.5 deg.C, 300-.

And (3) centrifugal bacterial concentration: the ratio of the cell weight to the cell volume after the cell liquid is centrifuged (centrifugation parameters: 10ml of fermentation liquid is taken and centrifuged at 3000rpm for 10 minutes) is shown.

Primary seed culture medium: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin.

(2) Preparation of Secondary seeds

Inoculating the primary seed in a secondary seed culture medium according to the inoculation amount of 5%, culturing for 55h under the conditions of pH of 6.5, 28 +/-0.5 ℃, 300-1.2 of aeration ratio, taking 10ml of fermentation liquor, centrifuging for 10 minutes at 3000rpm, and taking the secondary seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Secondary seed culture medium: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate.

(3) Preparation of tertiary seeds

Inoculating the second-level seeds into a third-level seed culture medium according to the inoculation amount of 10%, culturing for 54h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the speed is 300-1.2, and the aeration ratio is 0.8-1.2, centrifuging 10ml of fermentation liquor at 3000rpm for 10 min, and taking the third-level seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Third-level seed culture medium: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

(4) Fermenting in a fermentation tank

Inoculating the third-level seeds into a fermentation medium according to the inoculation amount of 25%, culturing for 128h under the conditions that the pH is 5.6, the temperature is 28 +/-0.5 ℃, the rpm is 50-100 and the aeration ratio is 0.8-1.5, obtaining the fermentation liquor of the de-ethoxyl de-cephalosporanic acid (DAOC), and supplementing soybean oil, ammonium sulfate (22% w/v), liquid sugar (65% w/v) and ammonia water (23% w/v) in the fermentation process. The oil supplementing time is any time from 20 h to 128h after fermentation is started. The sugar supplementing time is any time from 20 h to 127h after fermentation is started. The time for supplementing ammonium sulfate is any time from 20 h to 80h after fermentation is started. Ammonia water is used for controlling the pH value of fermentation liquor in the whole fermentation process to be 5.6 +/-0.2.

Fermentation tank culture medium: 15 parts of corn steep liquor, 12 parts of peanut powder, 8 parts of glucose, 5 parts of dextrin, 2 parts of methionine, 8 parts of vegetable oil, 0.01 part of defoaming agent, 5 parts of ammonium sulfate, 0.01 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 5 parts of wheat gluten.

Example 2 fermentative production of Deacetoxy-Decephalosporanic acid according to the invention

(1) Preparation of first seed

Taking the Acremonium terricola strain eggplant bottle inclined plane verified by a shake flask, scraping the inclined plane strain by using sterile water to prepare 25 percent (weight parts/volume part) of bacterial suspension. Inoculating to seed culture medium according to 1% inoculum size, culturing at pH of 7.0, 28 + -0.5 deg.C, 300-.

Primary seed culture medium: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin.

(2) Preparation of Secondary seeds

Inoculating the first-stage seed in a second-stage seed culture medium according to the inoculation amount of 5%, culturing for 55h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the air flow ratio is 0.8-1.2, taking 10ml of fermentation liquor, centrifuging for 10 minutes at 3000rpm, and taking the second-stage seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Secondary seed culture medium: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate.

(3) Preparation of tertiary seeds

Inoculating the second-level seeds into a third-level seed culture medium according to the inoculation amount of 10%, culturing for 54h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the speed is 300-1.2, and the aeration ratio is 0.8-1.2, centrifuging 10ml of fermentation liquor at 3000rpm for 10 minutes, and taking the third-level seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Third-level seed culture medium: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

(4) Fermenting in a fermentation tank

Inoculating the third-level seeds into a fermentation medium according to the inoculation amount of 25%, culturing for 128h under the conditions that the pH is 5.6, the temperature is 28 +/-0.5 ℃, the rpm is 50-100 and the aeration ratio is 0.8-1.5, and obtaining the fermentation liquor of the de-ethoxyl de-cephalosporanic acid (DAOC), and in the fermentation process, adding soybean oil, ammonium sulfate (22% w/v)), liquid sugar (65% w/v) and ammonia water (23% w/v). The oil supplementing time is any time from 20 h to 128h after fermentation is started. The sugar supplementing time is any time from 20 h to 127h after fermentation is started. The time for supplementing ammonium sulfate is any time from 20 h to 80h after fermentation is started. Ammonia water is used for controlling the pH value of fermentation liquor in the whole fermentation process to be 5.6 +/-0.2.

Fermentation tank culture medium: 14 parts of corn steep liquor, 13 parts of peanut powder, 8 parts of glucose, 6 parts of dextrin, 1 part of methionine, 7 parts of vegetable oil, 0.01 part of defoaming agent, 4 parts of ammonium sulfate, 0.01 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 5 parts of wheat gluten.

Example 3 Process for fermentative production of Deacetoxy-Decephalosporanic acid according to the invention

(1) Preparation of first seed

Taking the Acremonium terricola strain eggplant bottle inclined plane verified by a shake flask, scraping the inclined plane strain by using sterile water to prepare 25 percent (weight parts/volume part) of bacterial suspension. Inoculating to seed culture medium according to 1% inoculum size, culturing at pH of 7.0, 28 + -0.5 deg.C, 300-.

Primary seed culture medium: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin.

(2) Preparation of Secondary seeds

Inoculating the first-stage seed in a second-stage seed culture medium according to the inoculation amount of 5%, culturing for 55h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the air flow ratio is 0.8-1.2, taking 10ml of fermentation liquor, centrifuging for 10 minutes at 3000rpm, and taking the second-stage seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Secondary seed culture medium: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate.

(3) Preparation of tertiary seeds

Inoculating the second-level seeds into a third-level seed culture medium according to the inoculation amount of 10%, culturing for 54h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the speed is 300-1.2, and the aeration ratio is 0.8-1.2, centrifuging 10ml of fermentation liquor at 3000rpm for 10 minutes, and taking the third-level seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Third-level seed culture medium: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

(4) Fermenting in a fermentation tank

Inoculating the third-level seeds into a fermentation medium according to the inoculation amount of 25%, culturing for 128h under the conditions that the pH is 5.6, the temperature is 28 +/-0.5 ℃, the rpm is 50-100rpm and the aeration ratio is 0.8-1.5, obtaining the fermentation liquor of the de-ethoxyl de-cephalosporanic acid (DAOC), and supplementing soybean oil, ammonium sulfate (22% w/v), liquid sugar (65% w/v) and ammonia water (23% w/v) in the fermentation process. The oil supplementing time is any time from 20 h to 128h after fermentation is started. The sugar supplementing time is any time from 20 h to 127h after fermentation is started. The time for supplementing ammonium sulfate is any time from 20 h to 80h after fermentation is started. Ammonia water is used for controlling the pH value of fermentation liquor in the whole fermentation process to be 5.6 +/-0.2.

Fermentation tank culture medium: 14 parts of corn steep liquor, 14 parts of peanut powder, 9 parts of glucose, 7 parts of dextrin, 2 parts of methionine, 8 parts of vegetable oil, 0.02 part of defoaming agent, 5 parts of ammonium sulfate, 0.02 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 5 parts of wheat gluten.

Example 4 Process for fermentative production of Deacetoxy-Decephalosporanic acid according to the invention

(1) Preparation of first seed

Taking the Acremonium terricola strain eggplant bottle inclined plane verified by a shake flask, scraping the inclined plane strain by using sterile water to prepare 25 percent (weight parts/volume part) of bacterial suspension. Inoculating to seed culture medium according to 1% inoculum size, culturing at pH of 7.0, 28 + -0.5 deg.C, 300-.

Primary seed culture medium: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin.

(2) Preparation of Secondary seeds

Inoculating the first-stage seed in a second-stage seed culture medium according to the inoculation amount of 5%, culturing for 55h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the air flow ratio is 0.8-1.2, taking 10ml of fermentation liquor, centrifuging for 10 minutes at 3000rpm, and taking the second-stage seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Secondary seed culture medium: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate.

(3) Preparation of tertiary seeds

Inoculating the second-level seeds into a third-level seed culture medium according to the inoculation amount of 10%, culturing for 54h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the speed is 300-1.2, and the aeration ratio is 0.8-1.2, centrifuging 10ml of fermentation liquor at 3000rpm for 10 minutes, and taking the third-level seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Third-level seed culture medium: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

(4) Fermenting in a fermentation tank

Inoculating the third-level seeds into a fermentation medium according to the inoculation amount of 25%, culturing for 128h under the conditions that the pH is 5.6, the temperature is 28 +/-0.5 ℃, the rpm is 50-100 and the aeration ratio is 0.8-1.5, obtaining the fermentation liquor of the de-ethoxyl de-cephalosporanic acid (DAOC), and supplementing soybean oil, ammonium sulfate (22% w/v), liquid sugar (65% w/v) and ammonia water (23% w/v) in the fermentation process. The oil supplementing time is any time from 20 h to 128h after fermentation is started. The sugar supplementing time is any time from 20 h to 127h after fermentation is started. The time for supplementing ammonium sulfate is any time from 20 h to 80h after fermentation is started. Ammonia water is used for controlling the pH value of fermentation liquor in the whole fermentation process to be 5.6 +/-0.2.

Fermentation tank culture medium: 13 parts of corn steep liquor, 16 parts of peanut powder, 10 parts of glucose, 8 parts of dextrin, 3 parts of methionine, 8 parts of vegetable oil, 0.02 part of defoaming agent, 5 parts of ammonium sulfate, 0.02 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 5 parts of wheat gluten.

Example 5 Process for fermentative production of Deacetoxy-Decephalosporanic acid according to the invention

(1) Preparation of first seed

Taking the Acremonium terricola strain eggplant bottle inclined plane verified by a shake flask, scraping the inclined plane strain by using sterile water to prepare 25 percent (weight parts/volume part) of bacterial suspension. Inoculating to seed culture medium according to 1% inoculum size, culturing at pH of 7.0, 28 + -0.5 deg.C, 300-.

Primary seed culture medium: 38 parts of corn steep liquor, 4.5 parts of soybean oil, 8 parts of glucose, 1.1 parts of cane sugar, 0.02 part of antifoaming agent, 0.22 part of calcium carbonate, 1.2 parts of soybean cake powder, 1.2 parts of peanut cake powder and 0.45 part of dextrin.

(2) Preparation of Secondary seeds

Inoculating the first-stage seed in a second-stage seed culture medium according to the inoculation amount of 5%, culturing for 55h under the conditions that the pH is 6.5, the temperature is 28 +/-0.5 ℃, the air flow ratio is 0.8-1.2, taking 10ml of fermentation liquor, centrifuging for 10 minutes at 3000rpm, and taking the second-stage seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Secondary seed culture medium: 9 parts of corn steep liquor, 1 part of soybean oil, 3 parts of glucose, 1.5 parts of dextrin, 3 parts of white granulated sugar, 3 parts of soybean cake powder, 0.06 part of defoaming agent and 0.5 part of calcium carbonate.

(3) Preparation of tertiary seeds

Inoculating the second-level seeds into a third-level seed culture medium according to the inoculation amount of 10%, culturing for 54h under the conditions that the pH is 5.6, the temperature is 28 +/-0.5 ℃, the speed is 300-1.2, and the aeration ratio is 0.8-1.2, centrifuging 10ml of fermentation liquor at 3000rpm for 10 minutes, and taking the third-level seed liquor when the concentration of the centrifuged bacteria reaches 26%.

Third-level seed culture medium: 3 parts of corn steep liquor, 2.5 parts of soybean oil, 1.6 parts of glucose, 1.5 parts of dextrin, 1.5 parts of peanut powder, 1 part of ammonium sulfate, 0.5 part of calcium sulfate, 0.3 part of methionine, 1.05 parts of wheat gluten, 0.03 part of defoaming agent and 0.5 part of calcium carbonate.

(4) Fermenting in a fermentation tank

Inoculating the third-level seeds into a fermentation medium according to the inoculation amount of 25%, culturing for 128h under the conditions that the pH is 5.6, the temperature is 28 +/-0.5 ℃, the rpm is 50-100rpm and the aeration ratio is 0.8-1.5, and obtaining the fermentation liquor of the desethoxy-Desalinic Acid (DAOC), wherein in the fermentation process, ammonium sulfate (22% w/v), liquid sugar (65% w/v) and ammonia water (23% w/v) are added in the fermentation process. The oil supplementing time is any time from 20 h to 128h after fermentation is started. The sugar supplementing time is any time from 20 h to 127h after fermentation is started. The time for supplementing ammonium sulfate is any time from 20 h to 80h after fermentation is started. Ammonia water is used for controlling the pH value of fermentation liquor in the whole fermentation process to be 5.6 +/-0.2.

Fermentation tank culture medium: 12 parts of corn steep liquor, 15 parts of peanut powder, 11 parts of glucose, 9 parts of dextrin, 3 parts of methionine, 8 parts of vegetable oil, 0.01 part of defoaming agent, 4 parts of ammonium sulfate, 0.02 part of ferrous sulfate, 0.01 part of manganese sulfate, 0.01 part of zinc sulfate, 0.01 part of copper sulfate, 3 parts of calcium carbonate and 5 parts of wheat gluten.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 detection of DAOC

The fermentation product prepared by the method is subjected to Infrared (IR), H-NMR, C-NMR, gCOSY, gHSQC, gHMBC and mass spectrum detection, and the result is shown in figures 1-7.

The above results demonstrate the success of the process of the present invention for the preparation of DAOC.

Experimental example 2 titer detection of fermentation broth of the present invention

1. Experimental methods

And filtering the fermentation liquor through a microporous filter membrane after the fermentation is finished to obtain filtrate. And (3) performing potency detection, namely adding water into a 1g fermentation liquor and a 50mL volumetric flask until the water is scaled and shaken uniformly to prepare DAOC mixed liquor, and taking the prepared DAOC to detect the potency.

The chromatographic conditions were as follows: a chromatographic column: hypersil ODS 5 μm × 4.6mm × 250mm, wavelength: 254nm, flow rate: 1 volume part/min, sample amount: 20 uL.

2. Results of the experiment

As shown in table 1:

the method can reach the highest fermentation tank-discharging titer of 29300 mu g/mL, and has great application value.

In conclusion, the invention provides a method for producing DAOC by fermentation, which can be used for preparing DAOC under the fermentation condition of the invention by taking the specific strain of the invention as a fermentation strain, can realize industrial production, has product titer as high as 29300 mug/mL, is beneficial to further preparing and synthesizing 7-ADCA, and has great application value.