CN102808013B - Method for preparing cephalosporin C through fermentation - Google Patents
Method for preparing cephalosporin C through fermentation Download PDFInfo
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- CN102808013B CN102808013B CN 201210310800 CN201210310800A CN102808013B CN 102808013 B CN102808013 B CN 102808013B CN 201210310800 CN201210310800 CN 201210310800 CN 201210310800 A CN201210310800 A CN 201210310800A CN 102808013 B CN102808013 B CN 102808013B
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Abstract
The invention discloses a method for preparing cephalosporin C through fermentation. The method comprises the following steps of: taking cephalosporium acremonium and preparing first-grade seeds and second-grade seeds; 2) inoculating the second-grade seeds prepared in the step 1) to a cephalosporium acremonium fermentation medium and fermenting, wherein in the fermenting process, methionine is supplemented at the feeding speed of 3 to 7 g/L.h from the 60th hour to the 80th hour, is supplemented at the feeding speed of 5 to 9 g/L.h from the 80th hour to the 120th hour, and is supplemented at the feeding speed of 4 to 8 g/L.h from the 120th hour to the 140th hour, and ammonium sulfate is supplemented at the feeding speed of 1 to 5 g/L.h from 50th hour to the 90th hour, is supplemented at the speed of 3 to 7 g/L.h from the 90th hour to the 120th hour and is supplemented at the feeding speed of 4 to 6 g/L.h from 120th hour to 140th hour; and 3) filtering fermentation broth which is obtained in the step 2) to obtain supernatant, and separating and purifying to obtain the cephalosporin C. The method for preparing the cephalosporin C through fermentation is convenient to operate, and theprepared cephalosporin C is high in valence and has good market application prospect.
Description
Technical field
The present invention relates to the fermentation process of cephalosporin.
Background technology
Cephalosporin is produced by cephalosporium acremonium (Cephalosporium acremonium); it is another important beta-lactam antibiotics after penicillin; the structural similitude of its structure and penicillin; there is the amino on an acyl side-chain and the dicyclo parent nucleus to join; the same with penicillin all have a quaternary lactam nucleus; but cephalosporin has hexa-atomic the second light industry bureau thiazine ring, has replaced five yuan of thiazole rings of penicillin feature.The molecular formula C of cephalosporin
16H
21O
8N
3S, molecular weight are 415.4, and structural formula is:
The characteristics of cephalosporin C a little less than because of its has a broad antifungal spectrum and supersensitivity are clinically widely used anti-inflammatory, antibacterials, also are the basic raw materials of producing the semi-synthetic cynnematin of various injections.
In the process of fermentation preparation cephalosporin, fermention medium is the important factor that affects cephalosporin output, usually add methyllanthionine or methionine(Met) in the fermention medium, the S element in its molecule is significant to the height of the conversion of the different cycles of formation cephalosporin, the length of producing the anti-phase and fermentation level.
Ren Lu etc., " the methionine(Met) dosage is on mycelia form and the impact of tiring in the Cephalosporin C fermentation process ", the Hebei chemical industry, 2010,33 (1): 56-57, utilize cephalosporin to produce the bacterium fermentation, fermention medium is with the wheat flour strength flour, groundnut meal, Semen Maydis powder, corn steep liquor, the high temperature soybean cake powder, soya-bean oil, glucose, fruit grape slurry, calcium sulfate, methionine(Met) (being methionine(Met)) is raw material, the dosage of methionine(Met) is divided into 5 gradients, be respectively 2 ‰, 4 ‰, 6 ‰, 8 ‰, 10 ‰, experimental result finds that the dosage of control methionine(Met) is at 8 ‰ o'clock, and the output of cephalosporin is the highest, be 32.55g/L, tiring is 32550U/mL.Wang Juan etc., " Cephalosporin C fermentation Sulfur During Process acid ammonium dosage is on mycelia form and the impact of tiring ", seek medical advice and medicine, 2011,9:40 discloses and has utilized cephalosporin to produce bacterium-HC bacterial classification, fermentation prepares the method for cephalosporin in the fermention medium that W-Gum, dextrin, corn steep liquor, methionine(Met), ammonium sulfate, potassium primary phosphate, sal epsom, calcium carbonate, sucrose, glucose form, wherein, when the ammonium sulfate add-on is 4%, the output of cephalosporin is the highest, be 28.39g/L, tiring is 28390U/mL.
Tiring of the cephalosporin of the fermentation process preparation of existing bibliographical information is low, is difficult to well satisfy the demand of industrial applications.
Summary of the invention
In order to address the above problem, the present invention improves kind and the sulphur source adding mode in sulphur source, and a kind of fermentation process of new cephalosporin is provided.
The fermentation process of cephalosporin of the present invention, it comprises the steps:
(1) gets cephalosporium acremonium, preparation first order seed, secondary seed;
(2) get the secondary seed that step (1) prepares, be seeded in the cephalosporium acremonium fermention medium and ferment, fill into methionine(Met) and ammonium sulfate within the following time period of fermenting process, technique is as follows:
Methionine(Met): 60-80h, stream adds 4-6g/Lh; 80-120h, stream adds 6-8g/Lh; 120-140h, stream adds 5-7g/Lh; Ammonium sulfate: 50-90h, stream adds 2-4g/Lh; 90-120h, stream adds 4-6g/Lh; 120-140h, stream adds 3-5g/Lh;
(3) with step (2) gained filtering fermentation liquor, get supernatant, separation and purification namely gets cephalosporin.
Wherein, the described cephalosporium acremonium of step (1) is that preserving number is the cephalosporium acremonium of ATCC 36225.
Wherein, first order seed and secondary seed can prepare according to this area ordinary method in the step (1), and wherein, the cephalosporium acremonium bacterial concentration for the preparation of first order seed after the activation should be not less than 25%(mg/ml).All the other steps can be according to Ji Zhixia etc., " Cephalosporin C fermentation process metabolic characteristics ", the method preparation of East China University of Science's journal (natural science is reported 2006-11 the 32nd volume 1275-1279) 1.2.6 three grade fermemtation record; Also can prepare as follows: a, first order seed: get cephalosporium acremonium, according to 1-2%(v/v) inoculum size is seeded to seed culture medium, be 6.5-7.5,27-29 ℃ at pH and cultivate 50-65h, described seed culture medium contains following composition: corn steep liquor 20-30 weight part, glucose 13-20 weight part, sucrose 10-20 weight part, vegetables oil 35-50 volume, calcium carbonate 1-1.5 weight part, foam killer 0.5-1.0 parts by volume; B, secondary seed: get first order seed, according to 8-10%(v/v) inoculum size is seeded to seed culture medium, be 6.0-7.0,27-29 ℃ at pH and cultivate 50-65h, described seed culture medium contains following composition and is: corn steep liquor 16-25 weight part, peanut powder 4-7 weight part, analysis for soybean powder 4-7 weight part, calcium sulfate 5-10 weight part, glucose 6-10 weight part, vegetables oil 40-50 parts by volume, calcium carbonate 4-7 weight part, foam killer 0.5-1 weight part.
Wherein, the described fermention medium of step (2) is: corn steep liquor 40-75 weight part, peanut powder 10-20 weight part, glucose 5-12 weight part, hydrolyzed starch 20-30 weight part, methionine(Met) 3-8 weight part, vegetables oil 50-70 parts by volume, foam killer 1.5-3 parts by volume, MgSO
42-5 weight part, (NH
4)
2SO
48-15 weight part, FeSO
40.05-0.1 weight part, MnSO
40.01-0.04 weight part, ZnSO
40.01-0.04 weight part, CuSO
40.01-0.04 weight part, CaCO
3The 5-10 weight part.
The ratio of aforementioned weight and parts by volume is g/mL.
Wherein, aforementioned described vegetables oil is soybean oil or Semen Maydis oil.
Wherein, the condition of the described fermentation of step (2) is: inoculum size is 18 ~ 22%(v/v); Leavening temperature: 0-40h is 28 ℃, and 40-140h is 24-25 ℃; Fermentation pH is 5.5-5.8; Fermentation time: 140h.
Preferably, the technique that fills into methionine(Met) is: 60-80h, and stream adds 4-6g/Lh; 80-120h, stream adds 6-8g/Lh; 120-140h, stream adds 5-7g/Lh; The technique that fills into ammonium sulfate is: 50-90h, and stream adds 2-4g/Lh; 90-120h, stream adds 4-6g/Lh; 120-140h, stream adds 3-5g/Lh.
Further preferably, the technique that fills into methionine(Met) is: 60-80h, and stream adds 6g/Lh, 80-120h, stream adds 8g/Lh, 120-140h, stream adds 7g/Lh; The technique that fills into ammonium sulfate is: 50-90h, and stream adds 4g/Lh, 90-120h, stream adds 6g/Lh, 120-140h, stream adds 5g/Lh.
Further preferably, the technique that fills into methionine(Met) is: 60-80h, and stream adds 5g/Lh, 80-120h, stream adds 7g/Lh, 120-140h, stream adds 6g/Lh; The technique that fills into ammonium sulfate is: 50-90h, and stream adds 3g/Lh, 90-120h, stream adds 5g/Lh, 120-140h, stream adds 4g/Lh.
Tiring of the cephalosporin that fermentation process of the present invention prepares reached 29400 ~ 423100U/mL, is significantly higher than tiring of cephalosporin that prior art prepares, significantly reduces production costs, and has a good application prospect.
Obviously, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
The embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
The present invention tires according to Liu Xuehui etc., and " utilize in the HPLC method Fast Measurement fermented liquid cephalosporin tire ", the method for Heilungkiang medicine the 9th phase 138-139 page or leaf record in 1996 is measured.
The fermentation process of embodiment 1 cephalosporin of the present invention
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 25%(mg/ml) bacterium liquid, according to 1%(v/v) inoculum size is seeded to seed culture medium, be 7.0,27 ℃ at pH and cultivate 50h.
First order seed substratum: corn steep liquor 20g, glucose 13g, sucrose 10g, vegetables oil 35ml, calcium carbonate 1g, foam killer 0.5ml.
(2) preparation secondary seed
Get the first order seed of step (1) preparation, according to 8%(v/v) inoculum size is seeded to seed culture medium, is 6.5,27 ℃ of cultivation 50h at pH.
Secondary seed medium: corn steep liquor 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, calcium carbonate 4g, foam killer 0.5g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 18% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 24 ℃, and pH is 5.6.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 6g/Lh, 80-120h, and stream adds 8g/Lh, 120-140h, stream adds 7g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 4g/Lh, 90-120h, and stream adds 6g/Lh, 120-140h, stream adds 5g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 42310U/mL.
The fermentation process of embodiment 2 cephalosporins of the present invention
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 26%(mg/ml) bacterium liquid, according to 2%(v/v) inoculum size is seeded to seed culture medium, be 7.0,29 ℃ at pH and cultivate 65h.
First order seed substratum: corn steep liquor 30g, glucose 20g, sucrose 20g, vegetables oil 50ml, calcium carbonate 1.5g, foam killer 1.0ml.
(2) preparation secondary seed
Get first order seed, according to 10%(v/v) inoculum size is seeded to seed culture medium, is 6.0,29 ℃ of cultivation 65h at pH.
Secondary seed medium: corn steep liquor 25g, peanut powder 7g, analysis for soybean powder 7g, calcium sulfate 10g, glucose 10g, vegetables oil 50ml, calcium carbonate 7g, foam killer 1g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 22% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 25 ℃, and pH is 5.8.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 5g/Lh, 80-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 3g/Lh, 90-120h, and stream adds 5g/Lh, 120-140h, stream adds 4g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 40380U/mL.
The fermentation process of embodiment 3 cephalosporins of the present invention
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 27%(mg/ml) bacterium liquid, according to 1.5%(v/v) inoculum size is seeded to seed culture medium, be 6.5,28 ℃ at pH and cultivate 55h.
First order seed substratum: corn steep liquor 25g, glucose 15g, sucrose 15g, vegetables oil 40ml, calcium carbonate 1.2g, foam killer 0.8ml.
(2) preparation secondary seed
Get first order seed, according to 9%(v/v) inoculum size is seeded to seed culture medium, is 7.0,28 ℃ of cultivation 55h at pH.
Secondary seed medium: corn steep liquor 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, calcium carbonate 4g, foam killer 0.5g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 19% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 24.5 ℃, and pH is 5.5.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 5g/Lh, 80-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 4g/Lh, 90-120h, and stream adds 6g/Lh, 120-140h, stream adds 5g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 41900U/mL.
The fermentation process of embodiment 4 cephalosporins of the present invention
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 28%(mg/ml) bacterium liquid, according to 1.5%(v/v) inoculum size is seeded to seed culture medium, be 7.5,28 ℃ at pH and cultivate 60h.
Seed culture medium: corn steep liquor 20g, glucose 13g, sucrose 10g, vegetables oil 35ml, calcium carbonate 1g, foam killer 0.5ml.
(2) preparation secondary seed
Get first order seed, according to 9%(v/v) inoculum size is seeded to seed culture medium, is 6.5,28 ℃ of cultivation 60h at pH.
Secondary seed medium: corn steep liquor 20g, peanut powder 5g, analysis for soybean powder 5g, calcium sulfate 8g, glucose 8g, vegetables oil 45ml, calcium carbonate 5g, foam killer 0.8g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 20% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 24.5 ℃, and pH is 5.6.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 4g/Lh, 80-120h, and stream adds 6g/Lh, 120-140h, stream adds 5g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 5g/Lh, 90-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 34000U/mL.
The fermentation process of embodiment 5 cephalosporins of the present invention
(1) preparation first order seed
First order seed substratum: corn steep liquor 30g, glucose 20g, sucrose 20g, vegetables oil 50ml, calcium carbonate 1.5g, foam killer 1.0ml.
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 29%(mg/ml) bacterium liquid, according to 2%(v/v) inoculum size is seeded to seed culture medium, be 7.0,29 ℃ at pH and cultivate 65h.
(2) preparation secondary seed
Secondary seed medium: corn steep liquor 25g, peanut powder 7g, analysis for soybean powder 7g, calcium sulfate 10g, glucose 10g, vegetables oil 50ml, calcium carbonate 7g, foam killer 1g.
Get first order seed, according to 10%(v/v) inoculum size is seeded to seed culture medium, is 6.0,29 ℃ of cultivation 65h at pH.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 22% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 25 ℃, and pH is 5.8.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 7g/Lh, 80-120h, and stream adds 9g/Lh, 120-140h, stream adds 8g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 5g/Lh, 90-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 42310U/mL.
The fermentation process of embodiment 6 cephalosporins of the present invention
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 30%(mg/ml) bacterium liquid, according to 1.5%(v/v) inoculum size is seeded to seed culture medium, be 6.5,28 ℃ at pH and cultivate 55h.
First order seed substratum: corn steep liquor 25g, glucose 15g, sucrose 15g, vegetables oil 40ml, calcium carbonate 1.2g, foam killer 0.8ml.
(2) preparation secondary seed
Get first order seed, according to 9%(v/v) inoculum size is seeded to seed culture medium, is 7.0,28 ℃ of cultivation 55h at pH.
Secondary seed medium: corn steep liquor 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, calcium carbonate 4g, foam killer 0.5g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 19% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 24.5 ℃, and pH is 5.5.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 3g/Lh, 80-120h, and stream adds 5g/Lh, 120-140h, stream adds 4g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 5g/Lh, 90-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 33200U/mL.
The fermentation process of embodiment 7 cephalosporins of the present invention
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 31%(mg/ml) bacterium liquid, according to 1.5%(v/v) inoculum size is seeded to seed culture medium, be 6.5,28 ℃ at pH and cultivate 55h.
First order seed substratum: corn steep liquor 25g, glucose 15g, sucrose 15g, vegetables oil 40ml, calcium carbonate 1.2g, foam killer 0.8ml.
(2) preparation secondary seed
Get first order seed, according to 9%(v/v) inoculum size is seeded to seed culture medium, is 7.0,28 ℃ of cultivation 55h at pH.
Secondary seed medium: corn steep liquor 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, calcium carbonate 4g, foam killer 0.5g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 19% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 24.5 ℃, and pH is 5.5.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 7g/Lh, 80-120h, and stream adds 9g/Lh, 120-140h, stream adds 8g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 1g/Lh, 90-120h, and stream adds 3g/Lh, 120-140h, stream adds 2g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 29400U/mL.
The fermentation process of comparative example's cephalosporin
1, fermentation process
(1) preparation first order seed
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is 26%(mg/ml) bacterium liquid, according to 1.5%(v/v) inoculum size is seeded to seed culture medium, be 7.5,28 ℃ at pH and cultivate 60h.
Seed culture medium: corn steep liquor 20g, glucose 13g, sucrose 10g, vegetables oil 35ml, calcium carbonate 1g, foam killer 0.5ml.(2) preparation secondary seed
Get first order seed, according to 9%(v/v) inoculum size is seeded to seed culture medium, is 6.5,28 ℃ of cultivation 60h at pH.
Secondary seed medium: corn steep liquor 20g, peanut powder 5g, analysis for soybean powder 5g, calcium sulfate 8g, glucose 8g, vegetables oil 45ml, calcium carbonate 5g, foam killer 0.8g.
(3) fermentation
Get the secondary seed of step (2) preparation, (v/v) inoculum size according to 20% is seeded in the fermention medium ferments, fermentation 140h, and 0-40h is 28 ℃, and 40-140h is 24.5 ℃, and pH is 5.6.
Fermention medium: corn steep liquor 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, soybean oil 55ml, foam killer 2ml, MgSO
44g, (NH
4)
2SO
413g, FeSO
40.09g, MnSO
40.03g, ZnSO
40.025g, CuSO
40.025g, CaCO
36g.
During this time, fill into methionine(Met) and ammonium sulfate:
Fill into the technique of methionine(Met): 60-80h, stream adds 3g/Lh, 80-120h, and stream adds 5g/Lh, 120-140h, stream adds 4g/Lh;
Fill into the technique of ammonium sulfate: 50-90h, stream adds 1g/Lh, 90-120h, and stream adds 3g/Lh, 120-140h, stream adds 2g/Lh;
(4) get fermented liquid after the fermentation ends by filtering with microporous membrane, get filtrate.Bioactivity: get 1mL filtrate and in the volumetric flask of 50mL, add water to scale and shake up, prepare the cephalosporin mixed solution, get the cephalosporin that makes, detect respectively it and tire;
(5) separation and purification: the filtrate of step (4) is used the adsorbent type macroporous resin adsorption, saturated macroporous resin is with ethanol or acetone desorption, the ethyl acetate that obtains (or acetone) solution adsorbs with resin anion(R.A), saturated resin anion(R.A) carries out desorption with sodium-acetate, the sodium acetate soln that obtains carries out drying, then carry out crystallization reaction, with washing with alcohol, drying, obtain at last the cephalosporin zinc salt.
2, experimental result
After testing, tiring of cephalosporin is 21400U/mL.
Comprehensive embodiment 1 ~ 7 and comparative example's result sees Table 1:
Table 1 is tired and the feed supplement cost
As can be seen from Table 1:
In the additional way and using dosage scope of methionine(Met) of the present invention and ammonium sulfate, tiring all on 29400U/mL of cephalosporin tired far above the cephalosporin of existing method preparation; In preferable range, the tiring all on 40000U/mL of the cephalosporin of preparation; The best processing condition under, cephalosporin is up to 42310U/mL, is 1.49 times that the cephalosporin of existing method preparation is tired.
By embodiment 1 and embodiment 5, the embodiment 4 relatively discovery with embodiment 6, tiring of cephalosporin is not that consumption with ammonium sulfate and methionine(Met) is simple linear relationship, and the amount of filling into and the fed-batch mode of itself and ammonium sulfate and methionine(Met) are all relevant.
Therefore, consider and tire and production cost, each processing parameter value of embodiment 2 is more suitable for industrial production.
To sum up, in the fermentation process of the present invention, use simultaneously two kinds of sulphur sources of ammonium sulfate and methionine(Met), and additional amount and additional time are carried out special selection, improved significantly tiring of cephalosporin, reduced production cost, prospects for commercial application is good.
Claims (7)
1. the fermentation process of a cephalosporin, it is characterized in that: it comprises the steps:
(1) gets cephalosporium acremonium, preparation first order seed, secondary seed;
(2) get the secondary seed that step (1) prepares, be seeded in the cephalosporium acremonium fermention medium and ferment, fill into methionine(Met) and ammonium sulfate within the following time period of fermenting process, technique is as follows:
Methionine(Met): 60-80 h, stream add 3-7 g/Lh; 80-120 h, stream add 5-9 g/Lh; 120-140 h, stream add 4-8 g/Lh; Ammonium sulfate: 50-90 h, stream add 1-5 g/Lh; 90-120 h, stream add 3-7 g/Lh; 120-140 h, stream add 4-6 g/Lh;
(3) with step (2) gained filtering fermentation liquor, get supernatant, separation and purification namely gets cephalosporin;
The described fermention medium of step (2) comprises following composition: corn steep liquor 40-75 weight part, peanut powder 10-20 weight part, glucose 5-12 weight part, hydrolyzed starch 20-30 weight part, methionine(Met) 3-8 weight part, vegetables oil 50-70 parts by volume, foam killer 1.5-3 parts by volume, MgSO
42-5 weight part, (NH
4)
2SO
48-15 weight part, FeSO
40.05-0.1 weight part, MnSO
40.01-0.04 weight part, ZnSO
40.01-0.04 weight part, CuSO
40.01-0.04 weight part, CaCO
3The 5-10 weight part;
Described weight part: parts by volume is g/ml.
2. method according to claim 1, it is characterized in that: the described cephalosporium acremonium of step (1) is that preserving number is the cephalosporium acremonium of ATCC 36225.
3. method according to claim 1, it is characterized in that: in the described step (2), the technique that fills into methionine(Met) is: 60-80 h, stream add 4-6 g/Lh; 80-120 h, stream add 6-8 g/Lh; 120-140 h, stream add 5-7 g/Lh; The technique that fills into ammonium sulfate is: 50-90 h, stream add 2-4 g/Lh; 90-120 h, stream add 4-6 g/Lh; 120-140 h, stream add 3-5 g/Lh.
4. method according to claim 3, it is characterized in that: the described technique that fills into methionine(Met) is: 60-80 h, stream add 6 g/Lh, and 80-120 h, stream add 8 g/Lh, and 120-140 h, stream add 7 g/Lh; The described technique that fills into ammonium sulfate is: 50-90 h, stream add 4 g/Lh, and 90-120 h, stream add 6 g/Lh, and 120-140 h, stream add 5 g/Lh.
5. method according to claim 3, it is characterized in that: the described technique that fills into methionine(Met) is: 60-80 h, stream add 5 g/Lh, and 80-120 h, stream add 7 g/Lh, and 120-140 h, stream add 6 g/Lh; The described technique that fills into ammonium sulfate is: 50-90 h, stream add 3 g/Lh, and 90-120 h, stream add 5 g/Lh, and 120-140 h, stream add 4 g/Lh.
6. method according to claim 1, it is characterized in that: the condition of the described fermentation of step (2) is: leavening temperature: 0-40 h is 28 ℃, and 40-140 h is 24-25 ℃; Fermentation pH is 5.5-5.8; Fermentation time: 140h.
7. method according to claim 1, it is characterized in that: described vegetables oil is soybean oil or Semen Maydis oil.
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| US3139388A (en) * | 1963-01-04 | 1964-06-30 | Olin Mathieson | Method of producing cephalosporinc |
| US3825473A (en) * | 1971-08-13 | 1974-07-23 | Alfa Farmaceutici Spa | Production of cephalosporin c |
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| KR910004371B1 (en) * | 1989-08-17 | 1991-06-26 | 제일제당 주식회사 | Method for preparation of cephalosporin -c |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55144896A (en) * | 1979-04-24 | 1980-11-12 | Takeda Chem Ind Ltd | Preparation of cephalosporin antibiotic |
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| US3139388A (en) * | 1963-01-04 | 1964-06-30 | Olin Mathieson | Method of producing cephalosporinc |
| US3825473A (en) * | 1971-08-13 | 1974-07-23 | Alfa Farmaceutici Spa | Production of cephalosporin c |
| US3929577A (en) * | 1973-09-28 | 1975-12-30 | Takeda Chemical Industries Ltd | Production of cephalosporin C |
| US3948726A (en) * | 1973-10-01 | 1976-04-06 | Takeda Chemical Industries, Ltd. | Production of cephalosporin C |
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