CN114032265A - Fermentation method to produce thymidine - Google Patents
Fermentation method to produce thymidine Download PDFInfo
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- CN114032265A CN114032265A CN202111470737.6A CN202111470737A CN114032265A CN 114032265 A CN114032265 A CN 114032265A CN 202111470737 A CN202111470737 A CN 202111470737A CN 114032265 A CN114032265 A CN 114032265A
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- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 title claims abstract description 79
- 238000000855 fermentation Methods 0.000 title claims abstract description 65
- 230000004151 fermentation Effects 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 40
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 title claims abstract description 36
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 229940104230 thymidine Drugs 0.000 title claims abstract description 35
- 239000012528 membrane Substances 0.000 claims abstract description 23
- 235000015097 nutrients Nutrition 0.000 claims abstract description 18
- 230000003321 amplification Effects 0.000 claims abstract description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 17
- 230000008569 process Effects 0.000 claims abstract description 17
- 238000012216 screening Methods 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 238000011081 inoculation Methods 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 6
- 239000006185 dispersion Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 13
- 241000588724 Escherichia coli Species 0.000 claims description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 238000005273 aeration Methods 0.000 claims description 4
- 238000001728 nano-filtration Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims 3
- 230000001580 bacterial effect Effects 0.000 claims 2
- 239000002609 medium Substances 0.000 claims 2
- 238000005138 cryopreservation Methods 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- 238000012856 packing Methods 0.000 claims 1
- 238000010257 thawing Methods 0.000 claims 1
- 238000002255 vaccination Methods 0.000 claims 1
- 238000012545 processing Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 238000012423 maintenance Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- XNKLLVCARDGLGL-JGVFFNPUSA-N Stavudine Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1C=C[C@@H](CO)O1 XNKLLVCARDGLGL-JGVFFNPUSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- IQFYYKKMVGJFEH-BIIVOSGPSA-N 2'-deoxythymidine Natural products O=C1NC(=O)C(C)=CN1[C@@H]1O[C@@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-BIIVOSGPSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 108010025899 gelatin film Proteins 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229960001203 stavudine Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960002555 zidovudine Drugs 0.000 description 1
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/385—Pyrimidine nucleosides
-
- 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
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a process for producing thymidine by a fermentation method, and relates to the technical field of preparation of beta-thymidine. The thymidine production process based on fermentation method comprises inoculation, screening, amplification, fermentation, extraction and purification. Through the secondary culture amplification processing, a large amount of strain bags convenient to quantitatively and directly use are prepared through a gel membrane, and when initial fermentation, tank body maintenance and fermentation nutrient solution replacement are carried out, the strain bags are convenient to directly carry out proportioning use, the working efficiency is improved, and the filtered residual solution flows back to a fermentation tank by utilizing circulating equipment, so that nutrient substances left over by the residual solution can be reused, the utilization rate of the nutrient solution is improved, the processing cost is reduced, and the practicability of the invention is enhanced.
Description
Technical Field
The invention relates to the technical field of preparation of beta-thymidine, in particular to a process for producing thymidine by a fermentation method.
Background
Beta-thymidine, also known as 2 '-deoxythymidine, is a precursor substance against aids stavudine (3' -deoxy-2 ', 3' -didehydro thymidine) and azidothymidine. There are three major methods for thymidine production. Firstly, the chemical synthesis method for producing thymidine is very long in process, and the formation of glycosidic bonds lacks stereospecificity, so that the finally obtained thymidine is relatively expensive. And secondly, the biological enzyme method has the advantage of strong specificity, but the period is long, and the product is not easy to separate. And thirdly, the biological fermentation method has simple process and low subsequent separation cost, and is a production method with great prospect, so the research on the method for extracting and purifying thymidine from the fermentation liquor obtained by preparing thymidine by the biological fermentation method is popular.
The existing technology for producing thymidine by a biological fermentation method has the advantages of low utilization rate of culture solution, production cost improvement and low production efficiency.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a fermentation process for producing thymidine, and solves the problems of low utilization rate of culture solution, high production cost and low production efficiency of the existing biological fermentation process for producing thymidine.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: the process for producing thymidine by a fermentation method comprises the following steps of inoculation, screening, amplification, fermentation, extraction and purification, and comprises the following specific process flows:
s1, inoculation
In a sterile environment, coating and inoculating escherichia coli strains implanted with related genes on a culture medium, and putting the culture medium into a culture room for colony cultivation;
s2, screening
After the colony culture is finished, in a sterile environment, screening and breeding fast colonies, cutting off, preparing a dispersion liquid, diluting 2ml of the dispersion liquid by 1000 times, and detecting the number of escherichia coli in unit volume of the dispersion liquid;
s3. amplification
Uniformly mixing the prepared dispersion liquid into a liquid culture medium according to the proportion of 1:100, putting the mixture into a culture chamber for culture for 3-5 days for amplification, subpackaging the mixture into a plurality of strain packets through gel membranes after the amplification is finished, and freezing and preserving the strain packets;
s4, fermentation
Filling nutrient solution according to the specification of a fermentation tank, unfreezing the strain packets, putting the strain packets in corresponding proportion into the fermentation tank, performing primary aeration culture for 10-24h, starting an extraction process, and continuously adding the nutrient solution to perform continuous fermentation treatment;
s5, extracting
A microporous filter membrane is arranged at the bottom of a fermentation tank to form a filtering chamber, fermentation solution without escherichia coli is filtered out, the fermentation solution in the chamber is extracted, thymidine solution is filtered out through ultramicropore filter membrane equipment, and the residual solution flows back to the fermentation tank through circulating equipment;
s6, purifying
Decolorizing thymidine solution with activated carbon, vacuum concentrating thymidine solution by evaporation to obtain crystalline state, lyophilizing to obtain beta-thymidine powder, and packaging.
Preferably, in the first step, the culture dish is GYT culture medium, and the colony cultivation time is 24-36 h.
Preferably, in the third step, the volume of the strain bag is 50ml, and the colony density is 3000-4000 per ml.
Preferably, in the fourth package, the ratio of the nutrient solution to the strain bag is 1: 2000-3500.
Preferably, the pore size of the microporous filter membrane in the step five is 0.1-0.3 μm.
Preferably, the ultra-microporous filter membrane equipment in the fifth step performs double-layer filtration through an ultrafiltration membrane with the molecular weight cutoff of 3500-20000 Da and a nanofiltration membrane with the molecular weight cutoff of 150-300 Da.
In the fifth step, the whole circulation frequency of the solution in the fermentation tank is 8-10 times.
(III) advantageous effects
The present invention provides a process for producing thymidine by a fermentation method. The method has the following beneficial effects:
1. according to the invention, through secondary culture amplification processing, a large amount of strain bags convenient to quantitatively use directly are prepared through a gel film, and are convenient to directly carry out proportioning use when initial fermentation, tank body maintenance and fermentation nutrient solution replacement are carried out, so that the working efficiency is improved.
2. According to the invention, the residual solution after filtration is returned to the fermentation tank by using the circulating equipment, so that the nutrient substances left by the residual solution can be reused, the utilization rate of the nutrient solution is improved, the processing cost is reduced, and the practicability of the invention is enhanced.
Drawings
FIG. 1 is a schematic process flow diagram 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 drawings in the embodiments of the present invention, and it is obvious 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.
The first embodiment is as follows:
as shown in fig. 1, the embodiment of the present invention provides a process for producing thymidine by a fermentation method, which comprises inoculation, screening, amplification, fermentation, extraction and purification, and the specific process flow is as follows:
s1, inoculation
In a sterile environment, coating and inoculating escherichia coli strains implanted with related genes on a culture medium, putting the culture medium into a culture room, and culturing colonies, wherein a culture dish is a GYT culture medium, and the colony culturing time is 24;
s2, screening
After the colony culture is finished, in a sterile environment, screening and breeding fast colonies, cutting off, preparing a dispersion liquid, diluting 2ml of the dispersion liquid by 1000 times, and detecting the number of escherichia coli in unit volume of the dispersion liquid;
s3. amplification
Uniformly mixing the prepared dispersion liquid into a liquid culture medium according to the proportion of 1:100, putting the mixture into a culture room for culture for 3 days for amplification, subpackaging the mixture into a plurality of strain packets through gel membranes after the amplification is finished, wherein the capacity of each strain packet is 50ml, the colony density is 3000 per ml, and freezing and storing the strain packets;
s4, fermentation
Adding nutrient solution according to the specification of a fermentation tank, unfreezing the strain package, putting the strain package with a corresponding proportion into the fermentation tank, carrying out preliminary aeration culture for 24 hours with the proportion of the nutrient solution to the strain package being 1:2000, starting an extraction process, and continuously adding the nutrient solution at the same time to carry out continuous fermentation treatment;
s5, extracting
A microporous filter membrane is arranged at the bottom of a fermentation tank, the pore size of the microporous filter membrane is 0.1-0.3 mu m, a filtering chamber is formed, fermentation solution without escherichia coli is filtered out, and the fermentation solution in the chamber is extracted; carrying out double-layer filtering on a thymidine solution by using an ultra-microporous filter membrane device through an ultrafiltration membrane with the molecular weight cutoff of 3500-20000 Da and a nanofiltration membrane with the molecular weight cutoff of 150-300 Da, and refluxing the residual solution to a fermentation tank through circulation equipment, wherein the integral circulation frequency of the solution in the fermentation tank is 10 times;
s6, purifying
Decolorizing thymidine solution with activated carbon, vacuum concentrating thymidine solution by evaporation to obtain crystalline state, lyophilizing to obtain beta-thymidine powder, and packaging.
Example two
The difference between the present embodiment and the first embodiment is: the process for producing thymidine by a fermentation method comprises the following steps of inoculation, screening, amplification, fermentation, extraction and purification, and comprises the following specific process flows:
s1, inoculation
In a sterile environment, coating and inoculating escherichia coli strains implanted with related genes on a culture medium, putting the culture medium into a culture room, and culturing colonies, wherein a culture dish is a GYT culture medium, and the colony culturing time is 36 hours;
s2, screening
After the colony culture is finished, in a sterile environment, screening and breeding fast colonies, cutting off, preparing a dispersion liquid, diluting 2ml of the dispersion liquid by 1000 times, and detecting the number of escherichia coli in unit volume of the dispersion liquid;
s3. amplification
Uniformly mixing the prepared dispersion liquid into a liquid culture medium according to the proportion of 1:100, putting the dispersion liquid into a culture chamber for culture for 3-5 days for amplification, after the amplification is finished, subpackaging the dispersion liquid into a plurality of strain packets through gel membranes, wherein the capacity of each strain packet is 50ml, the colony density is 4000 per ml, and freezing and storing the strain packets;
s4, fermentation
Adding nutrient solution according to the specification of a fermentation tank, unfreezing the strain package, putting the strain package with a corresponding proportion into the fermentation tank, carrying out primary aeration culture for 10 hours with the proportion of the nutrient solution to the strain package being 1:3500, starting an extraction process, continuously adding the nutrient solution at the same time, and carrying out continuous fermentation treatment;
s5, extracting
A microporous filter membrane is arranged at the bottom of a fermentation tank, the pore size of the microporous filter membrane is 0.1-0.3 mu m, a filtering chamber is formed, fermentation solution without escherichia coli is filtered out, and the fermentation solution in the chamber is extracted; the method comprises the following steps of filtering thymidine solution through an ultra-microporous filter membrane device through an ultrafiltration membrane with the molecular weight cutoff of 3500-20000 Da and a nanofiltration membrane with the molecular weight cutoff of 150-300 Da in a double-layer mode, enabling the residual solution to flow back to a fermentation tank through circulation equipment, wherein the integral circulation frequency of the solution in the fermentation tank is 8 times;
s6, purifying
Decolorizing thymidine solution with activated carbon, vacuum concentrating thymidine solution by evaporation to obtain crystalline state, lyophilizing to obtain beta-thymidine powder, and packaging.
Compared with the first embodiment, the present embodiment has a larger number of bacteria, faster reaction, fewer cycles, higher production efficiency, but higher production cost.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225413A (en) * | 2007-01-18 | 2008-07-23 | 肇东爱尔乳酸有限公司 | Method for producing lacitc acid by non-calcium autocycle continuous fermentation salt fermentation |
| CN104560838A (en) * | 2013-10-09 | 2015-04-29 | 丁庆豹 | Recombinant thymidine kinase enzymatic synthesis method of 2'-deoxypyrimidine nucleotide |
| KR20160086659A (en) * | 2015-01-12 | 2016-07-20 | (주)포바이오코리아 | Fermentation process for preparing thymidine by the recombinant E. coli |
| CN107574201A (en) * | 2017-09-27 | 2018-01-12 | 江西诚志生物工程有限公司 | Using the method for fermentation method production β thymidines |
| CN110272461A (en) * | 2019-06-29 | 2019-09-24 | 赤峰蒙广生物科技有限公司 | A method of purifying beta-thymidine from fermentation liquid |
| US20200330984A1 (en) * | 2017-10-06 | 2020-10-22 | Kansas State University Research Foundation | Hydrogel membrane and methods for selective retrieval of microbial targets |
| CN112831086A (en) * | 2021-01-18 | 2021-05-25 | 刘永昶 | Preparation method and application of special solid strain for liquid fermentation culture |
-
2021
- 2021-12-03 CN CN202111470737.6A patent/CN114032265A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225413A (en) * | 2007-01-18 | 2008-07-23 | 肇东爱尔乳酸有限公司 | Method for producing lacitc acid by non-calcium autocycle continuous fermentation salt fermentation |
| CN104560838A (en) * | 2013-10-09 | 2015-04-29 | 丁庆豹 | Recombinant thymidine kinase enzymatic synthesis method of 2'-deoxypyrimidine nucleotide |
| KR20160086659A (en) * | 2015-01-12 | 2016-07-20 | (주)포바이오코리아 | Fermentation process for preparing thymidine by the recombinant E. coli |
| CN107574201A (en) * | 2017-09-27 | 2018-01-12 | 江西诚志生物工程有限公司 | Using the method for fermentation method production β thymidines |
| US20200330984A1 (en) * | 2017-10-06 | 2020-10-22 | Kansas State University Research Foundation | Hydrogel membrane and methods for selective retrieval of microbial targets |
| CN110272461A (en) * | 2019-06-29 | 2019-09-24 | 赤峰蒙广生物科技有限公司 | A method of purifying beta-thymidine from fermentation liquid |
| CN112831086A (en) * | 2021-01-18 | 2021-05-25 | 刘永昶 | Preparation method and application of special solid strain for liquid fermentation culture |
Non-Patent Citations (1)
| Title |
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| 李思梦等: ""代谢工程方法改造大肠杆菌生产胸苷"", 《生物工程学报》, 28 May 2014 (2014-05-28), pages 105 - 114 * |
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