CN102808013A - Method for preparing cephalosporin C through fermentation - Google Patents
Method for preparing cephalosporin C through fermentation Download PDFInfo
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- CN102808013A CN102808013A CN2012103108004A CN201210310800A CN102808013A CN 102808013 A CN102808013 A CN 102808013A CN 2012103108004 A CN2012103108004 A CN 2012103108004A CN 201210310800 A CN201210310800 A CN 201210310800A CN 102808013 A CN102808013 A CN 102808013A
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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 the prepared 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 penicillium mould; The structural similitude of its structure and penicillium mould has the amino on an acyl side-chain and the dicyclo parent nucleus to join, and the same with penicillium mould 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 penicillium mould characteristic.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 widely used clinically anti-inflammatory, antibacterials, also are the basic raw materials of producing the semi-synthetic cynnematin of various injections.
In the process of fermentative prepn cephalosporin; Fermention medium is the important factor that influences 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 to the mycelia form and the influence of tiring in the cephalosporin fermenting process ", Hebei chemical industry; 2010,33 (1): 56-57, utilize cephalosporin to produce the bacterium fermentation; Fermention medium is to be raw material with wheat flour strength flour, groundnut meal, Semen Maydis powder, steeping water, high temperature soybean cake powder, soya-bean oil, glucose, fruit grape slurry, calcium sulfate, methionine(Met) (being methionine(Met)), and the dosage of methionine(Met) is divided into 5 gradients, is respectively 2 ‰, 4 ‰, 6 ‰, 8 ‰, 10 ‰; Experimental result finds that the dosage of control methionine(Met) is at 8 ‰ o'clock; The output of cephalosporin is the highest, is 32.55g/L, tires to be 32550U/mL.Wang Juan etc., " the ammonium sulfate dosage is to the mycelia form and the influence of tiring in the cephalosporin fermenting process " seeks medical advice and medicine; 2011; 9:40 discloses and has utilized cephalosporin to produce bacterium-HC bacterial classification, the method for fermentative prepn cephalosporin in the fermention medium that W-Gum, dextrin, steeping water, methionine(Met), ammonium sulfate, potassium primary phosphate, sal epsom, lime carbonate, sucrose, glucose are formed, wherein; The ammonium sulfate add-on is 4% o'clock; The output of cephalosporin is the highest, is 28.39g/L, tires to be 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 the 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, in the following time period of fermenting process, mend methionine(Met) and ammonium sulfate, technology is following:
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 promptly gets cephalosporin.
Wherein, the said 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 that is used to prepare 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 fermenting 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; Be seeded to seed culture medium according to 1-2% (v/v) inoculum size; At pH is 6.5-7.5,27-29 ℃ cultivation 50-65h, and said seed culture medium contains following composition: steeping water 20-30 weight part, glucose 13-20 weight part, sucrose 10-20 weight part, vegetables oil 35-50 volume, lime carbonate 1-1.5 weight part, foam killer 0.5-1.0 parts by volume; B, secondary seed: get first order seed; Be seeded to seed culture medium according to 8-10% (v/v) inoculum size; At pH is 6.0-7.0,27-29 ℃ cultivation 50-65h, and said seed culture medium contains following composition and is: steeping water 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, lime carbonate 4-7 weight part, foam killer 0.5-1 weight part.
Wherein, the said fermention medium of step (2) is: steeping water 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 said vegetables oil is VT 18 or Semen Maydis oil.
Wherein, the condition of the said 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 technology of mending 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 technology of mending 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 technology of mending methionine(Met) is: 60-80h, and stream adds 6g/Lh, 80-120h, stream adds 8g/Lh, 120-140h, stream adds 7g/Lh; The technology of mending 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 technology of mending methionine(Met) is: 60-80h, and stream adds 5g/Lh, 80-120h, stream adds 7g/Lh, 120-140h, stream adds 6g/Lh; The technology of mending 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 for preparing obtains, significantly reduces production costs, and has a good application prospect.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.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 rapid determination fermented liquid cephalosporin tire ", the method for Heilungkiang medicine 1996 the 9th phases 138-139 page or leaf record 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 the bacterium liquid of 25% (mg/ml), is seeded to seed culture medium according to 1% (v/v) inoculum size, is 7.0,27 ℃ at pH and cultivates 50h.
First order seed substratum: steeping water 20g, glucose 13g, sucrose 10g, vegetables oil 35ml, lime carbonate 1g, foam killer 0.5ml.
(2) preparation secondary seed
Get the first order seed of step (1) preparation, be seeded to seed culture medium, be 6.5,27 ℃ at pH and cultivate 50h according to 8% (v/v) inoculum size.
Secondary seed medium: steeping water 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 6g/Lh, 80-120h, and stream adds 8g/Lh, 120-140h, stream adds 7g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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 the bacterium liquid of 26% (mg/ml), is seeded to seed culture medium according to 2% (v/v) inoculum size, is 7.0,29 ℃ at pH and cultivates 65h.
First order seed substratum: steeping water 30g, glucose 20g, sucrose 20g, vegetables oil 50ml, lime carbonate 1.5g, foam killer 1.0ml.
(2) preparation secondary seed
Get first order seed, be seeded to seed culture medium, be 6.0,29 ℃ at pH and cultivate 65h according to 10% (v/v) inoculum size.
Secondary seed medium: steeping water 25g, peanut powder 7g, analysis for soybean powder 7g, calcium sulfate 10g, glucose 10g, vegetables oil 50ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 5g/Lh, 80-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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 the bacterium liquid of 27% (mg/ml), is seeded to seed culture medium according to 1.5% (v/v) inoculum size, is 6.5,28 ℃ at pH and cultivates 55h.
First order seed substratum: steeping water 25g, glucose 15g, sucrose 15g, vegetables oil 40ml, lime carbonate 1.2g, foam killer 0.8ml.
(2) preparation secondary seed
Get first order seed, be seeded to seed culture medium, be 7.0,28 ℃ at pH and cultivate 55h according to 9% (v/v) inoculum size.
Secondary seed medium: steeping water 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 5g/Lh, 80-120h, and stream adds 7g/Lh, 120-140h, stream adds 6g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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 the bacterium liquid of 28% (mg/ml), is seeded to seed culture medium according to 1.5% (v/v) inoculum size, is 7.5,28 ℃ at pH and cultivates 60h.
Seed culture medium: steeping water 20g, glucose 13g, sucrose 10g, vegetables oil 35ml, lime carbonate 1g, foam killer 0.5ml.
(2) preparation secondary seed
Get first order seed, be seeded to seed culture medium, be 6.5,28 ℃ at pH and cultivate 60h according to 9% (v/v) inoculum size.
Secondary seed medium: steeping water 20g, peanut powder 5g, analysis for soybean powder 5g, calcium sulfate 8g, glucose 8g, vegetables oil 45ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 4g/Lh, 80-120h, and stream adds 6g/Lh, 120-140h, stream adds 5g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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: steeping water 30g, glucose 20g, sucrose 20g, vegetables oil 50ml, lime carbonate 1.5g, foam killer 1.0ml.
Get the cephalosporium acremonium that preserving number is ATCC 36225, activation, getting concentration is the bacterium liquid of 29% (mg/ml), is seeded to seed culture medium according to 2% (v/v) inoculum size, is 7.0,29 ℃ at pH and cultivates 65h.
(2) preparation secondary seed
Secondary seed medium: steeping water 25g, peanut powder 7g, analysis for soybean powder 7g, calcium sulfate 10g, glucose 10g, vegetables oil 50ml, lime carbonate 7g, foam killer 1g.
Get first order seed, be seeded to seed culture medium, be 6.0,29 ℃ at pH and cultivate 65h according to 10% (v/v) inoculum size.
(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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 7g/Lh, 80-120h, and stream adds 9g/Lh, 120-140h, stream adds 8g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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 the bacterium liquid of 30% (mg/ml), is seeded to seed culture medium according to 1.5% (v/v) inoculum size, is 6.5,28 ℃ at pH and cultivates 55h.
First order seed substratum: steeping water 25g, glucose 15g, sucrose 15g, vegetables oil 40ml, lime carbonate 1.2g, foam killer 0.8ml.
(2) preparation secondary seed
Get first order seed, be seeded to seed culture medium, be 7.0,28 ℃ at pH and cultivate 55h according to 9% (v/v) inoculum size.
Secondary seed medium: steeping water 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 3g/Lh, 80-120h, and stream adds 5g/Lh, 120-140h, stream adds 4g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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 the bacterium liquid of 31% (mg/ml), is seeded to seed culture medium according to 1.5% (v/v) inoculum size, is 6.5,28 ℃ at pH and cultivates 55h.
First order seed substratum: steeping water 25g, glucose 15g, sucrose 15g, vegetables oil 40ml, lime carbonate 1.2g, foam killer 0.8ml.
(2) preparation secondary seed
Get first order seed, be seeded to seed culture medium, be 7.0,28 ℃ at pH and cultivate 55h according to 9% (v/v) inoculum size.
Secondary seed medium: steeping water 16g, peanut powder 4g, analysis for soybean powder 4g, calcium sulfate 5g, glucose 6g, vegetables oil 40ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 7g/Lh, 80-120h, and stream adds 9g/Lh, 120-140h, stream adds 8g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, 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 the bacterium liquid of 26% (mg/ml), is seeded to seed culture medium according to 1.5% (v/v) inoculum size, is 7.5,28 ℃ at pH and cultivates 60h.
Seed culture medium: steeping water 20g, glucose 13g, sucrose 10g, vegetables oil 35ml, lime carbonate 1g, foam killer 0.5ml.(2) preparation secondary seed
Get first order seed, be seeded to seed culture medium, be 6.5,28 ℃ at pH and cultivate 60h according to 9% (v/v) inoculum size.
Secondary seed medium: steeping water 20g, peanut powder 5g, analysis for soybean powder 5g, calcium sulfate 8g, glucose 8g, vegetables oil 45ml, lime 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: steeping water 50g, peanut powder 15g, glucose 8g, hydrolyzed starch 26g, methionine(Met) 4g, VT 18 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, mend methionine(Met) and ammonium sulfate:
Mend the technology of methionine(Met): 60-80h, stream adds 3g/Lh, 80-120h, and stream adds 5g/Lh, 120-140h, stream adds 4g/Lh;
Mend the technology 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 through filtering with microporous membrane, must filtrate.The detection of tiring: get 1mL and 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 it respectively and tire;
(5) separation and purification: the filtrating of step (4) is used the adsorbent type macroporous resin adsorption; Saturated macroporous resin is with ethanol or acetone desorption, and the ETHYLE ACETATE that obtains (or acetone) solution adsorbs with resin anion(R.A), and saturated resin anion(R.A) carries out desorption with sodium-acetate; The sodium acetate soln that obtains carries out drying; Carry out crystallization reaction then,, obtain the cephalosporin zinc salt at last with washing with alcohol, drying.
2, experimental result
Through detecting, tiring of cephalosporin is 21400U/mL.
Comprehensive embodiment 1 ~ 7 and comparative example's result, see table 1:
Table 1 is tired and the feed supplement cost
Can find out by 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.
Through 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 benefit of itself and ammonium sulfate and methionine(Met) goes into amount and fed-batch mode is all relevant.
Therefore, take all factors into consideration 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 two kinds of sulphur sources of ammonium sulfate and methionine(Met) simultaneously, and additional amount and additional time are carried out special selection, improved tiring of cephalosporin significantly, reduced production cost, prospects for commercial application is good.
Claims (8)
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, in the following time period of fermenting process, mend methionine(Met) and ammonium sulfate, technology is following:
Methionine(Met): 60-80h, stream adds 3-7g/Lh; 80-120h, stream adds 5-9g/Lh; 120-140h, stream adds 4-8g/Lh; Ammonium sulfate: 50-90h, stream adds 1-5g/Lh; 90-120h, stream adds 3-7g/Lh; 120-140h, stream adds 4-6g/Lh;
(3) with step (2) gained filtering fermentation liquor, get supernatant, separation and purification promptly gets cephalosporin.
2. method according to claim 1 is characterized in that: the said cephalosporium acremonium of step (1) is that preserving number is the cephalosporium acremonium of ATCC 36225.
3. method according to claim 1 is characterized in that: in the said step (2), the technology of mending 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 technology of mending 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.
4. method according to claim 3 is characterized in that: the technology that said benefit is gone into methionine(Met) is: 60-80h, and stream adds 6g/Lh, 80-120h, stream adds 8g/Lh, 120-140h, stream adds 7g/Lh; The technology that said benefit is gone into ammonium sulfate is: 50-90h, and stream adds 4g/Lh, 90-120h, stream adds 6g/Lh, 120-140h, stream adds 5g/Lh.
5. method according to claim 3 is characterized in that: the technology that said benefit is gone into methionine(Met) is: 60-80h, and stream adds 5g/Lh, 80-120h, stream adds 7g/Lh, 120-140h, stream adds 6g/Lh; The technology that said benefit is gone into ammonium sulfate is: 50-90h, and stream adds 3g/Lh, 90-120h, stream adds 5g/Lh, 120-140h, stream adds 4g/Lh.
6. method according to claim 1 is characterized in that: the condition of the said fermentation of step (2) is: leavening temperature: 0-40h is 28 ℃, and 40-140h is 24-25 ℃; Fermentation pH is 5.5-5.8; Fermentation time: 140h.
7. method according to claim 1 is characterized in that: the said fermention medium of step (2) comprises following composition: steeping water 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.
8. method according to claim 7 is characterized in that: said vegetables oil is VT 18 or Semen Maydis oil.
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Cited By (2)
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| CN108048519A (en) * | 2017-12-25 | 2018-05-18 | 伊犁川宁生物技术有限公司 | A kind of cephalosporin bacteria residue processing method, hydrolyzate and its application |
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