CN109371093A - A kind of method of streptomyces aureus fermenting and producing tetracycline - Google Patents
A kind of method of streptomyces aureus fermenting and producing tetracycline Download PDFInfo
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- CN109371093A CN109371093A CN201811382569.3A CN201811382569A CN109371093A CN 109371093 A CN109371093 A CN 109371093A CN 201811382569 A CN201811382569 A CN 201811382569A CN 109371093 A CN109371093 A CN 109371093A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P29/00—Preparation of compounds containing a naphthacene ring system, e.g. tetracycline
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Abstract
The present invention relates to a kind of methods of streptomyces aureus fermenting and producing tetracycline, its processing step are as follows: by streptomyces aureus original strain, with sterile water dissolved dilution, then it accesses in Spore cultivation base, it shakes up, under the conditions of 33-35 DEG C of temperature, cultivates 17-25 hours, seed culture and fermented and cultured are successively carried out later.The Spore cultivation base composition are as follows: wheat bran 1.0-1.5%, dipotassium hydrogen phosphate 0.003-0.006%, starch 1.0-1.6%, magnesium sulfate 0.01-0.03%, corn pulp 0.3-0.8%, yeast powder 0.03-0.06%, calcium carbonate 0.01-0.03%.The fermenting and producing tetracycline by the way of Spore cultivation is different from the prior art by the way of inclined-plane culture in the present invention, and using preparation culture medium culture spore, overcome the influence factor of agar in slant medium, it is not influenced by raw material with culture medium, material is easy to get, the stable advantage of quality, shake-flask seed is accessed with the strain of spore liquid culture, mycelia is sturdy, unfolds, adaptable for culture solutions at different levels, it is fast to move into fermentor accretion rate, tetracycline secretion is early, and fermentation period is short, and fermentation level is greatly improved.
Description
Technical field
The invention belongs to biology and pharmaceutical technology fields, more particularly to a kind of streptomyces aureus fermenting and producing tetracycline
Method.
Background technique
Tetracycline is a kind of macrolide antibiotics, mainly using streptomyces aureus as production bacterial strain.Traditional fermentation
Strain is that cryovial strain is prepared into slant pore, then again by spore inoculating in seed culture medium, by well-grown
Seed asepsis is inoculated in fermentation medium.Wherein slant medium does main carbon source and microelement supply using wheat bran,
Agar provides phosphorus and a small amount of nitrogen source as coagulator, with inorganic phosphate.By above-mentioned culture medium prescription preparation strain exist with
Lower defect:
(1) the sole carbon source supplier that wheat bran is grown as spore-germination, quality is by the Wheat Production place of production, processing method
Influence highly unstable, although agar has special influence as coagulator, to the growth of tetracycline slant pore, ingredient is multiple
Miscellaneous difficulty or ease accurately determine, so to be affected by it fluctuation larger for inclined-plane presentation quality.
(2) because in recent years, industrial development, environmental pollution is on the rise, wheat bran and agar quality are by growth conditions and environment
It influences, it is more and more unstable, cause the production strain quality of preparation unstable；
(3) raw material qualities such as wheat bran change, and the strain newly selected is caused to change, and newly using above-mentioned formula
Strain production is not suitable for, and the strain of preparation is affected from presentation quality to inherent conidium vitality.
Summary of the invention
The object of the invention is that the defect for overcoming the above-mentioned prior art, and provide one kind is not influenced by raw material, and material is easy
, quality is stablized, and obtained spore mycelia is sturdy, and culture solutions at different levels are adaptable, the gold that fermentation level significantly improves
Streptomycete fermentation produces tetracycline method.
The technical solution taken for achieving the above object are as follows:
A kind of method of streptomyces aureus fermenting and producing tetracycline, it is characterised in that its processing step are as follows: by golden strepto-
Then bacterium original strain is accessed in Spore cultivation base, is shaken up with sterile water dissolved dilution, under the conditions of 33-35 DEG C of temperature, training
It supports 17-25 hours, successively carries out seed culture and fermented and cultured later.
The Spore cultivation base composition are as follows: wheat bran 1.0-1.5%, dipotassium hydrogen phosphate 0.003-0.006%, starch 1.0-
1.6%, magnesium sulfate 0.01-0.03%, corn pulp 0.3-0.8%, yeast powder 0.03-0.06%, calcium carbonate 0.01-0.03%.
The seed culture and fermented and cultured use routine techniques.
The fermenting and producing Fourth Ring by the way of Spore cultivation is different from the prior art by the way of inclined-plane culture in the present invention
Element, and using culture medium culture spore is prepared, the influence factor of agar in slant medium is overcome, has culture medium not by original
Effect of Materials, material are easy to get, and the stable advantage of quality accesses shake-flask seed with the strain of spore liquid culture, and mycelia is sturdy, relax
Exhibition, adaptable for culture solutions at different levels, immigration fermentor accretion rate is fast, and tetracycline secretion is early, and fermentation period is short, fermentation
Level is greatly improved.
Specific embodiment
The present invention will be described below by way of examples, it should be understood that example is for illustrating rather than to this
The limitation of invention.The scope of the present invention is determined with core content according to claims.
Shake-flask seed culture medium forms in following embodiments are as follows:
Starch 2.0-4.0%, bean powder 1.5-2.5%, yeast powder 0.3-1.0%, peptone 0.3-1.0%, ammonium sulfate
0.1-0.5%, sodium bromide 0-0.5%, calcium carbonate 0.1-0.5%, magnesium sulfate 0.01-0.04%, potassium dihydrogen phosphate 0-0.03%.
Fermentation medium composition are as follows:
Starch 4.0-10.0%, bean powder 1.5-2.5%, yeast powder 0.3-1.0%, ammonium sulfate 0.3-1.0%, sodium bromide 0-
0.5%, calcium carbonate 0.3-0.8%, dextrin 1.5-3.0%, corn pulp 0.3-1.0%, M- promotor 0.001-0.003%
Tetracycline original strain (streptomyces aureus): selection is stored in the cryovial bacterial strain of -70 ± 5 DEG C of cryogenic refrigerator.
Embodiment 1 (repeats three groups)
1, prepared by Spore cultivation base:
Spore cultivation based formulas: wheat bran 1.0%, dipotassium hydrogen phosphate 0.003%, starch 1.0%, magnesium sulfate 0.01% are beautiful
Rice & peanut milk 0.3%, yeast powder 0.03%, calcium carbonate 0.01%.
Preparation method: it will be contained in fixed container after the above material (except calcium carbonate) dissolution, calcium carbonate addition filled
Divide dissolution, after mixing evenly, controls pH 7.2 or so, be sub-packed in the triangular flask of 500ml, every bottle of splendid attire 120ml.It goes out
Bacterium controls 117-121 DEG C of sterilising temp, pressure 0.08-0.12MPa, and sterilize 20-25min, and pH is in 6.4-6.7 after sterilizing.
2, Spore cultivation:
Tetracycline original strain (streptomyces aureus) 1 is taken, 1ml sterile water is added and shakes up absorption 0.5ml, in aseptic condition
It in the lower above-mentioned Spore cultivation base of access, shakes up, under the conditions of 33-35 DEG C of temperature, cultivates 17-25 hours.
3, shake-flask seed culture:
By process 2) gained spore liquid, be inoculated in sterilized shake-flask seed culture medium (starch 2.0%, bean powder 1.5%,
Yeast powder 0.3%, peptone 0.3%, ammonium sulfate 0.1%, sodium bromide 0.1%, calcium carbonate 0.1%, magnesium sulfate 0.01%) on,
32-33 DEG C of temperature is kept, is cultivated 22-28 hours, the thick yellow wall built-up of range estimation seed liquor appearance.
4, fermented and cultured:
Medium of shaking flask fermentation (starch 4.0%, the bean powder that the sterile immigration of cultured seed liquor has been sterilized
1.5%, yeast powder 0.3%, ammonium sulfate 0.3%, sodium bromide 0.1%, calcium carbonate 0.3%, dextrin 1.5%, corn pulp 0.3%,
M- promotor 0.001%) in, whole-process control temperature is at 31-33 DEG C, sterile culture 150-170 hours.
5, control batch:
5.1 traditional inclined-plane formulas: agar 2.0-3.0%, wheat bran 3.0-5.0%, potassium dihydrogen phosphate 0.005-
0.008%, diammonium hydrogen phosphate 0.008-0.02%.
5.2 culture mediums are prepared: weighing agar, wheat bran be respectively contained in eggplant-shape bottle according to the above ratio.
Potassium dihydrogen phosphate, diammonium hydrogen phosphate are calculated by dose volume and are weighed by 5.3, after being dissolved respectively with purified water, are contained
In graduated cylinder, it is diluted with water to scale, is sufficiently shaken up.
5.4 are sub-packed in 5.3 prepared solution in 5.2 eggplant-shape bottle equipped with agar and wheat bran.
5.5 sterile blank cultures preparations: 5.4 resulting eggplant-shape bottle culture mediums are put in autoclave, keep the temperature (118-122
DEG C) pressure maintaining (0.008-0.012MPa) 30min, after be cooled to 40-50 DEG C, apply bevel it is spare.
5.6 take freeze-drying pipe strain one, are prepared into bacteria suspension after sterile water dissolution is added, the appropriate bacteria suspension difference of picking is equal
It is even to be applied on 5.5 gained blank slant mediums, culture, refrigeration are terminated after cultivating 80-100 hours under 32-36 DEG C of environment
It is spare.
5.7 shake-flask seed cultures: the slant pore that 5.6 are obtained accesses shake-flask seed culture, the same embodiment of incubation
Article 3.
5.8 shake flask fermentation cultures: with embodiment Article 4.
6, fermentation results:
First group:
Second group:
Third group:
Embodiment 2 (repeats three groups)
1, prepared by Spore cultivation base:
Spore cultivation based formulas: wheat bran 1.2%, dipotassium hydrogen phosphate 0.005%, starch 1.2%, magnesium sulfate 0.02% are beautiful
Rice & peanut milk 0.5%, yeast powder 0.05%, calcium carbonate 0.02%.
Preparation method: it will be contained in fixed container after the above material (except calcium carbonate) dissolution, calcium carbonate addition filled
Divide dissolution, after mixing evenly, controls pH 7.2 or so, be sub-packed in the triangular flask of 500ml, every bottle of splendid attire 120ml.It goes out
Bacterium controls 117-121 DEG C of sterilising temp, pressure 0.08-0.12MPa, and sterilize 20-25min, and pH is in 6.4-6.7 after sterilizing.
2, prepared by spore:
Tetracycline original strain (streptomyces aureus) 1 is taken, 1ml sterile water is added and shakes up absorption 0.5ml, in aseptic condition
It in the lower above-mentioned Spore cultivation base of access, shakes up, under the conditions of 33-35 DEG C of temperature, cultivates 17-25 hours.
3, shake-flask seed culture:
By process 2) gained spore liquid, be inoculated in sterilized shake-flask seed culture medium (starch 3.0%, bean powder 2.0%,
Yeast powder 0.6%, peptone 0.5%, ammonium sulfate 0.2%, sodium bromide 0.3%, calcium carbonate 0.3%, magnesium sulfate 0.02%, phosphoric acid
Potassium dihydrogen 0.02%) on, 32-33 DEG C of temperature is kept, is cultivated 22-28 hours, the thick yellow wall built-up of range estimation seed liquor appearance.
4, fermented and cultured:
Medium of shaking flask fermentation (starch 6.0%, the bean powder that the sterile immigration of cultured seed liquor has been sterilized
2.0%, yeast powder 0.6%, ammonium sulfate 0.6%, sodium bromide 0.25%, calcium carbonate 0.5%, dextrin 2.0%, corn pulp 0.5%,
M- promotor 0.002%) in, whole-process control temperature is at 31-33 DEG C, sterile culture 150-170 hours.
5, control batch: (under following process preferably refines)
5.1 traditional inclined-plane formulas: agar 2.0-3.0%, wheat bran 3.0-5.0%, potassium dihydrogen phosphate 0.005-
0.008%, diammonium hydrogen phosphate 0.008-0.02%
5.2 culture mediums are prepared: weighing agar, wheat bran be respectively contained in eggplant-shape bottle according to the above ratio
Potassium dihydrogen phosphate, diammonium hydrogen phosphate are calculated by dose volume and are weighed by 5.3, after being dissolved respectively with purified water, are contained
In graduated cylinder, it is diluted with water to scale, is sufficiently shaken up.
5.4 are sub-packed in 5.3 prepared solution in 5.2 eggplant-shape bottle equipped with agar and wheat bran.
5.5 sterile blank cultures preparations: 5.4 resulting eggplant-shape bottle culture mediums are put in autoclave, keep the temperature (118-122
DEG C) pressure maintaining (0.008-0.012MPa) 30min, after be cooled to 40-50 DEG C, apply bevel it is spare.
5.6 take freeze-drying pipe strain one, are prepared into bacteria suspension after sterile water dissolution is added, the appropriate bacteria suspension difference of picking is equal
It is even to be applied on 5.5 gained blank slant mediums, culture, refrigeration are terminated after cultivating 80-100 hours under 32-36 DEG C of environment
It is spare.
5.7 shake-flask seed cultures: the slant pore that 5.6 are obtained accesses shake-flask seed culture, the same embodiment of incubation
Article 3.
5.8 shake flask fermentation cultures: with embodiment Article 4.
6, fermentation results:
First group:
Second group:
Third group:
Embodiment 3 (repeats three groups)
1, prepared by Spore cultivation base:
Spore cultivation based formulas: wheat bran 1.5%, dipotassium hydrogen phosphate 0.006%, starch 1.6%, magnesium sulfate 0.03% are beautiful
Rice & peanut milk 0.8%, yeast powder 0.06%, calcium carbonate 0.03%.
Preparation method: it will be contained in fixed container after the above material (except calcium carbonate) dissolution, calcium carbonate addition filled
Divide dissolution, after mixing evenly, controls pH 7.2 or so, be sub-packed in the triangular flask of 500ml, every bottle of splendid attire 120ml.It goes out
Bacterium controls 117-121 DEG C of sterilising temp, pressure 0.08-0.12MPa, and sterilize 20-25min, and pH is in 6.4-6.7 after sterilizing.
2, Spore cultivation:
Tetracycline original strain (streptomyces aureus) 1 is taken, 1ml sterile water is added and shakes up absorption 0.5ml, in aseptic condition
It in the lower above-mentioned Spore cultivation base of access, shakes up, under the conditions of 33-35 DEG C of temperature, cultivates 17-25 hours.
3, shake-flask seed culture:
By process 2) gained spore liquid, be inoculated in sterilized shake-flask seed culture medium (starch 4.0%, bean powder 2.5%,
Yeast powder 1.0%, peptone 1.0%, ammonium sulfate 0.5%, sodium bromide 0.5%, calcium carbonate 0.5%, magnesium sulfate 0.04%, phosphoric acid
Potassium dihydrogen 0.03%) on, 32-33 DEG C of temperature is kept, is cultivated 22-28 hours, the thick yellow wall built-up of range estimation seed liquor appearance.
4, fermented and cultured:
Medium of shaking flask fermentation (starch 10.0%, the bean powder that the sterile immigration of cultured seed liquor has been sterilized
2.5%, yeast powder 1.0%, ammonium sulfate 1.0%, sodium bromide 0.5%, calcium carbonate 0.8%, dextrin 3.0%, corn pulp 1.0%,
M- promotor 0.003%) in, whole-process control temperature is at 31-33 DEG C, sterile culture 150-170 hours.
5, control batch: (under following process preferably refines)
5.1 traditional inclined-plane formulas: agar 2.0-3.0%, wheat bran 3.0-5.0%, potassium dihydrogen phosphate 0.005-
0.008%, diammonium hydrogen phosphate 0.008-0.02%.
5.2 culture mediums are prepared: weighing agar, wheat bran be respectively contained in eggplant-shape bottle according to the above ratio
Potassium dihydrogen phosphate, diammonium hydrogen phosphate are calculated by dose volume and are weighed by 5.3, after being dissolved respectively with purified water, are contained
In graduated cylinder, it is diluted with water to scale, is sufficiently shaken up.
5.4 are sub-packed in 5.3 prepared solution in 5.2 eggplant-shape bottle equipped with agar and wheat bran.
5.5 sterile blank cultures preparations: 5.4 resulting eggplant-shape bottle culture mediums are put in autoclave, keep the temperature (118-122
DEG C) pressure maintaining (0.008-0.012MPa) 30min, after be cooled to 40-50 DEG C, apply bevel it is spare.
5.6 take freeze-drying pipe strain one, are prepared into bacteria suspension after sterile water dissolution is added, the appropriate bacteria suspension difference of picking is equal
It is even to be applied on 5.5 gained blank slant mediums, culture, refrigeration are terminated after cultivating 80-100 hours under 32-36 DEG C of environment
It is spare.
5.7 shake-flask seed cultures: the slant pore that 5.6 are obtained accesses shake-flask seed culture, the same embodiment of incubation
Article 3.
5.8 shake flask fermentation cultures: with embodiment Article 4.
6, fermentation results:
First group:
Second group:
Third group:
Claims (3)
1. a kind of method of streptomyces aureus fermenting and producing tetracycline, it is characterised in that its processing step are as follows: by streptomyces aureus
Then original strain is accessed in Spore cultivation base, is shaken up with sterile water dissolved dilution, under the conditions of 33-35 DEG C of temperature, culture
17-25 hours, seed culture and fermented and cultured were successively carried out later.
2. the method for streptomyces aureus fermenting and producing tetracycline described in accordance with the claim 1, it is characterised in that the spore training
Support base composition are as follows: wheat bran 1.0-1.5%, dipotassium hydrogen phosphate 0.003-0.006%, starch 1.0-1.6%, magnesium sulfate 0.01-0.03%,
Corn pulp 0.3-0.8%, yeast powder 0.03-0.06%, calcium carbonate 0.01-0.03%.
3. the method for streptomyces aureus fermenting and producing tetracycline described in accordance with the claim 1, it is characterised in that the seed training
It supports and fermented and cultured uses routine techniques.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699314A (en) * | 2019-10-22 | 2020-01-17 | 河北圣雪大成制药有限责任公司 | Method for producing 6-demethyltetracycline by fermentation |
| CN111793668A (en) * | 2020-05-28 | 2020-10-20 | 河北圣雪大成制药有限责任公司 | Method for producing nortetracycline |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638623A (en) * | 2009-05-06 | 2010-02-03 | 厦门金达威集团股份有限公司 | Preparation method of seed culture medium for producing arachidonic acid by fermentation method |
| CN103602714A (en) * | 2013-11-20 | 2014-02-26 | 宁夏启元药业有限公司 | Method for producing tetracycline by fermentation of streptomyces aureus |
| CN104342477A (en) * | 2014-11-03 | 2015-02-11 | 金河生物科技股份有限公司 | Preparation method for tetracycline |
| CN107653292A (en) * | 2017-11-22 | 2018-02-02 | 宁夏启元药业有限公司 | The inclined-plane solid medium and cultural method of a kind of streptomyces aureus fermenting and producing tetracycline |
-
2018
- 2018-11-20 CN CN201811382569.3A patent/CN109371093A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638623A (en) * | 2009-05-06 | 2010-02-03 | 厦门金达威集团股份有限公司 | Preparation method of seed culture medium for producing arachidonic acid by fermentation method |
| CN103602714A (en) * | 2013-11-20 | 2014-02-26 | 宁夏启元药业有限公司 | Method for producing tetracycline by fermentation of streptomyces aureus |
| CN104342477A (en) * | 2014-11-03 | 2015-02-11 | 金河生物科技股份有限公司 | Preparation method for tetracycline |
| CN107653292A (en) * | 2017-11-22 | 2018-02-02 | 宁夏启元药业有限公司 | The inclined-plane solid medium and cultural method of a kind of streptomyces aureus fermenting and producing tetracycline |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699314A (en) * | 2019-10-22 | 2020-01-17 | 河北圣雪大成制药有限责任公司 | Method for producing 6-demethyltetracycline by fermentation |
| CN111793668A (en) * | 2020-05-28 | 2020-10-20 | 河北圣雪大成制药有限责任公司 | Method for producing nortetracycline |
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Application publication date: 20190222 |