CN112501222A - Fermentation method of glutamic acid - Google Patents
Fermentation method of glutamic acid Download PDFInfo
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- CN112501222A CN112501222A CN202011469070.3A CN202011469070A CN112501222A CN 112501222 A CN112501222 A CN 112501222A CN 202011469070 A CN202011469070 A CN 202011469070A CN 112501222 A CN112501222 A CN 112501222A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 115
- 230000004151 fermentation Effects 0.000 title claims abstract description 115
- 238000000034 method Methods 0.000 title claims abstract description 47
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 41
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 41
- 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 abstract description 33
- 239000008103 glucose Substances 0.000 claims abstract description 33
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 23
- 230000001954 sterilising effect Effects 0.000 claims abstract description 16
- 238000012807 shake-flask culturing Methods 0.000 claims abstract description 14
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims description 59
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 32
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 26
- 239000002253 acid Substances 0.000 claims description 22
- 238000010899 nucleation Methods 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 17
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 16
- 235000019764 Soybean Meal Nutrition 0.000 claims description 16
- 238000001816 cooling Methods 0.000 claims description 16
- 229960003390 magnesium sulfate Drugs 0.000 claims description 16
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 16
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 16
- 229930182817 methionine Natural products 0.000 claims description 16
- 229960004452 methionine Drugs 0.000 claims description 16
- 239000004455 soybean meal Substances 0.000 claims description 16
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
- 229960001781 ferrous sulfate Drugs 0.000 claims description 13
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 13
- 239000011790 ferrous sulphate Substances 0.000 claims description 13
- 239000000413 hydrolysate Substances 0.000 claims description 13
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 13
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 13
- 229940099596 manganese sulfate Drugs 0.000 claims description 13
- 235000007079 manganese sulphate Nutrition 0.000 claims description 13
- 239000011702 manganese sulphate Substances 0.000 claims description 13
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 10
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 10
- 240000008042 Zea mays Species 0.000 claims description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 10
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- 230000029087 digestion Effects 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- 239000001103 potassium chloride Substances 0.000 claims description 7
- 235000011164 potassium chloride Nutrition 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 239000001384 succinic acid Substances 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- 239000002518 antifoaming agent Substances 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 235000006109 methionine Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 229960002816 potassium chloride Drugs 0.000 claims description 4
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229910000831 Steel Inorganic materials 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 239000010959 steel Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007640 basal medium Substances 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 24
- 238000006243 chemical reaction Methods 0.000 abstract description 12
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- 230000009286 beneficial effect Effects 0.000 abstract description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 32
- 229960002989 glutamic acid Drugs 0.000 description 32
- 230000000052 comparative effect Effects 0.000 description 17
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 14
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- 239000002126 C01EB10 - Adenosine Substances 0.000 description 7
- 229960005305 adenosine Drugs 0.000 description 7
- YVBGRQLITPHVOP-UHFFFAOYSA-L disodium;[hydroxy-[hydroxy(oxido)phosphoryl]oxyphosphoryl] hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])(=O)OP(O)(=O)OP(O)([O-])=O YVBGRQLITPHVOP-UHFFFAOYSA-L 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 description 6
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- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- XKGUZGHMWUIYDR-UHFFFAOYSA-N benzyl n-(3-fluoro-4-morpholin-4-ylphenyl)carbamate Chemical compound C=1C=C(N2CCOCC2)C(F)=CC=1NC(=O)OCC1=CC=CC=C1 XKGUZGHMWUIYDR-UHFFFAOYSA-N 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
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- 238000006555 catalytic reaction Methods 0.000 description 2
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- 238000007405 data analysis Methods 0.000 description 2
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- 230000037361 pathway Effects 0.000 description 2
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- 238000002834 transmittance Methods 0.000 description 2
- 108091006112 ATPases Proteins 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 241000186145 Corynebacterium ammoniagenes Species 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- -1 hexose phosphate Chemical class 0.000 description 1
- UJKDYMOBUGTJLZ-RUCXOUQFSA-N ksc605q1h Chemical compound OC(=O)[C@@H](N)CCC(O)=O.OC(=O)[C@@H](N)CCC(O)=O UJKDYMOBUGTJLZ-RUCXOUQFSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
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- 239000010452 phosphate Substances 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/14—Glutamic acid; Glutamine
-
- 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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- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses a fermentation method of glutamic acid, belonging to the technical field of glutamic acid fermentation, comprising a shake flask culture stage of a fermentation strain, an expanded culture stage of a first-stage seed tank, an expanded culture stage of a second-stage seed tank and a fermentation stage, wherein the fermentation stage adopts a fermentation medium with zero base sugar concentration and replaces the traditional fermentation mode of initial addition of high-concentration glucose in a mode of feeding in a flowing manner at first; the invention has the beneficial effects that: the problems that high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process and the liquid ammonia with over-high local concentration and the high-concentration glucose can generate side reaction are solved, and the fermentation time is further shortened compared with the conventional fermentation method for initially adding the high-concentration glucose.
Description
The technical field is as follows:
the invention belongs to the technical field of glutamic acid fermentation, and particularly relates to a fermentation method of glutamic acid.
Background art:
the fermentation production of glutamic acid is a biochemical process of decomposing and metabolizing nutrient substances, synthesizing required products and glutamic acid by glutamic acid producing bacteria in the life activity process. Currently, there are Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium scatterum, Brevibacterium flavum, Brevibacterium ammoniagenes, and the like, which are industrially used. In the fermentation production process, a plurality of factors influencing the growth, reproduction, metabolism and synthesis of the glutamic acid producing strain are controlled purposefully through artificial intervention, so that the metabolic synthesis requirement of the glutamic acid strain is finally met, and the aim of increasing the product and reducing the consumption can be achieved.
The glutamic acid producing bacteria are the main body of the reaction process and the biocatalyst of the reaction process, take in the nutrition of the raw materials, and carry out complex biochemical reaction through specific enzyme systems in cells. The reactant in the substrate enters the cell body through the cell wall and the cell membrane, and is subjected to catalytic reaction under the action of enzyme to be converted into a product and released, and the inherent characteristics and the metabolic rule of the cell are key factors influencing biochemical reaction.
The glutamic acid fermentation stage is mainly divided into: a glutamic acid producing bacterium proliferation stage and a glutamic acid producing bacterium acid production stage, wherein a biosynthetic pathway of glutamic acid in the acid production stage of the glutamic acid producing bacterium is approximately: glucose is subjected to glycolysis (EMP pathway) and hexose phosphate shunt (HMP pathway) to produce pyruvate, which is then oxidized to acetyl-CoA (acetyl COA), which then enters the tricarboxylic acid cycle to produce alpha-ketoglutarate. Catalysis of alpha-ketoglutarate in glutamate dehydrogenase and with NH4 +In the presence of a catalyst, glutamic acid is produced.
In the proliferation stage of the glutamic acid producing bacteria, the glutamic acid producing bacteria need to reach a certain OD value in a short time, the fermentation culture medium of the glutamic acid fermentation process of the traditional process contains 10-15% or even higher initial sugar content to meet the requirement of quick growth of the glutamic acid producing bacteria in the initial growth stage, so high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process, a large amount of pigment is generated to burden subsequent processes, a certain amount of glucose is wasted, especially, liquid ammonia needs to be added for regulation in the subsequent process, the local overhigh liquid ammonia and the high-concentration glucose can generate side reaction to generate byproducts to cause product quality reduction, the generated byproducts are similar to the physicochemical property of glutamic acid, and the difficulty of subsequent extraction and purification is increased.
The invention content is as follows:
in order to solve the problems and overcome the defects of the prior art, the invention provides a fermentation method of glutamic acid, which can effectively solve the problems that high-concentration glucose can generate serious caramelization reaction in a high-temperature sterilization process and the high-concentration glucose and liquid ammonia with over-high local concentration can generate side reaction.
The specific technical scheme for solving the technical problems comprises the following steps: the fermentation method of the glutamic acid comprises a shake flask culture stage of a fermentation strain, an amplification culture stage of a first-stage seed tank, an amplification culture stage of a second-stage seed tank and a fermentation stage;
the method comprises the following specific steps:
(1) shake flask culture stage of fermentation strain: activating the low-temperature preserved strain; inoculating the activated fermentation strain into shake flask culture medium, wherein the initial sugar content is 0%, feeding sugar to control the residual sugar concentration to be maintained at 0.6-1.0%, the temperature is controlled at 32 deg.C, the rotation speed of shaking table is 100rpm, shaking table culturing is carried out for 8-10h, and the OD reaches 0.8-1.0, and storing for later use;
(2) the first-level seeding tank enlargement culture stage: adding a first-stage seeding tank culture medium into a tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating with flame in a steel cylinder of a strain chamber, fermenting at 32 deg.C, controlling initial sugar content to be 0%, feeding sugar to control residual sugar concentration to be 0.6-1.0%, maintaining for 24 hr, and terminating fermentation until OD reaches 0.5-0.8; cooling, and storing at 4 deg.C;
(3) and (3) a secondary seeding tank amplification culture stage: adding the culture medium of the secondary seeding tank into the tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating to the primary seeding tank, adding sugar at 32 deg.C to control residual sugar concentration to be 0.6-1.0%, fermenting for 12-16h and residual sugar to be 0.1%, stopping fermentation, cooling to 0.8-1.0 according to OD, and storing at 4 deg.C;
the shake flask culture medium is a bottomless sugar culture medium and comprises 1% of soybean meal hydrolysate, 0.1% of succinic acid, 0.05% of methionine, 0.15% of magnesium sulfate, 0.13% of betaine, 0.4% of monopotassium phosphate, 0.2% of corn steep liquor, 10mg/L of ferrous sulfate, 10mg/L of manganese sulfate, 300 mu g/L of biotin, 200 mu g/L of VB1, 0.55% of urea and 0.5% of yeast powder.
The first-stage seeding tank culture medium is a bottomless sugar culture medium and comprises 0% of glucose, 0% of phosphoric acid: 0.2-0.3%, magnesium sulfate: 0.1-0.2%, potassium dihydrogen phosphate: 0.1-0.2%, succinic acid: 0.2-0.4%, soybean meal hydrolysate: 3-5%, methionine: 0.04-0.05% and the balance of water.
The secondary seed tank culture medium is a non-glucose culture medium and comprises 0% of glucose and soybean meal hydrolysate in percentage by mass: 2.5-5% and succinic acid: 0.3-0.5%, magnesium sulfate: 0.1-0.2%, phosphoric acid: 0.25-0.5%, monopotassium phosphate 0.1-0.2%, methionine: 0.0125-0.02%, ferrous sulfate: 0.0008-0.001%, manganese sulfate: 0.0008 to 0.001 percent.
(4) The fermentation stage is as follows:
i: performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;
II: transferring the second-stage seed tank when the temperature of the culture medium is reduced to 38 ℃, fermenting when inoculation is finished, adding sugar, liquid ammonia and a defoaming agent in the fermentation process, keeping the concentration of residual sugar in the culture medium at about 0.6-1.0% all the time, fermenting for about 8-10h, increasing OD to 0.8-1.0, raising the temperature of fermentation to 37 ℃, and transferring the fermentation to an acid production stage;
III: fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.
The culture medium in the fermentation stage is a bottomless sugar culture medium and comprises 5-8% of corn steep liquor and soybean meal hydrolysate by mass percent: 0.3-0.5%, phosphoric acid: 0.1-0.2%, sugar: 0%, potassium chloride: 0.1-0.3%, magnesium sulfate: 0.1-0.2%, betaine: 0.1-0.2%, methionine: 0.02-0.03%, manganese sulfate: 0.0008-0.001%, ferrous sulfate: 0.0008 to 0.001 percent.
The invention has the beneficial effects that:
the invention creatively sets the fermentation medium with zero-base sugar concentration, and replaces the traditional fermentation mode of high-concentration glucose initial addition by the mode of feeding at the beginning, thereby avoiding the problems that the high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process and the liquid ammonia with over-high local concentration and the high-concentration glucose can generate side reaction;
meanwhile, the invention also unexpectedly discovers that the fermentation medium with zero-base sugar concentration is adopted, and the feeding is carried out at a specific flow rate, so that the early growth of the glutamic acid bacteria is not slow due to unilateral reduction of the glucose concentration, and the fermentation time is further shortened compared with the conventional fermentation method for initially adding the high-concentration glucose.
The specific implementation mode is as follows:
in the description of the invention, specific details are given only to enable a full understanding of the embodiments of the invention, but it should be understood by those skilled in the art that the invention is not limited to these details for the implementation. In other instances, well-known structures and functions have not been described or shown in detail to avoid obscuring the points of the embodiments of the invention. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
The specific implementation mode of the invention is as follows:
the first embodiment is as follows:
the fermentation method of the glutamic acid comprises a shake flask culture stage of a fermentation strain, an amplification culture stage of a first-stage seed tank, an amplification culture stage of a second-stage seed tank and a fermentation stage;
the method comprises the following specific steps:
(1) shake flask culture stage of fermentation strain: activating the low-temperature preserved strain; inoculating the activated fermentation strain into shake flask culture medium, wherein the initial sugar content is 0%, feeding sugar to control the residual sugar concentration to be maintained at 0.6-1.0%, the temperature is controlled at 32 deg.C, the rotation speed of shaking table is 100rpm, shaking table culturing is carried out for 8-10h, and the OD reaches 0.8-1.0, and storing for later use;
(2) the first-level seeding tank enlargement culture stage: adding a first-stage seeding tank culture medium into a tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating with flame in a steel cylinder of a strain chamber, fermenting at 32 deg.C, controlling initial sugar content to be 0%, feeding sugar to control residual sugar concentration to be 0.6-1.0%, maintaining for 24 hr, and terminating fermentation until OD reaches 0.5-0.8; cooling, and storing at 4 deg.C;
(3) and (3) a secondary seeding tank amplification culture stage: adding the culture medium of the secondary seeding tank into the tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating to the primary seeding tank, adding sugar at 32 deg.C to control residual sugar concentration to be 0.6-1.0%, fermenting for 12-16h and residual sugar to be 0.1%, stopping fermentation, cooling to 0.8-1.0 according to OD, and storing at 4 deg.C;
the shake flask culture medium is a bottomless sugar culture medium and comprises 1% of soybean meal hydrolysate, 0.1% of succinic acid, 0.05% of methionine, 0.15% of magnesium sulfate, 0.13% of betaine, 0.4% of monopotassium phosphate, 0.2% of corn steep liquor, 10mg/L of ferrous sulfate, 10mg/L of manganese sulfate, 300 mu g/L of biotin, 200 mu g/L of VB1, 0.55% of urea and 0.5% of yeast powder.
The first-stage seeding tank culture medium is a bottomless sugar culture medium and comprises 0% of glucose, 0% of phosphoric acid: 0.2-0.3%, magnesium sulfate: 0.1-0.2%, potassium dihydrogen phosphate: 0.1-0.2%, succinic acid: 0.2-0.4%, soybean meal hydrolysate: 3-5%, methionine: 0.04-0.05% and the balance of water.
The secondary seed tank culture medium is a non-glucose culture medium and comprises 0% of glucose and soybean meal hydrolysate in percentage by mass: 2.5-5% and succinic acid: 0.3-0.5%, magnesium sulfate: 0.1-0.2%, phosphoric acid: 0.25-0.5%, monopotassium phosphate 0.1-0.2%, methionine: 0.0125-0.02%, ferrous sulfate: 0.0008-0.001%, manganese sulfate: 0.0008 to 0.001 percent.
(4) The fermentation stage is as follows:
i: performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;
II: transferring the second-stage seed tank when the temperature of the culture medium is reduced to 38 ℃, fermenting when inoculation is finished, adding sugar, liquid ammonia and a defoaming agent in the fermentation process, keeping the concentration of residual sugar in the culture medium at about 0.6-1.0% all the time, fermenting for about 8-10h, increasing OD to 0.8-1.0, raising the temperature of fermentation to 37 ℃, and transferring the fermentation to an acid production stage;
III: fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.
The culture medium in the fermentation stage is a bottomless sugar culture medium and comprises 5-8% of corn steep liquor and soybean meal hydrolysate by mass percent: 0.3-0.5%, phosphoric acid: 0.1-0.2%, sugar: 0%, potassium chloride: 0.1-0.3%, magnesium sulfate: 0.1-0.2%, betaine: 0.1-0.2%, methionine: 0.02-0.03%, manganese sulfate: 0.0008-0.001%, ferrous sulfate: 0.0008 to 0.001 percent.
In order to more intuitively show the process advantages of the zero initial sugar concentration culture medium, the zero initial sugar concentration culture medium fermentation method is compared with the method of the comparative example,
comparative example one:
the preparation method is the same as the first embodiment except that: in the preparation process of the comparative example, the fermentation medium adopts a high-concentration base sugar culture medium, and the concentration of the base sugar is 15 percent; wherein the temperature in the early stage of fermentation is maintained at about 33 ℃, the fermentation lasts for about 6-8h, the initial sugar consumption is almost the same, and the sugar feeding is started to ensure that the concentration of residual sugar in the culture medium is always maintained at about 1.0%;
comparative example two:
the preparation method is the same as the first embodiment except that: in the preparation process of the comparative example, the fermentation medium adopts a low-concentration base sugar medium, and the concentration of the base sugar is 8 percent; wherein the temperature in the early stage of fermentation is maintained at about 33 ℃, the fermentation lasts for about 6-8h, the initial sugar consumption is almost the same, and the sugar feeding is started to ensure that the concentration of residual sugar in the culture medium is always maintained at about 1.0%;
the transparency and purity of the glutamic acid were determined according to the quality requirements for the semi-finished product L-glutamic acid (glutamic acid) in the monosodium glutamate industry manual (second edition):
table 1: comparison of influence of zero initial sugar concentration culture medium process on product quality
| Fermentation medium | Sterilization | Fermentation time/h | Light transmittance | Purity/%) | |
| Example one | Zero concentration base sugar | Lianxiao medicine for curing diabetes | 26 | 8-10 | 97 |
| Comparative example 1 | High concentration base sugar | Lianxiao medicine for curing diabetes | 32 | 1-3 | 95 |
| Comparative example No. two | Low-concentration base sugar | Lianxiao medicine for curing diabetes | 30 | 2-5 | 95 |
According to the data analysis in the table 1, the following results are obtained:
compared with a high-concentration base sugar culture medium process and a low-concentration base sugar culture medium process, the zero-initial sugar concentration culture medium process has the advantages that the light transmittance of the fermentation liquid is substantially improved, and because the caramelization reaction in the continuous digestion process is avoided due to the reduction of the glucose concentration;
compared with a high-concentration and low-concentration substrate culture medium process and a low-concentration substrate culture medium process, the zero-initial-sugar-concentration culture medium process has the advantages that the content is improved to a certain extent, probably because the zero-initial-sugar-concentration culture medium avoids the reaction of glucose and liquid ammonia to generate ammonium gluconate, and the ammonium gluconate can influence the purity of the product;
it is worth noting that: compared with the high-concentration and low-concentration base sugar culture medium processes, the zero-initial sugar concentration culture medium process has the effect of further shortening the fermentation time, and the difference of the fermentation time is small between the high-concentration base sugar culture medium process and the low-concentration base sugar culture medium process.
In order to solve the problem of how to further shorten the fermentation time, the method also comprises a method for shortening the fermentation time of glutamic acid, wherein in the early stage of the fermentation stage, on the basis of a zero-initial-sugar-concentration culture medium process, sugar, liquid ammonia and an antifoaming agent are added in a flowing manner, and meanwhile, disodium adenosine triphosphate is added in a flowing manner, and the flowing amount is 5-8 g/L; the method comprises the following specific steps:
example two
The fermentation method of the glutamic acid comprises a shake flask culture stage of a fermentation strain, an amplification culture stage of a primary seed tank, an amplification culture stage of a secondary seed tank and a fermentation stage, and is characterized in that the fermentation stage comprises:
(1) performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;
(2) when the temperature of the culture medium is reduced to 38 ℃, transplanting common glutamic acid bacteria in a secondary seed tank, fermenting when inoculation is finished, wherein sugar, liquid ammonia, adenosine disodium triphosphate and a defoaming agent are added in the fermentation process, and the adding amount of the adenosine disodium triphosphate is 5-8 g/L; and the concentration of residual sugar in the culture medium is maintained at about 0.6-1.0% all the time, the fermentation lasts for about 8-10h, the OD is increased to 0.8-1.0, the fermentation temperature is raised to 37 ℃, and the fermentation is shifted to the acid production stage;
(3) fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.
Comparative example three:
the preparation method is the same as the second embodiment, except that: in the preparation process of the comparative example, no disodium adenosine triphosphate is fed;
comparative example four:
the preparation method is the same as the third embodiment except that: in the preparation process of the comparative example, no disodium adenosine triphosphate is fed;
comparing the fermentation time and the comprehensive acid yield when the OD of the fermentation liquor reaches 0.8-1.0;
table 2: comparison of the influence of medium with zero initial sugar concentration and adenosine disodium triphosphate on the product quality
| Fermentation medium | Adenosine disodium triphosphate | Fermentation time/h | Acid yield/% | Sugar acid conversion/% | |
| Example one | Zero concentration base sugar | Is free of | 26 | 18-20.5 | 70-72 |
| Example two | Zero concentration base sugar | Is provided with | 23 | 19-21 | 70-72.5 |
| Comparative example 1 | High concentration base sugar | Is free of | 32 | 16-19 | 67-70 |
| Comparative example No. two | Low-concentration base sugar | Is free of | 30 | 17-20 | 69-72 |
| Comparative example No. three | High concentration base sugar | Is provided with | 31 | 16.5-18.5 | 68-70 |
| Comparative example No. four | Low-concentration base sugar | Is provided with | 29 | 18-20 | 68-71 |
According to the data analysis of the table 2, the following results are obtained:
the disodium adenosine triphosphate is fed in the early stage of fermentation, the fermentation time can be improved to a certain extent for the above examples and comparative examples, but the effect is small for a high-concentration primary sugar culture medium, wherein the effect is more remarkable along with the reduction of the glucose concentration, and particularly, the effect can be shortened by nearly 3 hours for a zero-primary sugar culture medium;
by adding disodium adenosine triphosphate, the difference between the acid yield and the sugar acid conversion rate is not large, but the high-concentration primary sugar culture medium has poor effect on both the acid yield and the sugar acid conversion rate compared with a zero-primary sugar culture medium.
Therefore, the invention creatively adds the adenosine disodium triphosphate in the initial fermentation stage, can quickly improve the absorption of the strains on the glucose and achieves the aim of quick growth of the strains;
this is probably because disodium adenosine triphosphate is capable of powering many of the vital activities of the fermenting energy-producing microorganisms, which require the motive force of transmembrane proton gradient production, and therefore these microorganisms also have ATPases to consume ATP to produce a transmembrane proton gradient.
Particularly, the magical discovery shows that the acid yield and the fermentation time are further improved by adding the adenosine disodium triphosphate in the zero-substrate-sugar-concentration fermentation medium process of the invention compared with the traditional high-concentration substrate fermentation method in the comparative example I.
In summary, the following steps:
(1) the invention creatively sets the fermentation culture medium with zero base sugar concentration, and replaces the traditional fermentation mode of the initial addition of high-concentration glucose by the mode of feeding at the beginning, thereby avoiding the problems that the high-concentration glucose can generate serious caramelization reaction in the high-temperature sterilization process and the liquid ammonia with over-high local concentration and the high-concentration glucose can generate side reaction;
(2) the fermentation medium with zero-base sugar concentration is adopted, and the feeding mode is started at a specific flow rate, so that the effect that the early growth of the glutamic acid bacteria is slow because the glucose concentration is reduced unilaterally is surprisingly found, and the fermentation time is further shortened compared with the conventional fermentation mode in which the high-concentration glucose is initially added;
(3) the invention creatively adds the adenosine disodium triphosphate in the initial fermentation stage, can quickly improve the absorption of the strains on glucose, and achieves the aim of further and quickly growing the strains.
Claims (5)
1. A fermentation method of glutamic acid comprises a shake flask culture stage of a fermentation strain, a first-stage seeding tank amplification culture stage, a second-stage seeding tank amplification culture stage and a fermentation stage, and is characterized by comprising the following specific steps:
(1) shake flask culture stage of fermentation strain: activating the low-temperature preserved strain; inoculating the activated fermentation strain into shake flask culture medium, wherein the initial sugar content is 0%, feeding sugar to control the residual sugar concentration to be maintained at 0.6-1.0%, the temperature is controlled at 32 deg.C, the rotation speed of shaking table is 100rpm, shaking table culturing is carried out for 8-10h, and the OD reaches 0.8-1.0, and storing for later use;
(2) the first-level seeding tank enlargement culture stage: adding a first-stage seeding tank culture medium into a tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating with flame in a steel cylinder of a strain chamber, fermenting at 32 deg.C, controlling initial sugar content to be 0%, feeding sugar to control residual sugar concentration to be 0.6-1.0%, maintaining for 24 hr, and terminating fermentation until OD reaches 0.5-0.8; cooling, and storing at 4 deg.C;
(3) and (3) a secondary seeding tank amplification culture stage: adding the culture medium of the secondary seeding tank into the tank at one time, sterilizing at 126 deg.C for 25min, cooling, adjusting pH to 7.0 with liquid ammonia, inoculating to the primary seeding tank, adding sugar at 32 deg.C to control residual sugar concentration to be 0.6-1.0%, fermenting for 12-16h and residual sugar to be 0.1%, stopping fermentation, cooling to 0.8-1.0 according to OD, and storing at 4 deg.C;
(4) the fermentation stage is as follows:
i: performing air digestion in a fermentation tank, and then adding potassium chloride, magnesium sulfate, betaine, methionine, manganese sulfate and ferrous sulfate which are sterilized in a continuous digestion system; controlling the concentration of the base sugar to be 0%, cooling, adding corn steep liquor, soybean meal hydrolysate and phosphoric acid which are sterilized by a continuous sterilization system, and adjusting the pH value to 7.0 by liquid ammonia when the temperature is reduced to 55 ℃;
II: transferring the second-stage seed tank when the temperature of the culture medium is reduced to 38 ℃, fermenting when inoculation is finished, adding sugar, liquid ammonia and a defoaming agent in the fermentation process, keeping the concentration of residual sugar in the culture medium at about 0.6-1.0% all the time, fermenting for about 8-10h, increasing OD to 0.8-1.0, raising the temperature of fermentation to 37 ℃, and transferring the fermentation to an acid production stage;
III: fermenting for 28-32h, slowing down sugar consumption and acid production in the final stage of fermentation, stopping sugar feeding, and measuring the acid production amount of glutamic acid fermentation after residual sugar is consumed to about 0.2%.
2. The glutamic acid fermentation method according to claim 1, wherein the shake flask culture medium is a bottomless medium comprising 1% soybean meal hydrolysate, 0.1% succinic acid, 0.05% methionine, 0.15% magnesium sulfate, 0.13% betaine, 0.4% potassium dihydrogen phosphate, 0.2% corn steep liquor, 10mg/L ferrous sulfate, 10mg/L manganese sulfate, 300 μ g/L biotin, 200 μ g/L VB1, 0.55% urea, and 0.5% yeast powder.
3. The glutamic acid fermentation method according to claim 1, wherein the first-stage seed tank medium is a non-basal medium comprising, by mass, 0% of glucose, phosphoric acid: 0.2-0.3%, magnesium sulfate: 0.1-0.2%, potassium dihydrogen phosphate: 0.1-0.2%, succinic acid: 0.2-0.4%, soybean meal hydrolysate: 3-5%, methionine: 0.04-0.05% and the balance of water.
4. The glutamic acid fermentation method according to claim 2, wherein the secondary seed tank culture medium is a non-substrate medium comprising, by mass, 0% of glucose, a soybean meal hydrolysate: 2.5-5% and succinic acid: 0.3-0.5%, magnesium sulfate: 0.1-0.2%, phosphoric acid: 0.25-0.5%, monopotassium phosphate 0.1-0.2%, methionine: 0.0125-0.02%, ferrous sulfate: 0.0008-0.001%, manganese sulfate: 0.0008 to 0.001 percent.
5. The glutamic acid fermentation method according to claim 2, wherein the medium in the fermentation stage is a bottomless medium comprising, by mass, 5-8% of corn steep liquor, soybean meal hydrolysate: 0.3-0.5%, phosphoric acid: 0.1-0.2%, sugar: 0%, potassium chloride: 0.1-0.3%, magnesium sulfate: 0.1-0.2%, betaine: 0.1-0.2%, methionine: 0.02-0.03%, manganese sulfate: 0.0008-0.001%, ferrous sulfate: 0.0008 to 0.001 percent.
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