CN105861343A - Hansenula polymorpha for preparing high-lysine single-cell protein through methyl alcohol and application of Hansenula polymorpha - Google Patents
Hansenula polymorpha for preparing high-lysine single-cell protein through methyl alcohol and application of Hansenula polymorpha Download PDFInfo
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- CN105861343A CN105861343A CN201610343015.7A CN201610343015A CN105861343A CN 105861343 A CN105861343 A CN 105861343A CN 201610343015 A CN201610343015 A CN 201610343015A CN 105861343 A CN105861343 A CN 105861343A
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- methanol
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- protein
- hansenula polymorpha
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 255
- 239000004472 Lysine Substances 0.000 title claims abstract description 31
- 108010027322 single cell proteins Proteins 0.000 title claims abstract description 25
- 241000320412 Ogataea angusta Species 0.000 title claims abstract description 11
- 238000000855 fermentation Methods 0.000 claims abstract description 51
- 230000004151 fermentation Effects 0.000 claims abstract description 51
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 34
- 239000002609 medium Substances 0.000 claims description 33
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 230000001580 bacterial effect Effects 0.000 claims description 20
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 17
- 229920001817 Agar Polymers 0.000 claims description 16
- 239000008272 agar Substances 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 13
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 239000007789 gas Substances 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000011573 trace mineral Substances 0.000 claims description 7
- 235000013619 trace mineral 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
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- PASHVRUKOFIRIK-UHFFFAOYSA-L calcium sulfate dihydrate Chemical compound O.O.[Ca+2].[O-]S([O-])(=O)=O PASHVRUKOFIRIK-UHFFFAOYSA-L 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 4
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 4
- 235000011151 potassium sulphates Nutrition 0.000 claims description 4
- 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 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 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
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 3
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 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 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 239000001117 sulphuric acid Substances 0.000 claims description 3
- 235000011149 sulphuric acid Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- 235000005074 zinc chloride Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- 235000015393 sodium molybdate Nutrition 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 1
- 239000001110 calcium chloride Substances 0.000 claims 1
- 229910001628 calcium chloride Inorganic materials 0.000 claims 1
- QSOMFNQEXNFPNU-UHFFFAOYSA-L magnesium;hydrogen sulfate;hydroxide;hydrate Chemical compound O.O.[Mg+2].[O-]S([O-])(=O)=O QSOMFNQEXNFPNU-UHFFFAOYSA-L 0.000 claims 1
- 239000012533 medium component Substances 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 102000004169 proteins and genes Human genes 0.000 abstract description 25
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 21
- 241000894006 Bacteria Species 0.000 abstract description 14
- 238000004321 preservation Methods 0.000 abstract description 4
- 230000014616 translation Effects 0.000 abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 abstract description 2
- 238000011161 development Methods 0.000 abstract description 2
- 239000000654 additive Substances 0.000 abstract 1
- 231100000350 mutagenesis Toxicity 0.000 abstract 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 241000235648 Pichia Species 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 238000012216 screening Methods 0.000 description 8
- 239000011521 glass Substances 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 235000013360 fish flour Nutrition 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 230000000394 mitotic effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000589220 Acetobacter Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940036811 bone meal Drugs 0.000 description 1
- 239000002374 bone meal Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000021321 essential mineral Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- -1 pplication Species 0.000 description 1
- 230000003946 protein process Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/78—Hansenula
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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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
- C12N1/36—Adaptation or attenuation of cells
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- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention relates to Hansenula polymorpha for preparing high-lysine single-cell protein through methyl alcohol and application of the Hansenula polymorpha. The classification name of the Hansenula polymorpha is Hansenula polymorpha AP23, the Hansenula polymorpha is preserved in the China Center for Type Culture Collection (CCTCC) on April 5th, 2016, and the preservation number is CCTCC No: M 2016173. By means of the plasma induced mutation technology, the strain capable of producing high-lysine single-cell protein is screened out with methyl alcohol as the only carbon source, and on this basis, the Hansenula polymorpha for efficiently utilizing methyl alcohol is screened out in a gradient mode through the further combination of methyl alcohol. By means of the strain, high-density fermentation can be achieved, methyl alcohol protein production is conducted through a 5 L fermentation tank, the final bacterium wet weight reaches 350-400 g/L, the methyl alcohol protein dry weight reaches 115-130 g/L, the protein content reaches 68%, and the lysine content reaches up to 60 mg/g. The Hansenula polymorpha has great social significance and economic value in development of animal feed protein additives.
Description
Technical field
The present invention relates to bioengineering field, particularly one strain utilizes methanol production many rich in lysine single cell protein
Shape Hansenula yeast bacterium and application thereof.
Background technology
Protein resource shortage is the key subjects that the whole world faces, and each state is all paying attention to reinforcement exploitation novel protein resource
Research.Wherein, what industrialization was the fastest is, and single cell protein produces, and is considered as to solve human protein's scarcity of resources to have most
One of approach of effect.
Single cell protein (single cell protein, SCP), refers to that yeast, mycete, fungus, non-pathogenic are thin
Produced bacterium protein in the unicellular microorganism bodies such as bacterium, its protein content typically constitutes from the 40% ~ 60% of thalline dry.
It is referred to as second filial generation single cell protein for raw material production Methanol Protein out with industrial methanol, compared with native protein, its
Crude protein content, will be high than fish flour and Semen sojae atricolor more than 70%, and containing abundant essential amino acids, minerals and vitamins,
Can part Peru Fish Dietary, Semen sojae atricolor, bone meal, meat and defatted milk powder, and have not against arable land, weatherproof, egg
The advantages such as white matter steady quality.
From the sixties in 20th century, just there are many countries to carry out the research of Methanol Protein, and develop into worldwide
Popular research topic.Wherein account for the ICI company of Yao Shu Britain of leading position, the device just having scale to be 100,000 t/a in 1980
It is constructed and put into operation.The Methanol Protein trade name " Pruteen " that ICI company produces." Pruteen " product contains the thick egg of 72%
In vain, methionine and lysine content are the most close with fish flour, as the feedstuff rich in heat, vitamin, mineral and high protein
Commercially sell.Nineteen eighty-three U.S. Phillips, oil company developed again Methanol Protein production new technique, improved fermentation tank
Heat exchange and oxygen condition of transmitting, can produce high density Methanol Protein.Subsequently, Germany's Hoechst-Uhde, Mitsubishi gas
The companies such as, Sweden Norprotein and France IFP also establish pilot-plant.Since 20 century 70s, China also begins to
The research of Methanol Protein.But up to the present, the industrialization of Methanol Protein is still at an early stage in China, domestic also do not have work
The Methanol Protein process units of industry, some units also stopped the research and development to Methanol Protein.
Methanol is a kind of platform chemicals, its wide material sources, and the methanol production capacity of China alreadys more than 45,000,000 t/a, and
In continuing increase trend, and domestic methanol demands amount is only 10,000,000 t/a, and most methanol Business survivals are hard to carry on, are badly in need of
Exploitation Downstream Products of Methanol, and with methanol for raw material production protein resource, the imbalance between supply and demand of methanol can be alleviated.At present, methanol
Cheap, Methanol Protein production cost is low, replaces the albumen such as traditional fish flour and beans with Methanol Protein, it will reduce feedstuff
Cost, improves feeding efficiency, accelerates the development of China feed industry, is the most also to solve the shortage of current albumen feedstuff, rely on into
The effective way of this contradiction of mouth.
Methanol Protein produces bacterial strain antibacterial and yeast etc., and saccharomycetic acid resistance is the best, it is possible to reduce extensive
The risk polluted during cultivation, energy high-density growth in cheap synthesis or semisynthetic medium, multiple-shaped nuohan inferior yeast is again in addition
There is of a relatively high optimal growth temperature, the speed of growth can be improved, shorten fermentation time, reduce Zymolysis Equipment simultaneously
Refrigeration requirement, thus beneficially large scale fermentation produces.But yeast how can be utilized to realize efficiently utilizing methanol production
Also do not have been reported that rich in certain amino acid whose single cell protein.
Summary of the invention
It is inferior that the first object of the present invention is to provide a strain to utilize methanol to prepare the multiform Chinese of high-lysine single cell protein
Yeast.
For realizing the first object of the present invention, the technical solution used in the present invention is as follows:
One plant height effect utilizes methanol production rich in the multiple-shaped nuohan inferior yeast of lysine single cell protein, and its Classification And Nomenclature is the multiform Chinese
Inferior yeast Hansenula polymorphaAP23, its deposit number is: CCTCC NO:M 2016173.Preservation day is 2016
On April 5, in.
The screening technique of multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 of the present invention is, by the multiform Chinese
Inferior yeast starting strain (ATCC34438) is after plasma mutation, using methanol as sole carbon source, utilizes high concentration methanol to put down
Screen choosing obtains the bacterial strain that methanol tolerance is stronger, then obtains high content of protein and high-load lysine through shake flask fermentation screening
Bacterial strain, after tame through high concentration methanol, it is thus achieved that can efficiently utilize methanol production rich in the multiform of lysine single cell protein
Hansenula yeast.
The screening of multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 specifically comprises the following steps that
(1) plasma mutation: by multiple-shaped nuohan inferior yeast original strain activation culture, 100 ml triangular flask liquid amounts are 20
Ml, in 37 DEG C of shaking tables, 200 rpm shaken cultivation 24 h, obtains being in the bacterium solution of exponential phase, is diluted to by the cell of cultivation
OD600=3-4, drips on the slide glass after sterilizing cools down, dries up with filtrated air；With helium as discharge gas, make with 80-120W
For radio-frequency power, using 10-30SLM as gas flow, as exposure time, bacterial strain is carried out plasma inducing using 10-1800s
Become.
(2) high concentration methanol flat board primary dcreening operation: the slide glass after mutation is placed in the tool plug test tube equipped with 1-2 mL normal saline
In, acutely shake, by the bacterial strain eluting on slide glass, be diluted to variable concentrations and coat the culture medium flat plate containing 500 mM methanol
On, cultivate 2-3 days, pick out eugonic bacterium colony for 37 DEG C.
(3) rich in the screening of lysine single cell protein bacterial strain: choose eugonic single bacterium colony and be inoculated in respectively and comprise
In 200 ml fermentation medium of 500 mM methanol, 37 DEG C, 200 rpm cultivate 48h, centrifugal tropina of collecting, detection thalline
The bacterial strain that the content of middle albumen and the content of lysine, selection protein content and lysine content are the highest.
(4) high concentration methanol domestication: protein content in selecting step (3) and the highest bacterial strain of lysine content continue
It is seeded on the Selective agar medium containing 500 mM methanol, after it grows to logarithmic (log) phase, is forwarded to the selection containing 1M methanol and cultivates
On base；It is forwarded on the Selective agar medium containing 1.5 M by same method afterwards, the most again bacterium solution is transferred to containing 2 M methanol
In Selective agar medium, after repeating to tame 2 times, take 0.2 ml bacterium solution and be coated on the Selective agar medium flat board comprising 2 M methanol, 37
DEG C constant temperature culture, obtains multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23.
The second object of the present invention is that multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 relies ammonia at preparation height
Application in acid single cell protein.The invention provides one and utilize multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23
In preparing high-lysine single cell protein, the method for application, specifically comprises the following steps that
(1) polymorpha AP23 ferments at 5 L fermentation tank middle-high densities: polymorpha AP23 ferments at 5 L
Using fermentation medium to carry out tropina production in tank, wherein, fermentation medium liquid amount is 2L, 121 DEG C of sterilizing 15 min,
Inoculation pipeline, ammonia and methanol feeding pipeline, sample tap pipeline, air filter are carried out sterilizing simultaneously, treat fermentation medium
When being down to about 37 DEG C, regulating pH to 5.0 with ammonia, add seed liquor and ferment, fermentation temperature is 37 DEG C, controls dissolved oxygen
20%-60%, speed of agitator 250-500 rpm；After 24 hours, glycerol depletion in culture medium, start stream and add methanol, monitor by gas phase
Methanol concentration so that it is control between 0.5%-2%, every 8 hours sampling and measuring thalline OD600And weight in wet base, after 96-100 hour,
Fermentation ends, collects fermentation liquid.
(2) rich in the extraction of lysine Methanol Protein: the fermentation liquor that above-mentioned steps (1) is collected in 5 L fermentation tanks
Centrifuge 5000r/min continuous centrifugal concentrates and obtains thalline, weighs its weight in wet base after clear water washing thalline；By collect thalline in
Dry to constant mass for 55 DEG C, collect dry powder and be single-cell methanol protein.
The beneficial effects of the present invention is:
Using plasma mutation multiple-shaped nuohan inferior yeast of the present invention, utilizes methanol flat screen to select protein content and lysine content
Higher bacterial strain, utilizes high concentration methanol to tame on this basis so that this bacterial strain can utilize methanol production unicellular efficiently
Albumen.In 5L fermentation tank, utilize glycerol 30 g/L, methanol 500 ml/L can produce 115-130g/L tropina, Qi Zhonglai
Histidine content, up to not containing methanol in 60mg/g, and the Methanol Protein dry powder obtained, is the desirable feedstock substituting forage protein,
There is great social meaning and economic worth.
Biomaterial explanation
Biomaterial of the present invention, its Classification And Nomenclature is multiple-shaped nuohan inferior yeast (Hansenula polymorpha) AP23,
Being preserved in China typical culture collection center (being called for short CCTCC), deposit number is CCTCC NO:M 2016173, preservation day
Phase is: on April 5th, 2016, preservation address is: China. Wuhan. and Wuhan University.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
This example demonstrates that the method that multiple-shaped nuohan inferior yeast original strain is carried out first step plasma mutation.
The method that multiple-shaped nuohan inferior yeast original strain carries out first step plasma mutation is as follows:
By multiple-shaped nuohan inferior yeast original strain activation culture, 100 ml triangular flask liquid amounts are 20 ml, in 37 DEG C of shaking tables 200
Rpm shaken cultivation 24 h, obtains growing bacterium solution vigorous, that thalline is sturdy；The cell taking fresh cultured is diluted to cell concentration
OD600=3-4, drips on the slide glass after sterilizing cools down, dries up with filtrated air；With helium as discharge gas, using 100W as
Radio-frequency power, using 10SLM as gas flow, carries out plasma mutation as exposure time to bacterial strain using 10-1800s, lures
After change, the Mycoderma on carrier is eluted, calculate survival rate.Test result indicate that 450 s are optimal mutation exposure times.
Embodiment 2
The explanation screening of this example efficiently utilizes methanol production rich in the method for the multiple-shaped nuohan inferior yeast of lysine single cell protein.
Wherein, the culture medium prescription used:
(1) seed culture medium: yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L, remaining is water.
(2) Selective agar medium: methanol 500 mM, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.7 g/L, chlorine
Changing calcium 0.4 g/L, yeast extract 1 g/L, agar powder 1.5 g/L, remaining is water, and pH is natural.
(3) fermentation medium: 85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, two
Water magnesium sulfate 14.9 g/L, potassium hydroxide 4.13 g/L, glycerol 30 g/L, trace element solution 4 ml/L, water 1 L；
(4) fermentation medium 2:85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, two water sulfur
Acid magnesium 14.9 g/L, potassium hydroxide 4.13 g/L, methanol 20ml/L, trace element solution 4 ml/L, water 1 L, every 24h during cultivation
Add 20ml/L methanol.
Described trace element solution is: copper sulphate pentahydrate 6 g/L, potassium iodide 0.088 g/L, manganese sulfate monohydrate 3 g/L,
Sodium Molybdate Dihydrate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride hexahydrate 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65
G/L, biotin 0.2 g/L, mass concentration is concentrated sulphuric acid 5 ml/L of 98 %, and remaining is water.
Screening step is specific as follows:
(1) high concentration methanol flat board primary dcreening operation:
Slide glass after mutation in embodiment 1 is placed in equipped with in the tool plug test tube of 1-2ml normal saline, acutely shakes, by slide glass
On bacterial strain eluting, be diluted on the Selective agar medium flat board that variable concentrations is coated containing 500mM methanol, 37 DEG C cultivate 3-4 days,
Pick out eugonic bacterium colony.
(2) rich in the screening of lysine single cell protein bacterial strain: choose eugonic single bacterium colony and be inoculated in respectively and comprise
In 200 ml fermentation medium of 500 mM methanol, 37 DEG C, 200 rpm cultivate 48h, centrifugal tropina of collecting, detection thalline
The bacterial strain that the content of middle albumen and the content of lysine, selection protein content and lysine content are the highest.
(3) domestication of resisting high-concentration methanol: protein content in selecting step 2 and the highest bacterial strain of lysine content continue
Continue and be seeded on the Selective agar medium containing 500 mM methanol, after it grows to logarithmic (log) phase, be forwarded to the selection training containing 1M methanol
Support on base；It is forwarded on the Selective agar medium containing 1.5 M by same method afterwards, the most again bacterium solution is transferred to containing 2 M methanol
Selective agar medium in, after repeating to tame 2 times, take 0.2 ml bacterium solution and be coated on the Selective agar medium flat board comprising 2 M methanol,
37 DEG C of constant temperature culture, obtain multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23.
(4) shake flask fermentation screening:
Strains A P23 that step (3) is obtained and original strain access seed culture medium amplification culture, cultivation temperature 37 DEG C, 100
ML triangular flask liquid amount 20 mL, incubation time 24 h.Ferment the most in the fermentation medium, inoculum concentration 10%(v/v), fermentation
Temperature 37 DEG C, 1L triangular flask liquid amount 200 mL, after cultivating 24h, stand overnight, change fermentation medium 2, fermentation time 72 h
Protein content and the lysine content of each bacterial strain of rear detection are as shown in table 1:
Table 1
Embodiment 3
This example demonstrates that the hereditary stability test of the multiple-shaped nuohan inferior yeast AP23 of embodiment 2, strain passage fermenting experiment is such as
Shown in lower.
Utilize shake flask fermentation, the mitotic stability of detection mutant AP23.Strains A P23 passes on fermentation test result such as table 2
Shown in:
Table 2 strains A P23 passes on fermentation test result
As can be seen from the table, passing on through 7 times, strains A P23 albumen and lysine content are stable, and mitotic stability is good.
Embodiment 4
This example demonstrates that multiple-shaped nuohan inferior yeast AP23 efficiently utilizes methanol production rich in the application of lysine single cell protein.
Wherein, the culture medium prescription used:
(1) seed slant medium: yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L, agar powder 20 g/L, its
Yu Weishui.
(2) Selective agar medium: methanol 500 mM, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.7 g/L, chlorine
Changing calcium 0.4 g/L, yeast extract 1 g/L, remaining is water, and pH is natural.
(3) fermentation medium: 85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, two
Water magnesium sulfate 14.9 g/L, potassium hydroxide 4.13 g/L, glycerol 30 g/L, trace element solution 4 ml/L, water 1 L；
Described trace element solution is: copper sulphate pentahydrate 6 g/L, potassium iodide 0.088 g/L, manganese sulfate monohydrate 3 g/L, two water
Sodium molybdate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride hexahydrate 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L,
Biotin 0.2 g/L, mass concentration is concentrated sulphuric acid 5 ml/L of 98 %, and remaining is water.
Multiple-shaped nuohan inferior yeast AP23 bacterial strain is in the method for 5 L fermentation tank middle-high density fermenting and producing Methanol Proteins, and step is such as
Under:
(1) cultivation of seed: multiple-shaped nuohan inferior yeast AP23 bacterial strain method of scoring is seeded on seed slant medium, 37 DEG C of trainings
Support 48 hours, take 1 cultured inclined-plane seed, with 3 mL sterilized water, seed is rinsed, be seeded to comprise 200 mL choosings
Select in 1 L triangular flask of culture medium, 37 DEG C, 220 rpm shaken cultivation about 24 hours to OD=9-10.
(2) 5 L fermentation tank middle-high density fermentations: multiple-shaped nuohan inferior yeast AP23 bacterial strain uses fermentation training in 5 L fermentation tanks
Foster base carries out tropina production, and wherein, fermentation medium liquid amount is 2L, and 121 DEG C of sterilizing 15 min, simultaneously to inoculated tube
Road, ammonia and methanol feeding pipeline, sample tap pipeline, air filter carry out sterilizing, treat that fermentation medium is down to about 37 DEG C
Time, regulating pH to 5.0 with ammonia, add seed liquor and ferment, fermentation temperature is 37 DEG C, controls dissolved oxygen 20%-60%, and stirring turns
Speed 250-500 rpm；After 24 hours, glycerol depletion in culture medium, start stream and add methanol, monitor methanol concentration by gas phase so that it is
Control between 0.5%-2%, every 8 hours sampling and measuring thalline OD600And weight in wet base, after 96-100 hour, fermentation ends, collect
Fermentation liquid.Subsequently fermentation liquor centrifuge 5000 r/min continuous centrifugal is concentrated and obtain thalline, claim after clear water washing thalline
Measure its weight in wet base；The thalline collected is dried to constant mass in 55 DEG C, collects dry powder and be single-cell methanol protein.
Final thalline weight in wet base reaches 350-400 g/L, and Methanol Protein dry weight reaches 115-130 g/L, and protein content reaches
To 68%, wherein lysine content is up to 60 mg/g.
Claims (7)
1. a strain utilizes methanol to prepare the polymorpha of high-lysine single cell protein, it is characterised in that Classification And Nomenclature
For multiple-shaped nuohan inferior yeast (Hansenula polymorpha) AP23, it is preserved in Chinese Typical Representative culture on April 5th, 2016
Preservation center CCTCC, deposit number: CCTCC NO:M 2016173.
2. the preparation method of polymorpha described in claim 1, it is characterised in that preparation process includes using high concentration
Methanol flat board primary dcreening operation, then through shake flask fermentation, after tame through high concentration methanol.
The preparation method of polymorpha the most according to claim 2, it is characterised in that described primary dcreening operation and fermentation step
Middle methanol concentration is 500mM.
The preparation method of polymorpha the most according to claim 2, it is characterised in that described domestication step methanol is dense
Degree graded, is finally 2 M.
5. the application in prepared by high-lysine single cell protein of the polymorpha described in claim 1.
Polymorpha application in prepared by high-lysine single cell protein, its feature the most according to claim 5
It is, comprises the steps:
(1) seed culture: by multiple-shaped nuohan inferior yeast AP23 inoculation to seed slant medium, cultivate 48 hours for 37 DEG C
After, take in the triangular flask that cultured inclined-plane seed is seeded to comprise Selective agar medium, 37 DEG C, 220 rpm shaken cultivation about 24
Hour to OD=9-10；
(2) fermentation culture: multiple-shaped nuohan inferior yeast AP23 bacterial strain uses fermentation medium to carry out tropina production in fermentation tank,
121 DEG C of sterilizing 15 min, when fermentation medium is down to 37 DEG C, regulate pH to 5.0, add seed liquor and ferment, fermentation temperature
Degree is 37 DEG C, controls dissolved oxygen 20%-60%, speed of agitator 250-500 rpm；After 24 hours, glycerol depletion in culture medium, start stream
Add methanol, monitor methanol concentration by gas phase so that it is control between 0.5%-2%, every 8 hours sampling and measuring thalline OD600With wet
Weight, after 96-100 hour, fermentation ends, collect fermentation liquid；
(3) albumen is extracted: concentrated by fermentation liquor centrifuge 5000 r/min continuous centrifugal and obtain thalline, wash thalline with clear water
Its weight in wet base of rear weighing；The thalline collected is dried to constant mass in 55 DEG C, collects dry powder.
Polymorpha application in prepared by high-lysine single cell protein, its feature the most according to claim 6
It is, uses medium component as follows:
Seed slant medium: yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L, agar powder 20 g/L, remaining
For water；
Selective agar medium: methanol 500 mM, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.7 g/L, calcium chloride 0.4
G/L, yeast extract 1 g/L, remaining is water, and pH is natural；
Fermentation medium: 85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, sulfate dihydrate magnesium
14.9 g/L, potassium hydroxide 4.13 g/L, glycerol 30 g/L, trace element solution 4 ml/L, water 1 L；
Described trace element solution is: copper sulphate pentahydrate 6 g/L, potassium iodide 0.088 g/L, manganese sulfate monohydrate 3 g/L, two water
Sodium molybdate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride hexahydrate 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L,
Biotin 0.2 g/L, mass concentration is concentrated sulphuric acid 5 ml/L of 98 %, and remaining is water.
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| CN112646740A (en) * | 2020-11-27 | 2021-04-13 | 中国科学院天津工业生物技术研究所 | Formate single-cell protein strain MA5 and application thereof |
| RU2805960C1 (en) * | 2022-12-26 | 2023-10-24 | Общество с ограниченной ответственностью "Эй Пи Интернэйшнл" | Methylotrophic yeast strain ogataea polymorpha 16 ap (bkm y-3389d) for protein production |
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| RU2731517C1 (en) * | 2020-02-06 | 2020-09-03 | Андрей Дмитриевич Берков | Method of producing yeast biomass for production of fodder protein product |
| RU2742472C1 (en) * | 2020-08-06 | 2021-02-08 | Андрей Дмитриевич Берков | Nutrient medium for the cultivation of yeast biomass and a method for its production |
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