CN114717274A - Clean fermentation process of L-lysine - Google Patents
Clean fermentation process of L-lysine Download PDFInfo
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- CN114717274A CN114717274A CN202210321275.XA CN202210321275A CN114717274A CN 114717274 A CN114717274 A CN 114717274A CN 202210321275 A CN202210321275 A CN 202210321275A CN 114717274 A CN114717274 A CN 114717274A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 94
- 230000004151 fermentation Effects 0.000 title claims abstract description 94
- 239000004472 Lysine Substances 0.000 title claims abstract description 25
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 title claims description 35
- 235000019766 L-Lysine Nutrition 0.000 title claims description 11
- 239000002609 medium Substances 0.000 claims abstract description 19
- 241000319304 [Brevibacterium] flavum Species 0.000 claims abstract description 17
- 238000003756 stirring Methods 0.000 claims abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 238000009423 ventilation Methods 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 7
- 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 32
- 239000008103 glucose Substances 0.000 claims description 32
- 235000015097 nutrients Nutrition 0.000 claims description 16
- 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 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 14
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 14
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 14
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 claims description 10
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 9
- 229930182847 D-glutamic acid Natural products 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 7
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 7
- 239000002518 antifoaming agent Substances 0.000 claims description 7
- 229960002685 biotin Drugs 0.000 claims description 7
- 235000020958 biotin Nutrition 0.000 claims description 7
- 239000011616 biotin Substances 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 7
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 7
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 7
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 claims description 7
- 235000013379 molasses Nutrition 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000011691 vitamin B1 Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 14
- 235000018977 lysine Nutrition 0.000 abstract description 14
- 238000000605 extraction Methods 0.000 abstract description 4
- 235000001014 amino acid Nutrition 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 230000003204 osmotic effect Effects 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 12
- 239000000306 component Substances 0.000 description 12
- 210000002421 cell wall Anatomy 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 241001052560 Thallis Species 0.000 description 8
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 6
- 108010013639 Peptidoglycan Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000011049 filling Methods 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 239000012533 medium component Substances 0.000 description 5
- 230000002503 metabolic effect Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241000186146 Brevibacterium Species 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000004075 alteration Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 241000203809 Actinomycetales Species 0.000 description 1
- 241001297667 Candidatus Wallbacteria Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 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 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000035899 viability Effects 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
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- General Health & Medical Sciences (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention belongs to the technical field of amino acid fermentation, and discloses a clean fermentation process of L-lysine, which comprises the following steps: inoculating the brevibacterium flavum seed liquid into a fermentation culture medium, wherein the fermentation temperature is as follows: the fermentation time is 60 hours, the temperature is 31 ℃ after 0 to 18 hours, the temperature is 34 ℃ after 18 hours, the ventilation ratio is 1:0.7, the stirring speed is 200 r/min. The fermentation medium has simple and controllable components, is beneficial to the subsequent separation and extraction process, and realizes the clean fermentation production of lysine.
Description
Technical Field
The invention belongs to the technical field of amino acid fermentation, and particularly relates to a clean fermentation process of L-lysine.
Background
The yellow brevibacterium belongs to the genus brevibacterium, and is an obligate aerobic, catalase-positive, spore-free gram-positive brevibacterium in the order of Actinomycetales. Brevibacterium flavum is an important lysine-producing bacterium and is sensitive to osmotic pressure. In the process of fermenting lysine by brevibacterium flavum, the osmotic pressure exceeds 2000mosm, and the thallus activity in the later fermentation period is maintained. In general, it is necessary to increase the tolerance of the cells to osmotic pressure by adding N, N, N-trimethylglycine, sodium chloride or the like. However, under the condition of only relying on N, N, N-trimethylglycine, the thalli cannot well resist the environment with high external osmotic pressure. To increase the tolerance of the bacterial cells to high osmotic pressure from the outside, other mechanisms of bacterial cells against high osmotic pressure must be activated. The tolerance of the thalli to high osmotic pressure can be divided into two main mechanisms: 1) absorbing osmotic pressure compatible substances in the environment; 2) the cell body improves the cell wall strength, thereby improving the tolerance to external high osmotic pressure.
Metabolic flux analysis is an important method for analyzing microscopic metabolic characteristics in bacteria, and is used for analyzing through a metabolic pathway so as to find a new metabolic target or confirm the result of upstream metabolic modification. In the fermentation process, the thalli can be stimulated to generate osmotic pressure tolerance through external additives, so that the tolerance of the brevibacterium flavum to high osmotic pressure in the fermentation process is finally improved, and the fermentation intensity and the conversion rate are improved.
Bacteria resist high osmotic pressure mainly by bacterial cell wall bacteria. The cell wall, cell membrane, cytoplasm, nuclear region and the like belong to the general structure of bacterial cells, are thick and tough coats positioned at the outermost layer of the cell, and have various physiological functions of fixing the cell appearance, protecting the cell from being damaged and the like. The bacterial cell wall is composed of substances with different chemical components, such as peptidoglycan, teichoic acid, lipopolysaccharide, phospholipid, outer membrane protein and the like, peptidoglycan mainly plays a role in the physiological function of the cell wall in the components, and the content of the peptidoglycan in gram-positive bacteria is up to 30-95 percent, so that the strength of the cell wall can be increased by increasing the synthetic amount of the peptidoglycan, the tolerance of high osmotic pressure is increased, the thalli is protected, and the fermentation efficiency is improved. The synthetic route of the peptidoglycan involves various enzymes and intermediate substances, and how to intervene can effectively improve the content of the peptidoglycan and is difficult to improve the strength and the osmotic pressure resistance of cell walls. The prior art is also rarely reported.
Lysine, also known as L-lysine, lysine and the like, is one of the most important essential amino acids in human and animal nutrition, and currently, the global yield of lysine reaches more than 300 ten thousand tons, which is second to glutamic acid and becomes the second largest amino acid variety in the global yield. The main mode of production of lysine is biological fermentation. The clean fermentation process for improving the fermentation efficiency and the extraction efficiency is a technical problem which needs to be solved all the time.
Disclosure of Invention
The invention aims to overcome the defects of low lysine fermentation efficiency, difficult extraction process caused by complex culture medium components and the like in the prior art, and provides a clean fermentation process of L-lysine.
The invention is realized by the following technical scheme.
A clean fermentation process of L-lysine, which is characterized by comprising the following steps:
inoculating the brevibacterium flavum seed liquid into a fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation temperature is as follows: the fermentation time is 0-18h at 31 ℃, 18 h-end at 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding 300g/L ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.
Further, the glucose nutrient solution comprises glucose and N, N, N-trimethylglycine.
Further, the fermentation medium comprises the following components: d-alanine or/and D-glutamic acid is/are added on the basis of a conventional culture medium.
Specifically, the glucose nutrient solution comprises the following components: 500g/L glucose, 10g/L N, N, N-trimethyl glycine.
Specifically, the fermentation medium comprises the following raw materials: 80g/L glucose, 10g/L corn steep liquor, 12g/L molasses, 8g/L ammonium sulfate, 10g/L potassium dihydrogen phosphate, 1g/L D-alanine, 1g/L D-glutamic acid, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate tetrahydrate, 0.02g/L ferrous sulfate heptahydrate, and vitamin B10.01g/L and biotin 0.5 mg/L.
Preferably, the preparation method of the fermentation medium comprises the following steps:
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
Compared with the prior art, the research starting point and the obtained beneficial effects of the invention mainly comprise but are not limited to the following aspects:
the high osmotic pressure can inhibit the synthesis of lysine to a greater extent, and the research regulates and controls the synthesis of main components of bacterial cell walls from a metabolic synthesis way to achieve the purpose of improving the strength of the cell walls, so that thalli can be prevented from being damaged by the high osmotic pressure, and the higher fermentation efficiency is kept.
The N, N, N-trimethylglycine can enable the thalli to endure high osmotic pressure and avoid the damage of osmotic pressure, so that the growth of the thalli can be promoted, and the fermentation efficiency is improved; the fermentation efficiency of Brevibacterium flavum can be greatly improved by combining D-alanine and D-glutamic acid.
The fermentation medium has simple and controllable components, is beneficial to the subsequent separation and extraction process, and realizes the clean fermentation production of lysine.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The clean fermentation process of the L-lysine comprises the following steps:
the liquid filling amount of a 50L full-automatic fermentation tank is 30L of fermentation medium, and the strain selects Brevibacterium flavum XQ 90; brevibacterium flavum seed liquid (OD 600= 4.5) was inoculated into a fermentation medium at an inoculum size of 10%, fermentation temperature: the fermentation time is 0-18h at 31 ℃, 18 h-end at 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding 300g/L ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.
The glucose nutrient solution comprises the following components: 500g/L glucose, 10g/L N, N, N-trimethyl glycine;
fermentation medium components: 80g/L glucose and 10g/L corn steep liquor12g/L of molasses, 8g/L of ammonium sulfate, 10g/L of monopotassium phosphate, 1g/L of D-alanine, 1g/L of D-glutamic acid, 0.8g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate tetrahydrate, 0.02g/L of ferrous sulfate heptahydrate, and vitamin B10.01g/L and biotin 0.5 mg/L;
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
Comparative example 1
The clean fermentation process of the L-lysine comprises the following steps:
the liquid filling amount of a 50L full-automatic fermentation tank is 30L of fermentation medium, and the strain selects Brevibacterium flavum XQ 90; brevibacterium flavum seed liquid (OD 600= 4.5) was inoculated into a fermentation medium at an inoculum size of 10%, fermentation temperature: the temperature of 0-18h is 31 ℃, the temperature of 18 h-end is 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding 300g/L ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.
The glucose nutrient solution comprises the following components: 500g/L of glucose;
fermentation medium components: 80g/L glucose, 10g/L corn steep liquor, 12g/L molasses, 8g/L ammonium sulfate, 10g/L potassium dihydrogen phosphate, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate tetrahydrate, 0.02g/L ferrous sulfate heptahydrate, vitamin B10.01g/L and biotin 0.5 mg/L;
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
Comparative example 2
The clean fermentation process of the L-lysine comprises the following steps:
the liquid filling amount of a 50L full-automatic fermentation tank is 30L of fermentation medium, and the strain selects Brevibacterium flavum XQ 90; brevibacterium flavum seed liquid (OD 600= 4.5) was inoculated into a fermentation medium at an inoculum size of 10%, fermentation temperature: the fermentation time is 0-18h at 31 ℃, 18 h-end at 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding 300g/L ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.
The glucose nutrient solution comprises the following components: 500g/L glucose, 10g/L N, N, N-trimethyl glycine;
fermentation medium components: 80g/L glucose, 10g/L corn steep liquor, 12g/L molasses, 8g/L ammonium sulfate, 10g/L potassium dihydrogen phosphate, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate tetrahydrate, 0.02g/L ferrous sulfate heptahydrate, vitamin B10.01g/L and biotin 0.5 mg/L;
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
Comparative example 3
The clean fermentation process of the L-lysine comprises the following steps:
the liquid filling amount of a 50L full-automatic fermentation tank is 30L of fermentation medium, and the strain selects Brevibacterium flavum XQ 90; brevibacterium flavum seed liquid (OD 600= 4.5) was inoculated into a fermentation medium at an inoculum size of 10%, fermentation temperature: the fermentation time is 0-18h at 31 ℃, 18 h-end at 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding ammonium sulfate solution of 300g/L, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed.
The glucose nutrient solution comprises the following components: 500g/L of glucose;
fermentation medium components: 80g/L glucose, 10g/L corn steep liquor, 12g/L molasses, 8g/L ammonium sulfate, 10g/L potassium dihydrogen phosphate, 1g/L D-alanine, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate tetrahydrate, 0.02g/L ferrous sulfate heptahydrate, and vitamin B10.01g/L and biotin 0.5 mg/L;
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
Comparative example 4
The clean fermentation process of the L-lysine comprises the following steps:
the liquid filling amount of a 50L full-automatic fermentation tank is 30L of fermentation medium, and the strain selects Brevibacterium flavum XQ 90; brevibacterium flavum seed liquid (OD 600= 4.5) was inoculated into a fermentation medium at an inoculum size of 10%, fermentation temperature: the fermentation time is 0-18h at 31 ℃, 18 h-end at 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding 300g/L ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.
The glucose nutrient solution comprises the following components: 500g/L of glucose;
fermentation medium components: 80g/L glucose, 10g/L corn steep liquor, 12g/L molasses, 8g/L ammonium sulfate, 10g/L potassium dihydrogen phosphate, 1g/L D-glutamic acid, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate tetrahydrate, 0.02g/L ferrous sulfate heptahydrate, and vitamin B10.01g/L and biotin 0.5 mg/L;
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
Example 2
The results of the clean fermentation with L-lysine in example 1 and comparative examples 1 to 4 were analyzed from the main indicators such as the maximum cell content, lysine concentration, conversion rate, and the like, and are specifically shown in Table 1:
TABLE 1
| Group of | Maximum thallus content OD600 | Lysine concentration g/L | Conversion rate% | Osmotic pressure mOsm at 60h |
| Example 1 | 36.7 | 160.9 | 52.1 | 2394 |
| Comparative example 1 | 30.8 | 143.2 | 48.9 | 2326 |
| Comparative example 2 | 32.6 | 154.1 | 51.2 | 2387 |
| Comparative example 3 | 32.5 | 152.7 | 50.9 | 2380 |
| Comparative example 4 | 32.3 | 151.9 | 51.1 | 2365 |
And (4) conclusion: the maximum thallus content OD600 value, the lysine concentration and the conversion rate of each group are compared, the comparison example 1 is the lowest, and the comparison examples 2-4 groups added with N, N, N-trimethylglycine, D-alanine or D-glutamic acid are all improved compared with the comparison example 1, probably because the thallus can endure high osmotic pressure and is not damaged by adding the N, N, N-trimethylglycine, so that the thallus growth can be promoted, and the fermentation efficiency is improved; in the research, a racemization step with relatively poor synthesis activity in bacterial cells is selected as an entry point for research, and a proper amount of D-alanine and D-glutamic acid are added into a culture medium, so that the cell wall strength of the cells in a rapid division and proliferation period is greatly increased, the permeation is increased along with the rapid increase of the concentration of lysine, the high osmotic pressure environment possibly has great influence on the growth of thalli, the fermentation efficiency is slowed down, the increase of the cell wall strength can be prevented from being damaged by high osmotic pressure, and the high fermentation efficiency is maintained.
In addition, in this study, on the basis of comparative example 1, the addition of 50g/L sodium chloride to the fermentation medium to increase the osmotic pressure, although it could promote the formation of tolerance of the bacterial cells to the osmotic pressure, it was not good for the growth and viability of the bacterial cells, and thus the fermentation yield could not be increased. While sodium chloride at a lower concentration (20 g/L) does not significantly increase the osmotic pressure and does not make the strain tolerant.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. The clean fermentation process of the L-lysine is characterized by comprising the following steps:
inoculating the brevibacterium flavum seed liquid into a fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation temperature is as follows: the fermentation time is 0-18h at 31 ℃, 18 h-end at 34 ℃, the ventilation ratio is 1:0.7, the stirring speed is 200r/min, and the total fermentation time is 60 h; in the fermentation process, the residual sugar content is maintained at 10g/L by feeding glucose nutrient solution, the ammonia nitrogen content is maintained at 1g/L by feeding ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.
2. The process of claim 1, wherein the glucose nutrient solution comprises glucose and N, N-trimethylglycine.
3. The process of claim 1, wherein the fermentation medium comprises: d-alanine or/and D-glutamic acid is/are added on the basis of a conventional culture medium.
4. The process of claim 2, wherein the glucose nutrient solution comprises the following components: 500g/L glucose, 10g/L N, N, N-trimethyl glycine.
5. The process of claim 3, wherein the fermentation medium is prepared from: 80g/L glucose, 10g/L corn steep liquor, 12g/L molasses, 8g/L ammonium sulfate, 10g/L potassium dihydrogen phosphate, 1g/L D-alanine, 1g/L D-glutamic acid, 0.8g/L magnesium sulfate heptahydrate, 0.02g/L manganese sulfate tetrahydrate, 0.02g/L ferrous sulfate heptahydrate, and vitamin B10.01g/L and biotin 0.5 mg/L.
6. The process of claim 5, wherein the fermentation medium is prepared by:
sequentially adding the raw materials into water, stirring, adjusting pH to 7.5, and sterilizing at 115 deg.C for 10 min.
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| CN101423851A (en) * | 2008-11-07 | 2009-05-06 | 潍坊祥维斯化学品有限公司 | Method for fermentation preparation of L-aminoacid |
| CN101563453A (en) * | 2006-12-19 | 2009-10-21 | 味之素株式会社 | Process for production of L-amino acid |
| CN110777175A (en) * | 2019-12-01 | 2020-02-11 | 齐齐哈尔龙江阜丰生物科技有限公司 | Method for improving lysine fermentation efficiency |
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| CN101563453A (en) * | 2006-12-19 | 2009-10-21 | 味之素株式会社 | Process for production of L-amino acid |
| CN101423851A (en) * | 2008-11-07 | 2009-05-06 | 潍坊祥维斯化学品有限公司 | Method for fermentation preparation of L-aminoacid |
| CN110777175A (en) * | 2019-12-01 | 2020-02-11 | 齐齐哈尔龙江阜丰生物科技有限公司 | Method for improving lysine fermentation efficiency |
Non-Patent Citations (2)
| Title |
|---|
| HUBERT LAM等: "D-Amino Acids Govern Stationary Phase Cell Wall Remodeling in Bacteria", SCIENCE, pages 1552 - 1555 * |
| 洪铭等: "利用甜菜碱提升赖氨酸发酵及其代谢通量分析", 发酵科技通讯, pages 125 - 131 * |
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