CN112280812B - Method for improving fermentation yield of aureomycin A and ratio of aureomycin A to aureomycin B - Google Patents
Method for improving fermentation yield of aureomycin A and ratio of aureomycin A to aureomycin B Download PDFInfo
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- CN112280812B CN112280812B CN202011068674.7A CN202011068674A CN112280812B CN 112280812 B CN112280812 B CN 112280812B CN 202011068674 A CN202011068674 A CN 202011068674A CN 112280812 B CN112280812 B CN 112280812B
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- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 title claims abstract description 75
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 title claims abstract description 75
- 229960004475 chlortetracycline Drugs 0.000 title claims abstract description 75
- 235000019365 chlortetracycline Nutrition 0.000 title claims abstract description 75
- 238000000855 fermentation Methods 0.000 title claims abstract description 75
- 230000004151 fermentation Effects 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000005070 sampling Methods 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- 238000009423 ventilation Methods 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 238000003756 stirring Methods 0.000 claims abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 229920002472 Starch Polymers 0.000 claims description 6
- 239000008107 starch Substances 0.000 claims description 6
- 235000019698 starch Nutrition 0.000 claims description 6
- 229920002774 Maltodextrin Polymers 0.000 claims description 3
- 239000005913 Maltodextrin Substances 0.000 claims description 3
- 229940035034 maltodextrin Drugs 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 10
- 239000011259 mixed solution Substances 0.000 description 9
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 241000187180 Streptomyces sp. Species 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 235000013312 flour Nutrition 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 229930194765 Chrysomycin Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for improving fermentation yield of aureomycin A and the ratio of aureomycin A to aureomycin B, which comprises the following steps of 1: after the seeds are transferred in the fermentation tank, stirring and culturing at 28 ℃ with a certain ventilation amount and tank pressure; step 2: sampling and detecting the concentration of residual sugar in fermentation liquor at regular intervals after fermentation is started, and starting continuous flow sugar solution adding when the concentration of the residual sugar in the fermentation liquor reaches a preset value; and step 3: sampling at regular intervals to detect the content of the aureomycin A in the fermentation liquor and the content of the aureomycin A in the supernatant after the fermentation liquor is centrifuged; and 4, step 4: and judging whether the content of the aureomycin A in the fermentation liquor in the current time period is slowly increased or not and the content of the aureomycin A in the supernatant after the fermentation liquor in the current time period is centrifuged is rapidly increased, if so, putting the fermentation liquor in a tank. The method is simple and convenient to operate, the fermentation yield of the aureomycin A can reach 3.5 g/L, the ratio of the aureomycin A to the aureomycin B can reach 10: 1, and the separation and purification of the aureomycin A in the subsequent extraction process are greatly facilitated.
Description
Technical Field
The invention belongs to the field of biological fermentation production, and particularly relates to a method for improving the fermentation yield of aureomycin A, and more particularly relates to a method for improving the fermentation yield of aureomycin A and the ratio of aureomycin A to aureomycin B.
Background
Chrysomycins (chrysomycins) are produced by fermenting Streptomyces sp.891 and are divided into two components, namely aureomycin A and aureomycin B, wherein the aureomycin A has good activity of resisting drug-resistant staphylococcus aureus. The A, B structural formula is:
due to the extremely similar structures of the aureomycin A and the aureomycin B, post-extraction, separation and purification are extremely difficult. The method is particularly important for improving the yield of the aureomycin A and reducing the proportion of the aureomycin B from a fermentation source.
Disclosure of Invention
The invention aims to provide a method for improving the fermentation yield of the aureomycin A and the ratio of the aureomycin A to the aureomycin B so as to solve the problem of difficult extraction, separation and purification caused by similar structures of the aureomycin A and the aureomycin B in the background art.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for increasing the fermentation yield of aureomycin a and the ratio between aureomycin a and B, said method comprising the steps of:
step 1: after the seed is transferred to the fermentation tank, stirring and culturing at 28 ℃ with a certain ventilation amount and tank pressure, and executing the step 2;
step 2: after fermentation is started, sampling at regular intervals to detect the concentration of residual sugar in the fermentation liquor, and when the concentration of the residual sugar in the fermentation liquor reaches a preset value, starting continuous flow sugar adding solution and executing the step 3;
and step 3: sampling at regular intervals to detect the content of the aureomycin A in the fermentation liquor and the content of the aureomycin A in the supernatant after the fermentation liquor is centrifuged, and executing the step 4;
and 4, step 4: and (3) judging whether the ratio of the contents of the aureomycin A in the fermentation liquor in the current time period to the previous time period is less than or equal to 1.05 and whether the ratio of the contents of the aureomycin A in the supernatant after the fermentation liquor is centrifuged in the current time period to the previous time period is greater than 2, if so, putting the fermentation tank, and otherwise, returning to the step (3).
Preferably, in the step 1, the ventilation amount is 0-0.3 vvm.
Preferably, in the step 1, the tank pressure is 0-0.025 MPa.
Preferably, in the step 2, the sugar solution is one or more of a glucose solution, a soluble starch solution and a maltodextrin solution.
Preferably, in the step 2, the preset value of the residual sugar content is 0 to 10 g/l.
Preferably, in the step 2, the concentration of the sugar solution is 200-300 g/L.
Preferably, the interval in step 2 is every 4 hours.
Preferably, the interval in step 3 is every 8 hours.
Compared with the prior art, the invention has the beneficial effects that:
the method is simple and convenient to operate, the fermentation yield of the aureomycin A can reach 3.5 g/L, and the ratio of the aureomycin A to the aureomycin B can reach 10: 1, greatly facilitating the separation and purification of the aureomycin A in the subsequent extraction process.
Drawings
FIG. 1 is a flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the fermentation formula is as follows: dissolving the components in 20 liters of water to obtain a mixed solution, wherein the concentrations of the components in the mixed solution are respectively as follows: starch 5 g/l, glucose 20 g/l, soya flour 10 g/l, peptone 2 g/l, yeast extract 2 g/l, sodium chloride 4 g/l, K2HPO40.5 g/l, MgSO4·7H20.5 g/L of O and 2 g/L of calcium carbonate; pouring the mixed solution into a 50L fermentation tank, adjusting the pH value to 7.0-7.5, sterilizing at 121 ℃ for 30 minutes, and cooling to 28 ℃.
Transferring 5% of the cultured seeds to a fermentation tank for fermentation, wherein the ventilation rate of the fermentation tank is 0.25vvm, the tank pressure is 0.02MPa, and the seeds are stirred and cultured at 28 ℃; the dissolved oxygen is controlled to be not less than 40 percent by adjusting the rotating speed.
Sampling every 4 hours to detect the glucose content in the fermentation liquor, and continuously adding 300 g/L glucose solution when the residual sugar concentration reaches 10 g/L.
Sampling every 8 hours to detect the content of the aureomycin A in the fermentation broth and the content of the aureomycin A in the supernatant after the fermentation broth is centrifuged in the fermentation tank. After 160 hours, the fermentation yield of the aureomycin A reaches 3.5 g/l, and the ratio of the aureomycin A to the aureomycin B is 10: 1.
Example 2:
the fermentation formula is as follows: dissolving the components in 20 liters of water to obtain a mixed solution, wherein the concentrations of the components in the mixed solution are respectively as follows: starch 5 g/l, glucose 20 g/l, soya flour 10 g/l, peptone 2 g/l, yeast extract 2 g/l, sodium chloride 4 g/l, K2HPO40.5 g/l, MgSO4·7H20.5 g/L of O and 2 g/L of calcium carbonate; pouring the mixed solution into a 50L fermentation tank, adjusting the pH value to 7.0-7.5, sterilizing at 121 ℃ for 30 minutes, and cooling to 28 ℃.
Transferring 5% of the cultured seeds to a fermentation tank for fermentation, wherein the ventilation rate of the fermentation tank is 0.2vvm, the tank pressure is 0.025MPa, and the seeds are stirred and cultured at 28 ℃; the dissolved oxygen is controlled to be not less than 40 percent by adjusting the rotating speed.
Sampling every 4 hours to detect the glucose content in the fermentation liquor, and continuously adding 250 g/L soluble starch solution when the residual sugar concentration reaches 10 g/L.
Sampling every 8 hours to detect the content of the aureomycin A in the fermentation broth and the content of the aureomycin A in the supernatant after the fermentation broth is centrifuged in the fermentation tank. After 180 hours, the fermentation yield of the aureoflavomycin A reaches 3.15 g/L, and the ratio of the aureoflavomycin A to the aureoflavomycin B is 10: 3.
Example 3:
the fermentation formula is as follows: dissolving the components in 20 liters of water to obtain a mixed solution, wherein the concentrations of the components in the mixed solution are respectively as follows: starch 5 g/l, glucose 20 g/l, soya flour 10 g/l, peptone 2 g/l, yeast extract 2 g/l, sodium chloride 4 g/l, K2HPO40.5 g/l, MgSO4·7H20.5 g/L of O and 2 g/L of calcium carbonate; pouring the mixed solution into a 50L fermentation tank, adjusting the pH value to 7.0-7.5, sterilizing at 121 ℃ for 30 minutes, and cooling to 28 ℃.
Transferring 5% of the cultured seeds to a fermentation tank for fermentation, wherein the ventilation rate of the fermentation tank is 0.3vvm, the tank pressure is 0.02MPa, and the seeds are stirred and cultured at 28 ℃; the dissolved oxygen is controlled to be not less than 40 percent by adjusting the rotating speed.
Sampling every 4 hours to detect the glucose content in the fermentation liquor, and continuously adding 300 g/L of maltodextrin solution when the residual sugar concentration reaches 10 g/L.
Sampling every 8 hours to detect the content of the aureomycin A in the fermentation broth and the content of the aureomycin A in the supernatant after the fermentation broth is centrifuged in the fermentation tank. After 170 hours, the fermentation yield of the aureomycin A reaches 3.25 g/L, and the ratio of the aureomycin A to the aureomycin B is 10: 2.
The cultured seeds mentioned in the invention are liquid containing Streptomyces sp.891 obtained by seed tank culture. As for how to cultivate the seed tank to obtain the liquid containing the Streptomyces sp.891, the method is common knowledge in the field, and the skilled person can set the method according to the actual situation.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (8)
1. A method for increasing the fermentation yield of aureomycin a and the ratio between aureomycin a and B, characterized in that it comprises the following steps:
step 1: after the seeds are transferred from the fermentation tank, stirring and culturing at a certain temperature with a certain ventilation quantity and tank pressure, and executing the step 2;
step 2: after fermentation is started, sampling and detecting the concentration of residual sugar in the fermentation liquor at certain time intervals, starting continuous flow sugar solution adding when a preset value is reached, and executing the step 3;
and step 3: sampling and detecting the content of the aureomycin A in the fermentation liquor and the content of the aureomycin A in the supernatant after the fermentation liquor is centrifuged at intervals, and executing the step 4;
and 4, step 4: and (3) judging whether the ratio of the contents of the aureomycin A in the fermentation liquor in the current time period to the previous time period is less than or equal to 1.05 and whether the ratio of the contents of the aureomycin A in the supernatant after the fermentation liquor is centrifuged in the current time period to the previous time period is greater than 2, if so, putting the fermentation tank, and otherwise, returning to the step (3).
2. The method for increasing fermentation yield of aureomycin a and the ratio between aureomycin a and B according to claim 1, characterized in that in step 1 the aeration is 0-0.3 vvm.
3. The method for increasing fermentation yield of aureomycin A and the ratio between aureomycin A and B according to claim 1, characterized in that in step 1 the pot pressure is 0-0.025 MPa.
4. The method for improving fermentation yield of aureomycin A and ratio between aureomycin A and B according to claim 1, characterized in that in step 2 the sugar solution is one or more of glucose solution, soluble starch solution and maltodextrin solution.
5. The method for increasing fermentation yield of aureomycin A and the ratio between aureomycin A and B according to claim 1, characterized in that in step 2 the preset value of the residual sugar concentration is between 0 and 10 g/l.
6. The method for increasing fermentation yield of aureomycin A and the ratio between aureomycin A and B as claimed in claim 1, wherein in step 2, the concentration of the sugar solution is 200-300 g/l.
7. The method for increasing fermentation yield of aureomycin a and the ratio between aureomycin a and B according to claim 1, characterized in that the interval in step 2 is 4 hours.
8. The method for increasing fermentation yield of aureomycin a and the ratio between aureomycin a and B according to claim 1, characterized in that the interval in step 3 is every 8 hours.
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| US6030951A (en) * | 1996-11-22 | 2000-02-29 | Mercian Corporation | Chrysomycin derivative compounds and use as antitumor agents |
| CN102168034B (en) * | 2010-11-29 | 2012-12-05 | 中国科学院南海海洋研究所 | Marine streptomyces and method for preparing tirandamycin A and B by utilizing strain |
| ES2583093T3 (en) * | 2011-03-03 | 2016-09-19 | Denovamed Inc. | Antimicrobial compounds / adjuvants |
| CN107892680B (en) * | 2017-10-27 | 2020-11-10 | 北京大学 | Antituberculous compound and synthesis method and application thereof |
| CN109867663A (en) * | 2018-12-29 | 2019-06-11 | 浙江工业大学 | A method of with preparation chromatographic isolation chrysomycin A and chrysomycin B |
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Address after: 310000 Zhaohui District 6, Xiacheng District, Hangzhou City, Zhejiang Province Patentee after: JIANG University OF TECHNOLOGY Patentee after: ZHEJIANG LYUCHUANG BIOTECHNOLOGY Co.,Ltd. Patentee after: Sheng Yingtao Address before: 310014 No. 18 Chao Wang Road, Xiacheng District, Zhejiang, Hangzhou Patentee before: JIANG University OF TECHNOLOGY Patentee before: Zhejiang Lvchuang Biotechnology Co.,Ltd. Patentee before: Sheng Yingtao |
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