CN107653292A - The inclined-plane solid medium and cultural method of a kind of streptomyces aureus fermenting and producing tetracycline - Google Patents
The inclined-plane solid medium and cultural method of a kind of streptomyces aureus fermenting and producing tetracycline Download PDFInfo
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- CN107653292A CN107653292A CN201711169123.8A CN201711169123A CN107653292A CN 107653292 A CN107653292 A CN 107653292A CN 201711169123 A CN201711169123 A CN 201711169123A CN 107653292 A CN107653292 A CN 107653292A
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- solid medium
- plane solid
- plane
- tetracycline
- hydrogen phosphate
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The present invention relates to a kind of inclined-plane solid medium of streptomyces aureus fermenting and producing tetracycline and cultural method, culture medium composition is：Wheat bran 2.0~2.5%, dipotassium hydrogen phosphate 0.005~0.008%, diammonium hydrogen phosphate 0.008~0.013%, magnesium sulfate 0.003~0.006%, corn steep liquor 0.5~1.0%, calcium carbonate 0.003%, agar 1.8~2.5%, remaining is water, by weight percentage.The present invention is formed by changing the raw material in streptomyces aureus inclined-plane solid medium, strain quality caused by raw material quality fluctuation in original formulation is overcome to fluctuate, it ensure that the slant pore either outward appearance or spawn activity of preparation is all more stable, fermentation level also obtains stable raising.
Description
Technical field
The invention belongs to biology and pharmaceutical technology field, more particularly to a kind of streptomyces aureus fermenting and producing tetracycline
Inclined-plane solid medium and cultural method.
Background technology
Tetracycline is a kind of macrolide antibiotics, is mainly used as production strain fermentation production using streptomyces aureus.Pass
The fermentation process of system is to be prepared into bacteria suspension using lyophilized pipe strain, is directly inoculated on inclined-plane solid medium and cultivates, produces
Spore suspension, is then inoculated in seed culture medium by substantial amounts of spore again, finally connects well-grown seed liquor is sterile
Kind carries out fermenting and producing in fermentation medium.For a long time, the culture medium as streptomyces aureus inclined-plane culture is to use
Using wheat bran do main carbon source and trace element supply, inorganic phosphate as phosphorus and a small amount of nitrogen source supply and agar as coagulate
Gu the batching mode of agent.But in recent years, as commercial development, environmental pollution are on the rise, wheat bran and agar quality given birth to
The influence of elongate member and environment, it is more and more unstable, cause caused spore either mode of appearance or conidium vitality not
Enough ideals, quality is unstable, and then has influence on fermentation process below.
The content of the invention
The purpose of the present invention is that the defects of overcoming above-mentioned prior art, there is provided one kind can make up former material in original formulation
Material wheat bran is high to the place of production quality requirement of wheat, selection is difficult, easily causes the defects of fermenting and producing fluctuation, ensures to prepare oblique
Face spore outward appearance bacterium colony is full, and pure color, monospore protonatomic mass is good, and for fermenting and producing, thalline is energetic, to various unfavorable bars
Part strong adaptability, the inclined-plane solid medium of the stable streptomyces aureus fermenting and producing tetracycline improved of fermentation level.
It is a further object of the present invention to provide the cultural method using above-mentioned inclined-plane solid-substrate fermentation production tetracycline.
The technical scheme taken for achieving the above object is：
A kind of inclined-plane solid medium of streptomyces aureus fermenting and producing tetracycline, it is characterised in that the culture medium, which forms, is：Bran
Skin 2.0~2.5%, dipotassium hydrogen phosphate 0.005~0.008%, diammonium hydrogen phosphate 0.008~0.013%, magnesium sulfate 0.003~
0.006%, corn steep liquor 0.5~1.0%, calcium carbonate 0.003%, agar 1.8~2.1%, remaining is water, by weight percentage.
A kind of cultural method that tetracycline is produced using above-mentioned inclined-plane solid-substrate fermentation, it is characterised in that its technique walks
Suddenly it is：Bacteria suspension is made in tetracycline original strain first, is inoculated in inclined-plane solid medium, in 33~35 DEG C of temperature conditionss
Lower culture 80~100 hours, then adds sterilized water to be prepared into suspension cultured slant pore, is inoculated in seed culture medium
In, under 32~33 DEG C of temperature conditionss, shaking table culture 20~28 hours, cultured seed liquor is finally inoculated into fermented and cultured
In base, under 33~35 DEG C of temperature conditionss, shaking table culture 80~100 hours.
The shaking table frequency is 20 turns/min.
The present invention is formed by changing the raw material in streptomyces aureus inclined-plane solid medium, overcomes former material in original formulation
Strain quality fluctuates caused by expecting quality fluctuation, ensure that the slant pore either outward appearance or spawn activity of preparation all compares
Stable, fermentation level also obtains stable raising.Experiment shows, with the production strain of new inclined-plane formula preparation, inclined-plane outward appearance bacterium colony
Full, pure color, monospore is big, and shake-flask seed mycelia is sturdy, unfolds, and immigration fermentation tank accretion rate is fast, and fermentation level is good.
Fermentation process should not stably turn sour rubescent.
Embodiment
The present invention is explained with example, it should be understood that example is to be used to illustrate rather than to this below
The limitation of invention.The scope of the present invention is determined with core content according to claims.
Streptomyces aureus original strain in following embodiments：Selection is stored in the freezing of~70 ± 5 DEG C of cryogenic refrigerator
Pipe bacterial strain.
Embodiment 1（Repeat three groups）
1. the preparation on blank inclined-plane
Inclined-plane solid medium composition：Wheat bran 2.0%, dipotassium hydrogen phosphate 0.005%, diammonium hydrogen phosphate 0.008%, magnesium sulfate
0.003%, corn steep liquor 0.5%, calcium carbonate 0.003%, agar 1.8%, remaining is water.
It is prepared by inclined-plane solid medium：By above material（Except calcium carbonate）It is contained in after dissolving in fixed container, by carbon
Sour calcium adds fully dissolving, and after stirring, control pH is sub-packed in 250ml eggplant-shape bottle, every bottle contains 40 7.5 or so
~60ml.Sterilized, control 117~121 DEG C, 0.008~0.012MPa of pressure of sterilising temp, sterilize 20~25min, paved
Cooled down into inclined-plane standby.
2. inclined plane inoculating, culture
Take streptomyces aureus original strain 1, draw 0.2ml and aseptically pour into and bacterium is made in sterilized purified water hangs
Liquid, shake up and be inoculated in ramp solid medium, under 33~35 DEG C of temperature conditionss, to cultivate 80~100 hours.
3. seed culture
The gained slant pore of process 2 is prepared into suspension, is inoculated in sterilized seed culture medium（Starch 3.0~3.50%,
Soybean cake powder：1.5~1.8%, dusty yeast：0.2~0.5%, peptone：0.2~0.6%, ammonium sulfate：0~0.25%, sodium bromide：
0.10~0.15%, calcium carbonate：0.1~0.3%, magnesium sulfate：0~0.025%, potassium dihydrogen sulfate：0~0.01%）On, holding 32~
33 DEG C of temperature, 220 turns/min of shaking table frequency, shaking table culture 22~28 hours, estimate the thick yellow wall built-up of seed liquor outward appearance.
4. fermented and cultured
By the fermentation medium that the sterile immigration of cultured seed liquor has sterilized in process 3（Starch：6.0~7.5%, paste
Essence：1~1.1%, soybean cake powder：2~3.0%, dusty yeast：0.1~0.15%, peptone：0.5~0.8%, ammonium sulfate：0.5~
0.8%th, sodium bromide：0.1~0.1%, calcium carbonate：0.3~0.5%, M accelerator：0.001~0.0015%）In, whole-process control temperature
At 31~33 DEG C, 220 turns/min of shaking table frequency, sterile culture 168~170 hours.
5th, above-mentioned experimental group data（）Three groups of repetition, it is as a result as follows：
| Test number (TN) | Inclined-plane outward appearance | Micro- sem observation spore shape | Fermentation period hr | Shake flask fermentation potency u/ml |
| 1 | Bacterium colony is big, full | Spore is big, uniform coloring | 169 | 31096 |
| 2 | Bacterium colony is big, full | Spore is big, uniform coloring | 170 | 31282 |
| 3 | Bacterium colony is big, full | Spore is big, uniform coloring | 172 | 32767 |
Embodiment 2
1. the preparation on blank inclined-plane
Inclined-plane solid medium composition：Wheat bran 2.5%, dipotassium hydrogen phosphate 0.006%, diammonium hydrogen phosphate 0.01%, magnesium sulfate
0.005%, corn steep liquor 0.8%, calcium carbonate 0.003%, agar 2.0%, remaining is water.
It is prepared by inclined-plane solid medium：By above material（Except calcium carbonate）It is contained in after dissolving in fixed container, by carbon
Sour calcium adds fully dissolving, and after stirring, control pH is sub-packed in 250ml eggplant-shape bottle, every bottle contains 40 7.5 or so
~60ml.Sterilized, control 117~121 DEG C, 0.008~0.012MPa of pressure of sterilising temp, sterilize 20~25min, paved
Cooled down into inclined-plane standby.
2. inclined plane inoculating, culture are the same as embodiment 1.
3. seed culture and fermented and cultured are the same as embodiment 1.
4th, above-mentioned three groups of experimental group repetition, it is as a result as follows：
| Experiment number | Inclined-plane outward appearance | Micro- sem observation spore shape | Fermentation period hr | Shake flask fermentation potency u/ml |
| 1 | Bacterium colony is big, full | Spore is big, uniform coloring | 169 | 32023 |
| 2 | Bacterium colony is big, full | Spore is big, uniform coloring | 170 | 32636 |
| 3 | Bacterium colony is big, full | Spore is big, uniform coloring | 172 | 33887 |
Embodiment 3
1. the preparation on blank inclined-plane
Inclined-plane solid medium composition：Wheat bran 2.5%, dipotassium hydrogen phosphate 0.008%, diammonium hydrogen phosphate 0.013%, magnesium sulfate
0.006%, corn steep liquor 1.0%, calcium carbonate 0.003%, agar 2.2%, remaining is water.
It is prepared by inclined-plane solid medium：By above material（Except calcium carbonate）It is contained in after dissolving in fixed container, by carbon
Sour calcium adds fully dissolving, and after stirring, control pH is sub-packed in 250ml eggplant-shape bottle, every bottle contains 40 7.5 or so
~60ml.Sterilized, control 117~121 DEG C, 0.008~0.012MPa of pressure of sterilising temp, sterilize 20~25min, paved
Cooled down into inclined-plane standby.
2. inclined plane inoculating, culture are the same as embodiment 1.
3rd, the culture of seed and fermentation is the same as embodiment 1.
4th, above-mentioned three groups of experimental group repetition, it is as a result as follows：
| Experiment number | Inclined-plane outward appearance | Micro- sem observation spore shape | Fermentation period hr | Shake flask fermentation potency u/ml |
| 1 | Bacterium colony is big, full | Spore is big, uniform coloring | 169 | 32218 |
| 2 | Bacterium colony is big, full | Spore is big, uniform coloring | 170 | 32957 |
| 3 | Bacterium colony is big, full | Spore is big, uniform coloring | 172 | 33136 |
Check experiment
1. press traditional inclined-plane solid medium（Wheat bran：3.5%th, agar：2.5%th, magnesium sulfate：0.006%th, hydrogen sulfate dipotassium：
0.015%th, diammonium hydrogen phosphate：0.02%）, culture medium is prepared, and be sub-packed in inclined-plane bottle and sterilized.
2. taking lyophilized pipe strain one, bacteria suspension is prepared into after adding sterilized water dissolving.
3. bacteria suspension picking is uniformly applied on inclined-plane solid medium prepared by process 1 respectively in right amount, 33~35 DEG C
At a temperature of, cultivate 80~100 hours.
4. seed culture and fermented and cultured are the same as embodiment 1,2 and 3.
Above-mentioned three groups of experimental group repetition, it is as a result as follows：
| Inclined-plane outward appearance | Micro- sem observation spore shape | Fermentation period hr | Shake flask fermentation potency u/ml | |
| Control group one | Bacterium colony is small, flat | Spore is less than normal, and spore coloring is uneven | 169 | 29967 |
| Control group two | Bacterium colony is small, flat | Spore is less than normal, and spore coloring is uneven | 170 | 30483 |
| Control group three | Bacterium colony is small, flat | Spore is less than normal, and spore coloring is uneven | 172 | 31298 |
Claims (3)
1. a kind of inclined-plane solid medium of streptomyces aureus fermenting and producing tetracycline, it is characterised in that the culture medium, which forms, is：
Wheat bran 2.0~2.5%, dipotassium hydrogen phosphate 0.005~0.008%, diammonium hydrogen phosphate 0.008~0.013%, magnesium sulfate 0.003~
0.006%, corn steep liquor 0.5~1.0%, calcium carbonate 0.003%, agar 1.8~2.1%, remaining is water, by weight percentage.
2. a kind of cultural method of inclined-plane solid-substrate fermentation production tetracycline using described in claim 1, its feature exist
It is in its processing step：Bacteria suspension is made in tetracycline original strain first, is inoculated in inclined-plane solid medium, 33~35
Cultivated 80~100 hours under DEG C temperature conditionss, then add sterilized water to be prepared into suspension cultured slant pore, be inoculated in
In seed culture medium, under 32~33 DEG C of temperature conditionss, shaking table culture 20~28 hours, finally cultured seed liquor is inoculated with
Into fermentation medium, under 33~35 DEG C of temperature conditionss, shaking table culture 80~100 hours.
3. according to the cultural method described in claim 2, it is characterised in that the shaking table frequency is 20 turns/min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371093A (en) * | 2018-11-20 | 2019-02-22 | 宁夏启元药业有限公司 | A kind of method of streptomyces aureus fermenting and producing tetracycline |
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| WO2013148207A2 (en) * | 2012-03-30 | 2013-10-03 | Danisco Us Inc. | Direct starch to fermentable sugar |
| CN103773831A (en) * | 2014-02-20 | 2014-05-07 | 宁夏泰瑞制药股份有限公司 | Novel culture medium and culture method for producing aureomycin by fermenting 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 |
| CN104513845A (en) * | 2014-12-09 | 2015-04-15 | 宁夏启元药业有限公司 | Tetracycline fermentation feeding medium and feeding method |
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|---|---|---|---|---|
| WO2013148207A2 (en) * | 2012-03-30 | 2013-10-03 | Danisco Us Inc. | Direct starch to fermentable sugar |
| CN103773831A (en) * | 2014-02-20 | 2014-05-07 | 宁夏泰瑞制药股份有限公司 | Novel culture medium and culture method for producing aureomycin by fermenting 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 |
| CN104513845A (en) * | 2014-12-09 | 2015-04-15 | 宁夏启元药业有限公司 | Tetracycline fermentation feeding medium and feeding method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109371093A (en) * | 2018-11-20 | 2019-02-22 | 宁夏启元药业有限公司 | A kind of method of streptomyces aureus fermenting and producing tetracycline |
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