CN108796004A - A kind of technique of preparing lysine through fermentation - Google Patents
A kind of technique of preparing lysine through fermentation Download PDFInfo
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- CN108796004A CN108796004A CN201810634938.7A CN201810634938A CN108796004A CN 108796004 A CN108796004 A CN 108796004A CN 201810634938 A CN201810634938 A CN 201810634938A CN 108796004 A CN108796004 A CN 108796004A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 71
- 230000004151 fermentation Effects 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000004472 Lysine Substances 0.000 title claims abstract description 26
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 15
- 239000012530 fluid Substances 0.000 claims abstract description 11
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000002054 inoculum Substances 0.000 claims abstract description 10
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 21
- 241000194108 Bacillus licheniformis Species 0.000 claims description 20
- 239000000654 additive Substances 0.000 claims description 19
- 230000000996 additive effect Effects 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 18
- 241000223261 Trichoderma viride Species 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 15
- 229930195725 Mannitol Natural products 0.000 claims description 15
- 239000000594 mannitol Substances 0.000 claims description 15
- 235000010355 mannitol Nutrition 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 claims description 14
- 235000011092 calcium acetate Nutrition 0.000 claims description 14
- 239000001639 calcium acetate Substances 0.000 claims description 14
- 229960005147 calcium acetate Drugs 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 11
- 240000008042 Zea mays Species 0.000 claims description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 102000016943 Muramidase Human genes 0.000 claims description 9
- 108010014251 Muramidase Proteins 0.000 claims description 9
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 9
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 9
- 235000005822 corn Nutrition 0.000 claims description 9
- 229940088598 enzyme Drugs 0.000 claims description 9
- 239000004325 lysozyme Substances 0.000 claims description 9
- 229960000274 lysozyme Drugs 0.000 claims description 9
- 235000010335 lysozyme Nutrition 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 238000011218 seed culture Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 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
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate 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
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 108091005508 Acid proteases Proteins 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- 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 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 241000209140 Triticum Species 0.000 claims description 6
- 235000021307 Triticum Nutrition 0.000 claims description 6
- 229940085298 biotin 10 mg Drugs 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000011691 vitamin B1 Substances 0.000 claims description 6
- 235000015099 wheat brans Nutrition 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 241000223259 Trichoderma Species 0.000 claims description 4
- 235000013339 cereals Nutrition 0.000 claims description 4
- 230000009849 deactivation Effects 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000002604 ultrasonography Methods 0.000 claims description 4
- 238000010792 warming Methods 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 238000010564 aerobic fermentation Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 23
- 235000018977 lysine Nutrition 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
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- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 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 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 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
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- 239000000203 mixture Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 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 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- HFNUUHLSQPLBQI-UHFFFAOYSA-N acetic acid;calcium Chemical compound [Ca].CC(O)=O HFNUUHLSQPLBQI-UHFFFAOYSA-N 0.000 description 1
- 229940100228 acetyl coenzyme a Drugs 0.000 description 1
- 239000002154 agricultural waste Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229910001448 ferrous ion Inorganic materials 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 229960001855 mannitol Drugs 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- -1 saccharide compound Chemical class 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- 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
- 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
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention belongs to fermentation technical fields, disclose a kind of technique of preparing lysine through fermentation comprising following steps：Corynebacterium glutamicum seed liquor is seeded to according to the inoculum concentration of 5-10% in the fermentation tank containing fermentation medium, aerobic fermentation is carried out, fermentation temperature is 32-35 DEG C, and tank pressure is 0.05MPa, fermentation obtains zymotic fluid for 48-60 hours, persistently adds ammonium chloride as zymotic fluid neutralizer in fermentation process.Present invention process is simple, of low cost, and production lysine is efficient.
Description
Technical field
The invention belongs to amino acid fermentation technical fields, and in particular to a kind of technique of preparing lysine through fermentation.
Background technology
Lysine is a kind of amino acid that human body needs, a kind of indispensable nutriment.Protein is to constitute human body
The main ingredient of cell, the protein in food enter after human body by the first breaks down into amino acids of digestion, and then human body utilizes again
These amino acid synthesize new human body protein again, such as immune antiboidy, digestive ferment, plasma protein, growth hormone are all synthesis
Human body protein afterwards.In the various amino acid of synthetic protein, L-lysine is most important one kind, lacks lysine,
Other amino acid are just restricted or are not used, scientist it be referred to as the first essential amino acid of human body.Scientist also found,
It is also most required composition that L-lysine, which is most important in the long element of important substance suppression for control growth in humans, to the maincenter god of people
Through and peripheral nervous system all play an important role.Human body itself cannot synthesize L-lysine, it is necessary to be drawn from food and rely ammonia
Acid is to aid in other nutriments by the key substance that human body fully absorbs and utilizes, and human body only relies ammonia supplemented with enough L-
Acid could improve the absorption and utilization of food protein, reach balanced nutritious, enhancing development.Its effect mainly has：It improves
Intelligence promotes growth, builds up health；It improves a poor appetite, improve malnutritive situation；Improve insomnia, improves memory；Help generates
Antibody, hormone and enzyme improve immunity, increase hemochrome；The absorption of calcium, treatment is helped to prevent osteoporosis；It reduces in blood
The level of triglycerides prevents the generation of cardiovascular and cerebrovascular disease.
The method of production lysine mainly has fermentation method and enzyme process at present.Wherein, fermentation method is widest production method,
The major microorganisms of fermentation method production lysine have corynebacterium glutamicum, brevibacterium flavum etc..Most high acid in industrial production
Rate has been increased to every liter of zymotic fluid 100g or more, extraction rate reached to 80~90% or so.But the cost of culture medium restricts enterprise
Development, cost of material is high, and the profit of enterprise can accordingly reduce.
A kind of achievement in research " CN2018103846072, fermenting lysine culture medium " of applicant early period comprising corn
Skin processed material, corn steep liquor, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate heptahydrate, epsom salt, calcium acetate, mannitol,
Biotin, vitamin, water.This method is handled by primary raw material of maize peel, and acid production rate is high, reduce fermentation medium at
This；However, there remains addition other components, including corn steep liquor etc..
Invention content
To solve the above-mentioned problems, entreprise cost is reduced, resource utilization is improved, the present invention proposes a kind of preparing lysine through fermentation
Technique.
To achieve the goals above, the present invention is achieved by the following technical solution：
A kind of technique of preparing lysine through fermentation comprising following steps：
Corynebacterium glutamicum seed liquor is seeded to according to the inoculum concentration of 5-10% in the fermentation tank containing fermentation medium, into
It acts charitably aerobe fermentation, fermentation temperature is 32-35 DEG C, and tank pressure is 0.05MPa, and fermentation obtains zymotic fluid for 48-60 hours, in fermentation process
Persistently plus ammonium chloride is as zymotic fluid neutralizer.
Further,
The corynebacterium glutamicum seed liquor is prepared in accordance with the following steps：Choose corynebacterium glutamicum ATCC14997
As fermentation strain, corynebacterium glutamicum is accessed in seed culture medium, culture to density is 3-5 × 108The paddy of cfu/mL
Propylhomoserin corynebacteria seed liquor；The group of the seed culture medium is divided into：Glucose 80g/L, yeast extract 30g/L, corn steep liquor 10g/
L, potassium dihydrogen phosphate 2g/L, dipotassium hydrogen phosphate 2g/L, ferrous sulfate heptahydrate 0.2g/L, epsom salt 0.2g/L, biotin
10mg/L, vitamin B1 2mg/L。
Further, the preparation method of the fermentation medium includes the following steps：
Step 1）Wheat stalk is crushed, then with wheat bran according to 2：3 mass ratio mixing, then it is 50 mesh to be crushed to grain size, so
It is added to afterwards in the water of 4-5 times of weight, with the mixing speed warming while stirring of 200rpm to 60 DEG C, under heat-retaining condition, ultrasound
20-30min, ultrasonic power 500W are handled, then 121 DEG C of steam treatment 10min, cooled to room temperature is to get compound；
Step 2）By Trichoderma viride seed liquor and bacillus licheniformis seed liquor according to 1-2:The volume ratio of 2-3 mixes, and is mixed
Inoculation liquid is closed, by combined inoculation liquid according to 6-8%（Account for step 1）The volume ratio of gained compound）Inoculum concentration be linked into containing step
Rapid 1）It is cultivated in the fermentation tank of gained compound, temperature is 30 DEG C, and incubation time 72-96h obtains culture solution；
Step 3）It is ultrasonically treated culture solution, ultrasonic time 30-60min, ultrasonic power 500W, then it is 55 DEG C to adjust temperature,
It is 6 to add sulfuric acid adjustment pH, is separately added into lysozyme and acid protease, the additive amount of lysozyme is 20,000 U：1L solution, acid egg
The additive amount of white enzyme is 10,000 U：1L solution under heat-retaining condition, is digested 12-24 hours, is then filtered by filter screen, mistake
Aperture of filter screen is 5-10 microns, removes floccule, collects filtered solution, then 100 DEG C of enzyme deactivation 5min, cooled to room temperature, then
Water, appropriate calcium acetate and the mannitol for adding same volume, stir evenly, 121 DEG C of steam treatment 5min naturally cool to room
Temperature is to get fermentation medium.
Preferably, the preparation method of the Trichoderma viride seed liquor is：By Trichoderma viride streak inoculation in PDA culture medium
Culture, obtains single bacterium colony；Picking single bacterium colony is inoculated into PDA liquid medium and is cultivated, and obtains Trichoderma viride seed liquor.
Preferably, the preparation method of the bacillus licheniformis seed liquor is：Bacillus licheniformis is inoculated into LB solids
It is cultivated on culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into LB liquid medium and is cultivated, and obtains lichens gemma bar
Bacterium seed liquor.
Preferably, the additive amount of the calcium acetate is 100-200mg/L
Preferably, the additive amount of the mannitol is 50-100mg/L.
Specifically used bacterial strain is corynebacterium glutamicum ATCC14997, Trichoderma viride in embodiment of the present invention
ATCC9275, bacillus licheniformis ATCC12759；Other functionally similar bacterial strains in same kind can also be used.The present invention
Bacterial strain seed liquor can also be prepared according to other training methods that the prior art is recorded, and only need to reach suitable inoculum density i.e.
It can.
Compared with prior art, the advantages of culture medium that prepared by the present invention includes mainly the following aspects：
The present invention to wheat stalk and wheat bran by carrying out heating immersion and supersound process, the cavitation of the ultrasonic wave utilized,
It generates partial high pressure high temperature to be impacted, contributes to the separation of fiber, albumen and carbohydrate, it is easier to by bacterial strain profit
With；
Trichoderma viride can generate cellulase, and bacillus licheniformis can generate protease and amylase, and the present invention is using green
Color trichoderma and bacillus licheniformis are combined carries out fermentation enzymolysis processing to wheat stalk and wheat bran processed material, then digests thalline,
The culture medium containing saccharide compound, mycoprotein, polypeptide and inorganic mineral is obtained, uses, improves for Escherichia coli
Fermentation efficiency, and agricultural wastes are utilized, reduce cost.The cavitation for the ultrasonic wave that the present invention utilizes, generation office
Portion's high pressure-temperature, impacts somatic cells, and cytomorphosis and rupture, auxiliary lysozyme is caused to carry out broken wall dissolving, and
It is assisted using acid protease, improves the enzymolysis efficiency of mycoprotein.
Portion takes part in the synthesis of acetyl coenzyme A to calcium acetate in the cell, can participate in tricarboxylic acid cycle, to increase
The metabolism degree of cell, suitable calcium acetate can promote bar bacterium somatic cells to grow, to promoting the fermentation of lysine；It is sweet
Dew alcohol is able to maintain that osmotic pressure, while preventing the outflow of cell moisture and the invasion of salinity, improves cell to water shortage, high temperature is high
The tolerance of salt and hypertonic environment, stablizes the function of enzymatic activity and large biological molecule, and thalline can absorb the sweet dew in culture medium
Alcohol resists the pressure of Thief zone, and in lysine fermentation process, the addition of appropriate mannitol can play quickening growth
The effect of rate, sugar consumption rate and rate of producing acid.
Description of the drawings
Fig. 1：Influence of the calcium acetate additive amount to fermentation production lysine amount；
Fig. 2：Influence of the mannitol additive amount to fermentation production lysine amount.
Specific implementation mode
Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.The product and method of the present invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Product as described herein and method are modified or are suitably changed and combined in bright content, spirit and scope, to realize and answer
Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
A kind of technique of preparing lysine through fermentation comprising following steps：
Corynebacterium glutamicum ATCC14997 is chosen as fermentation strain；Corynebacterium glutamicum is accessed in seed culture medium,
Culture to density is 3 × 108The seed liquor of cfu/mL；The group of the seed culture medium is divided into：Glucose 80g/L, yeast extract
30g/L, corn steep liquor 10g/L, potassium dihydrogen phosphate 2g/L, dipotassium hydrogen phosphate 2g/L, ferrous sulfate heptahydrate 0.2g/L, epsom salt
0.2g/L, biotin 10mg/L, vitamin B12mg/L；
Seed liquor is seeded to according to 10% inoculum concentration in the fermentation tank containing fermentation medium, aerobic fermentation, fermentation are carried out
Temperature is 32 DEG C, and tank pressure is 0.05MPa, and fermentation obtains zymotic fluid for 48 hours, persistently adds ammonium chloride as fermentation in fermentation process
Liquid neutralizer makes zymotic fluid discharging pH controls 7.2；
The preparation method of the fermentation medium includes the following steps：
Step 1）Wheat stalk is crushed, then with wheat bran according to 2：3 mass ratio mixing, then it is 50 mesh to be crushed to grain size, so
It is added to afterwards in the water of 4 times of weight, with the mixing speed warming while stirring of 200rpm to 60 DEG C, under heat-retaining condition, at ultrasound
30min, ultrasonic power 500W are managed, then 121 DEG C of steam treatment 10min, cooled to room temperature is to get compound；
Step 2）By Trichoderma viride seed liquor and bacillus licheniformis seed liquor according to 2:3 volume ratio mixing, obtains mixing and connects
Kind liquid, by combined inoculation liquid according to 8%（Account for step 1）The volume ratio of gained compound）Inoculum concentration be linked into and contain step 1）Institute
It obtains and is cultivated in the fermentation tank of compound, temperature is 30 DEG C, and incubation time 72h obtains culture solution；
Step 3）It is ultrasonically treated culture solution, ultrasonic time 60min, ultrasonic power 500W, then it is 55 DEG C to adjust temperature, adds sulphur
Acid adjustment pH is 6, is separately added into lysozyme and acid protease, the additive amount of lysozyme is 20,000 U：1L solution, acid protease
Additive amount be 10,000 U：1L solution under heat-retaining condition, is digested 12 hours, is then filtered by filter screen, filter screen aperture
It is 5 microns, removes floccule, collects filtered solution, then 100 DEG C of enzyme deactivation 5min, cooled to room temperature, then add same volume
Water, the calcium acetate of final concentration of 200mg/L and the mannitol of 100mg/L, stir evenly, 121 DEG C of steam treatment 5min, it is natural
It is cooled to room temperature to get fermentation medium.
The preparation method of the Trichoderma viride seed liquor is：Trichoderma viride streak inoculation is cultivated in PDA culture medium, is obtained
To single bacterium colony；Picking single bacterium colony is inoculated into PDA liquid medium and is cultivated, and it is 1 × 10 to obtain cell concentration8Cfu/ml's is green
Color trichoderma seed liquor；
The preparation method of the bacillus licheniformis seed liquor is：Bacillus licheniformis is inoculated on LB solid mediums and is trained
It supports, obtains single bacterium colony；Picking single bacterium colony is inoculated into LB liquid medium and is cultivated, and it is 1 × 10 to obtain cell concentration8cfu/
The bacillus licheniformis seed liquor of ml.
Embodiment 2
A kind of technique of preparing lysine through fermentation comprising following steps：
Corynebacterium glutamicum ATCC14997 is chosen as fermentation strain；Corynebacterium glutamicum is accessed in seed culture medium,
Culture to density is 5 × 108The seed liquor of cfu/mL；The group of the seed culture medium is divided into：Glucose 80g/L, yeast extract
30g/L, corn steep liquor 10g/L, potassium dihydrogen phosphate 2g/L, dipotassium hydrogen phosphate 2g/L, ferrous sulfate heptahydrate 0.2g/L, epsom salt
0.2g/L, biotin 10mg/L, vitamin B12mg/L；
Seed liquor is seeded to according to 10% inoculum concentration in the fermentation tank containing fermentation medium, aerobic fermentation, fermentation are carried out
Temperature is 35 DEG C, and tank pressure is 0.05MPa, and fermentation obtains zymotic fluid for 60 hours, persistently adds ammonium chloride as fermentation in fermentation process
Liquid neutralizer makes zymotic fluid discharging pH controls 7.2；
The preparation method of the fermentation medium includes the following steps：
Step 1）Wheat stalk is crushed, then with wheat bran according to 2：3 mass ratio mixing, then it is 50 mesh to be crushed to grain size, so
It is added to afterwards in the water of 5 times of weight, with the mixing speed warming while stirring of 200rpm to 60 DEG C, under heat-retaining condition, at ultrasound
20min, ultrasonic power 500W are managed, then 121 DEG C of steam treatment 10min, cooled to room temperature is to get compound；
Step 2）By Trichoderma viride seed liquor and bacillus licheniformis seed liquor according to 1:2 volume ratio mixing, obtains mixing and connects
Kind liquid, by combined inoculation liquid according to 6%（Account for step 1）The volume ratio of gained compound）Inoculum concentration be linked into and contain step 1）Institute
It obtains and is cultivated in the fermentation tank of compound, temperature is 30 DEG C, and incubation time 96h obtains culture solution；
Step 3）It is ultrasonically treated culture solution, ultrasonic time 30-60min, ultrasonic power 500W, then it is 55 DEG C to adjust temperature,
It is 6 to add sulfuric acid adjustment pH, is separately added into lysozyme and acid protease, the additive amount of lysozyme is 20,000 U：1L solution, acid egg
The additive amount of white enzyme is 10,000 U：1L solution under heat-retaining condition, is digested 24 hours, is then filtered by filter screen, filter screen
Aperture is 10 microns, removes floccule, collects filtered solution, then 100 DEG C of enzyme deactivation 5min, cooled to room temperature, then add phase
The mannitol of the water of same volume, the calcium acetate of final concentration of 100mg/L and 50mg/L, stirs evenly, 121 DEG C of steam treatments
5min, cooled to room temperature is to get fermentation medium.
The preparation method of the Trichoderma viride seed liquor is：Trichoderma viride streak inoculation is cultivated in PDA culture medium, is obtained
To single bacterium colony；Picking single bacterium colony is inoculated into PDA liquid medium and is cultivated, and it is 1 × 10 to obtain cell concentration8Cfu/ml's is green
Color trichoderma seed liquor；
The preparation method of the bacillus licheniformis seed liquor is：Bacillus licheniformis is inoculated on LB solid mediums and is trained
It supports, obtains single bacterium colony；Picking single bacterium colony is inoculated into LB liquid medium and is cultivated, and it is 1 × 10 to obtain cell concentration8cfu/
The bacillus licheniformis seed liquor of ml.
Embodiment 3
Each Contents of Main Components in culture medium of the present invention：
Control group is set to examine influence of the bacterial strain to culture medium Contents of Main Components, control group 1：Only with Trichoderma viride；It is right
According to group 2：Only with bacillus licheniformis.Concrete outcome is shown in Table 1：
Table 1
| Component target | Saccharic composition g/L | High molecular weight protein（50000 Da or more） | Small molecular protein（50000 Da or less） | Calcium ion mg/L | Potassium ion mg/L | Ferrous ion mg/L |
| Embodiment 1 | 72.6 | 19.1 | 25.4 | 126.7 | 309.5 | 60.8 |
| Control group 1 | 61.5 | 14.5 | 14.1 | 85.8 | 221.6 | 41.8 |
| Control group 2 | 37.9 | 20.5 | 11.7 | 66.4 | 148.8 | 32.5 |
As shown in table 1, compared with the control group 1-2 using single bacterial strain, combined using Trichoderma viride and bacillus licheniformis
It is more rich comprehensively to handle the nutrient media components obtained, meets the use standard of Corynebacterium glutamicum fermentation and acid.
Embodiment 4
Fermentation medium product acid activity is tested：
Control group 1：Glucose 80g/L, yeast extract 30g/L, corn steep liquor 10g/L, potassium dihydrogen phosphate 2g/L, dipotassium hydrogen phosphate 2g/
L, ferrous sulfate heptahydrate 0.2g/L, epsom salt 0.2g/L, biotin 10mg/L, vitamin B12mg/L；
Control group 2：Glucose 80g/L, yeast extract 30g/L, corn steep liquor 10g/L, potassium dihydrogen phosphate 2g/L, dipotassium hydrogen phosphate 2g/
L, ferrous sulfate heptahydrate 0.2g/L, epsom salt 0.2g/L, calcium acetate 200mg/L, mannitol 50mg/L, biotin 10mg/
L, vitamin B12mg/L；
For specific zymotechnique with reference to embodiment 1, specific fermentation and acid amount is shown in Table 2：
Table 2
| Group | Embodiment 1 | Comparative example 1 | Comparative example 2 |
| Lysine production mg/100ml | 14.1 | 11.9 | 12.7 |
| Biomass g/L（Dry weight） | 14.7 | 13.1 | 13.5 |
As shown in table 2, control group 1 is common fermentation medium, and control group 2 is added to calcium acetate on the basis of common culture medium
And mannitol, compared with control group 1-2, the biomass and production acid of embodiment 1 measure equal highest, illustrate 1 fermentation medium of embodiment
Culture effect it is more preferable.
Embodiment 5
The influence of calcium acetate and mannitol additive amount to fermentation production lysine amount：
It is experimental example with embodiment 1, under the premise of maintaining other components constant, the concentration gradient that sets calcium acetate is respectively 0,
50,100,200,400,800mg/L, as shown in Figure 1, with the increase of acetic acid calcium concentration, lysine production is continuously improved, when adding
After dosage increases to 100mg/L, yield amplification is not obvious, and after 200mg/L, yield decreases, therefore, present invention selection
The additive amount of 100-200mg/L.
It is experimental example with embodiment 1, under the premise of maintaining other components constant, the concentration gradient for setting mannitol is respectively
0,25,50,100,200,400mg/L, as shown in Fig. 2, with the increase of mannitol concentration, lysine production also with raising,
When additive amount increases to 100mg/L, lysine production reaches maximum value, with the increase of mannitol additive amount, lysine production
Amount decreases, and therefore, the present invention selects the additive amount of selection 50-100mg/L.
The explanation of above example is only intended to facilitate the understanding of the method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill of technical field, without departing from the principle of the present invention, the present invention can also be carried out several
Improvement and modification, these improvement and modification are also fallen within the protection scope of the claims of the present invention.
Claims (7)
1. a kind of technique of preparing lysine through fermentation comprising following steps：
Corynebacterium glutamicum seed liquor is seeded to according to the inoculum concentration of 5-10% in the fermentation tank containing fermentation medium, into
It acts charitably aerobe fermentation, fermentation temperature is 32-35 DEG C, and tank pressure is 0.05MPa, and fermentation obtains zymotic fluid for 48-60 hours, in fermentation process
Persistently plus ammonium chloride is as zymotic fluid neutralizer.
2. technique according to claim 1, which is characterized in that the corynebacterium glutamicum seed liquor is in accordance with the following steps
It is prepared：Corynebacterium glutamicum is accessed in seed culture medium, culture to density is（3-5）×108The paddy ammonia of cfu/mL
Sour corynebacteria seed liquor；The group of the seed culture medium is divided into：Glucose 80g/L, yeast extract 30g/L, corn steep liquor 10g/L,
Potassium dihydrogen phosphate 2g/L, dipotassium hydrogen phosphate 2g/L, ferrous sulfate heptahydrate 0.2g/L, epsom salt 0.2g/L, biotin 10mg/
L, vitamin B1 2mg/L。
3. technique according to claim 1 or 2, which is characterized in that the preparation method of the fermentation medium includes as follows
Step：
Step 1）Wheat stalk is crushed, then with wheat bran according to 2：3 mass ratio mixing, then it is 50 mesh to be crushed to grain size, so
It is added to afterwards in the water of 4-5 times of weight, with the mixing speed warming while stirring of 200rpm to 60 DEG C, under heat-retaining condition, ultrasound
20-30min, ultrasonic power 500W are handled, then 121 DEG C of steam treatment 10min, cooled to room temperature is to get compound；
Step 2）By Trichoderma viride seed liquor and bacillus licheniformis seed liquor according to 1-2:The volume ratio of 2-3 mixes, and is mixed
Inoculation liquid is closed, combined inoculation liquid is linked into according to the inoculum concentration of 6-8% and contains step 1）It is carried out in the fermentation tank of gained compound
Culture, temperature are 30 DEG C, and incubation time 72-96h obtains culture solution；
Step 3）It is ultrasonically treated culture solution, ultrasonic time 30-60min, ultrasonic power 500W, then it is 55 DEG C to adjust temperature,
It is 6 to add sulfuric acid adjustment pH, is separately added into lysozyme and acid protease, the additive amount of lysozyme is 20,000 U：1L solution, acid egg
The additive amount of white enzyme is 10,000 U：1L solution under heat-retaining condition, is digested 12-24 hour, is then filtered, collection filtered solution, 100 DEG C
Enzyme deactivation 5min, cooled to room temperature, then the water, appropriate calcium acetate and mannitol of same volume are added, it stirs evenly, 121 DEG C
Steam treatment 5min, cooled to room temperature is to get fermentation medium.
4. technique according to claim 3, which is characterized in that the preparation method of the Trichoderma viride seed liquor is：It will be green
Color trichoderma streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into PDA liquid medium progress
Culture, obtains Trichoderma viride seed liquor.
5. technique according to claim 3, which is characterized in that the preparation method of the bacillus licheniformis seed liquor is：
Bacillus licheniformis is inoculated on LB solid mediums and is cultivated, single bacterium colony is obtained；Picking single bacterium colony is inoculated into LB Liquid Cultures
It is cultivated on base, obtains bacillus licheniformis seed liquor.
6. technique according to claim 3, which is characterized in that the additive amount of the calcium acetate is 100-200mg/L.
7. technique according to claim 3, which is characterized in that the additive amount of the mannitol is 50-100mg/L.
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| CN110885863A (en) * | 2019-12-01 | 2020-03-17 | 齐齐哈尔龙江阜丰生物科技有限公司 | Method for preparing lysine fermentation medium by using mycoprotein |
| CN110055296B (en) * | 2019-05-13 | 2021-02-26 | 南京牧奇亚生物科技有限公司 | Preparation method and application of lysine |
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| CN110885863A (en) * | 2019-12-01 | 2020-03-17 | 齐齐哈尔龙江阜丰生物科技有限公司 | Method for preparing lysine fermentation medium by using mycoprotein |
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