CN105861343B - Hansenula polymorpha for preparing high-lysine single-cell protein by using methanol and application thereof - Google Patents
Hansenula polymorpha for preparing high-lysine single-cell protein by using methanol and application thereof Download PDFInfo
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- CN105861343B CN105861343B CN201610343015.7A CN201610343015A CN105861343B CN 105861343 B CN105861343 B CN 105861343B CN 201610343015 A CN201610343015 A CN 201610343015A CN 105861343 B CN105861343 B CN 105861343B
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 243
- 239000004472 Lysine Substances 0.000 title claims abstract description 32
- 108010027322 single cell proteins Proteins 0.000 title claims abstract description 25
- 241000320412 Ogataea angusta Species 0.000 title claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 40
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 239000002609 medium Substances 0.000 claims description 31
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 26
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 230000001580 bacterial effect Effects 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 229920001817 Agar Polymers 0.000 claims description 17
- 239000008272 agar Substances 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 10
- 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
- 239000001963 growth medium Substances 0.000 claims description 8
- 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
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 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
- 238000002360 preparation method Methods 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
- 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
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- 229910000366 copper(II) sulfate Inorganic materials 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
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 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
- 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
- 239000000284 extract Substances 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
- 239000011684 sodium molybdate Substances 0.000 claims 1
- 235000015393 sodium molybdate Nutrition 0.000 claims 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 26
- 102000004169 proteins and genes Human genes 0.000 abstract description 26
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 23
- 238000002703 mutagenesis Methods 0.000 abstract description 12
- 231100000350 mutagenesis Toxicity 0.000 abstract description 12
- 238000012216 screening Methods 0.000 abstract description 7
- 230000014616 translation Effects 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 4
- 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
- 241000894006 Bacteria Species 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 8
- 239000011521 glass Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 235000019733 Fish meal Nutrition 0.000 description 4
- 241000235342 Saccharomycetes Species 0.000 description 4
- 239000000908 ammonium hydroxide Substances 0.000 description 4
- 239000004467 fishmeal Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241000235648 Pichia Species 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 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
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 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
- 238000005119 centrifugation Methods 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
- 238000001816 cooling Methods 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
- 235000010755 mineral Nutrition 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 230000000394 mitotic effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 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 2
- 238000004659 sterilization and disinfection Methods 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
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 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
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 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
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003698 anagen phase 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
- 230000008094 contradictory effect Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum 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
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 230000003946 protein process Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000000192 social effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
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- 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
- 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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- 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
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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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- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a Hansenula polymorpha strain for preparing high-lysine single-cell protein by using methanol and application thereof, wherein the strain is classified and named as Hansenula polymorpha (Hansenula polymorpha)Hansenula polymorpha) AP23, which has been preserved in China Center for Type Culture Collection (CCTCC) at 2016, 4 and 5 days, with the preservation number: CCTCC NO: M2016173. The invention utilizes a plasma mutagenesis technology, uses methanol as a unique carbon source to screen out a strain which can produce single-cell protein rich in lysine, and further combines with methanol gradient screening to obtain the Hansenula polymorpha which efficiently utilizes the methanol. The strain can realize high-density fermentation, and methanol protein production is carried out by using a 5L fermentation tank, so that the final wet weight of the strain reaches 350-400 g/L, the dry weight of the methanol protein reaches 115-130 g/L, and the protein content reaches 68 percent, wherein the lysine content reaches 60 mg/g. The invention has great social significance and economic value for the development of animal feed protein additives.
Description
Technical field
The present invention relates to bioengineering field, especially one plant to be rich in the more of lysine single cell protein using methanol production
Shape Hansenula yeast bacterium and its application.
Background technique
Protein resource shortage is the key subjects that the whole world faces, and each state all is paying attention to reinforcing exploitation novel protein resource
Research.Wherein, what industrialization was most fast is single cell protein production, and is considered as solving human protein's scarcity of resources most to have
One of approach of effect.
Single cell protein (single cell protein, SCP), refers to that saccharomycete, mould, fungi, non-pathogenic are thin
Generated bacterium protein in the unicellular microorganisms body such as bacterium, protein content typically constitute from the 40% ~ 60% of thallus dry matter.
It is referred to as second generation single cell protein by the Methanol Protein that raw material is produced of industrial methanol, compared with native protein,
Crude protein content, will be high than fish meal and soybean 70% or more, and essential amino acid rich in, minerals and vitamins,
Fish meal, soybean, bone meal, meat and skimmed milk power can be partially substituted, and is had not against arable land, weatherproof, egg
The advantages that white matter quality is stablized.
Since the 1960s, just there are many countries to carry out the research of Methanol Protein, and develops into worldwide
Hot research topic.Wherein account for leading position will belong to Britain's ICI Company, and just having within 1980 scale is the device of 100,000 t/a
It is constructed and put into operation.The Methanol Protein trade name " Pruteen " of ICI Company production.In " Pruteen " product containing 72% thick egg
White, methionine and lysine content and fish meal are very close, as the feed rich in heat, vitamin, minerals and high protein
It sells on the market.Nineteen eighty-three U.S. Phillips oil company develops Methanol Protein production new technique again, improves fermentor
Heat exchange and oxygen condition of transmitting, can produce high density Methanol Protein.Then, German Hoechst-Uhde, Mitsubishi gas
The companies such as, Sweden Norprotein and France IFP also establish pilot-plant.Since the 1970s, China also starts
The research of Methanol Protein.But up to the present, the industrialization of Methanol Protein is still at an early stage in China, domestic work not yet
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, from a wealth of sources, and the methanol production capacity in China alreadys exceed 45,000,000 t/a, and
In the trend that continues growing, 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
Downstream Products of Methanol is developed, and produces protein resource using methanol as raw material, the imbalance between supply and demand of methanol can be alleviated.Currently, methanol
Cheap, Methanol Protein production cost is low, replaces the albumen such as traditional fish meal and beans with Methanol Protein, it will reduce feed
Cost improves feeding efficiency, accelerates the development of China's feed industry, while being also to solve the shortage of current protein feed, rely on into
This contradictory effective way of mouth.
Methanol Protein production bacterial strain has bacterium and yeast etc., and the acid resistance of saccharomycete is very good, it is possible to reduce extensive
The risk polluted when culture, in cheap synthesis or semisynthetic medium can high-density growth, furthermore multiple-shaped nuohan inferior yeast is again
With relatively high optimal growth temperature, the speed of growth can be improved, shorten fermentation time, while reducing to Zymolysis Equipment
Refrigeration requires, to be conducive to large scale fermentation production.But how to be realized using saccharomycete and efficiently utilize methanol production
Single cell protein rich in certain amino acid does not have been reported that also.
Summary of the invention
It is inferior that the first object of the present invention is to provide the multiform Chinese that one plant prepares high-lysine single cell protein using methanol
Saccharomycete.
The first purpose to realize the present invention, The technical solution adopted by the invention is as follows:
One plant height effect is rich in the multiple-shaped nuohan inferior yeast of lysine single cell protein using methanol production, and classification naming is more
Shape Hansenula yeastHansenula polymorphaAP23, deposit number are as follows: CCTCC NO:M 2016173.Preservation day is
On April 5th, 2016.
Multiple-shaped nuohan inferior yeast of the present inventionHansenula polymorphaThe screening technique of AP23 is, by the multiform Chinese
Inferior yeast starting strain (ATCC34438) is after plasma mutagenesis, flat using high concentration methanol using methanol as sole carbon source
Screen is selected to obtain the stronger bacterial strain of methanol tolerance, then screens through shake flask fermentation and obtain high content of protein and high-content lysine
Bacterial strain, most tamed afterwards through high concentration methanol, obtain can efficiently using methanol production be rich in lysine single cell protein multiform
Hansenula yeast.
Multiple-shaped nuohan inferior yeastHansenula polymorphaSpecific step is as follows for the screening of AP23:
(1) plasma mutagenesis: by multiple-shaped nuohan inferior yeast original strain activation culture, 100 ml triangular flask liquid amounts are
20 ml, 200 rpm shaken cultivation, 24 h in 37 DEG C of shaking tables obtain the bacterium solution in logarithmic growth phase, and the cell of culture is diluted
To OD600=3-4 is added dropwise on slide glass after sterilization and cooling, is dried up with filtrated air；Using helium as discharge gas, with 80-120W
As radio-frequency power, using 10-30SLM as gas flow, plasma is carried out to bacterial strain using 10-1800s as irradiation time
Mutagenesis.
(2) slide glass after mutagenesis high concentration methanol plate primary dcreening operation: is placed in the tool plug test tube equipped with 1-2 mL physiological saline
In, it acutely shakes, the bacterial strain on slide glass is eluted, various concentration is diluted to and is coated on the culture medium flat plate containing 500 mM methanol
On, 37 DEG C are cultivated 2-3 days, and eugonic bacterium colony is picked out.
(3) it is rich in the screening of lysine single cell protein bacterial strain: choosing eugonic single colonie and be inoculated in respectively and include
In 200 ml fermentation mediums of 500 mM methanol, 37 DEG C, 200 rpm cultivate 48h, and thalline were collected by centrifugation albumen detects thallus
The content of middle albumen and the content of lysine select protein content and the higher bacterial strain of lysine content.
(4) high concentration methanol is tamed: the higher bacterial strain of protein content and lysine content in selecting step (3) continues
It is seeded on the Selective agar medium containing 500 mM methanol, after it grows to logarithmic phase, is forwarded to the selection culture of the methanol containing 1M
On base；It is forwarded on the Selective agar medium containing 1.5 M, finally bacterium solution is transferred to containing 2 M methanol again in the same way later
In Selective agar medium, after repeating domestication 2 times, 0.2 ml bacterium solution is taken to be coated on the Selective agar medium plate comprising 2 M methanol, 37
DEG C constant temperature incubation obtains multiple-shaped nuohan inferior yeastHansenula polymorphaAP23。
The second object of the present invention is multiple-shaped nuohan inferior yeastHansenula polymorphaAP23 is in the high bad ammonia of preparation
Application in sour single cell protein.Multiple-shaped nuohan inferior yeast is utilized the present invention provides a kind ofHansenula polymorphaAP23
The method applied in preparing high-lysine single cell protein, the specific steps are as follows:
(1) polymorpha AP23 ferments in 5 L fermentor middle-high densities: polymorpha AP23 is in 5 L
Mycoprotein production is carried out using fermentation medium in fermentor, wherein fermentation medium liquid amount is 2L, 121 DEG C of sterilizings 15
Min, while sterilizing to inoculation pipeline, ammonium hydroxide and methanol feeding pipeline, sample tap pipeline, air filter, wait training of fermenting
When feeding base is down to 37 DEG C or so, pH to 5.0 is adjusted with ammonium hydroxide, seed liquor is added and ferments, fermentation temperature is 37 DEG C, is controlled molten
Oxygen 20%-60%, speed of agitator 250-500 rpm；After 24 hours, glycerol depletion in culture medium starts stream plus methanol, is supervised with gas phase
Methanol concentration is surveyed, makes its control between 0.5%-2%, thallus OD is measured by sampling in every 8 hours600And weight in wet base, 96-100 hours
Afterwards, fermentation ends collect fermentation liquid.
(2) be rich in the extraction of lysine Methanol Protein: the fermentation liquid that above-mentioned steps (1) are collected in 5 L fermentors passes through
The concentration of centrifuge 5000r/min continuous centrifugal obtains thallus, with weighing its weight in wet base after clear water washing thalline；By the thallus of collection in
55 DEG C are dried to constant mass, and collecting dry powder is single-cell methanol protein.
The beneficial effects of the present invention are:
Using plasma mutagenesis multiple-shaped nuohan inferior yeast of the present invention selects protein content and lysine using methanol flat screen
The higher bacterial strain of content is tamed using high concentration methanol on this basis, and the bacterial strain is enabled efficiently to utilize methanol production list
Cell protein.In 5L fermentor, using 30 g/L of glycerol, 500 ml/L of methanol can produce 115-130g/L mycoprotein,
Middle lysine content is free of methanol up to 60mg/g in the Methanol Protein dry powder obtained, be the ideal for substituting forage protein
Raw material has great social effect and economic value.
Biomaterial explanation
Biomaterial of the present invention, classification naming be multiple-shaped nuohan inferior yeast (Hansenula polymorpha)
AP23 has been preserved in China typical culture collection center (abbreviation CCTCC), and deposit number is CCTCC NO:M 2016173,
Preservation date are as follows: on April 5th, 2016, preservation address are as follows: the Chinese Wuhan Wuhan University.
Specific embodiment
Only invention is further described in detail for following embodiments, 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 the mutagenesis of first step plasma.
The method that multiple-shaped nuohan inferior yeast original strain carries out the mutagenesis of first step plasma 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 thallus is sturdy；The cell of fresh cultured is taken to be diluted to cell dense
Spend OD600=3-4 is added dropwise on slide glass after sterilization and cooling, is dried up with filtrated air；Using helium as discharge gas, made with 100W
Plasma mutagenesis is carried out to bacterial strain using 10-1800s as irradiation time using 10SLM as gas flow for radio-frequency power,
After mutagenesis, the mycoderm on carrier is eluted, calculates survival rate.The experimental results showed that when 450 s are optimal mutagenesis irradiation
Between.
Embodiment 2
This example illustrates the side for screening the efficiently multiple-shaped nuohan inferior yeast using methanol production rich in lysine single cell protein
Method.
Wherein, used culture medium prescription:
(1) seed culture medium: 10 g/L of yeast powder, 20 g/L of peptone, 20 g/L of glucose, remaining is water.
(2) Selective agar medium: 500 mM of methanol, 1 g/L of ammonium sulfate, 1 g/L of potassium dihydrogen phosphate, 0.7 g/L of magnesium sulfate, chlorine
Change 0.4 g/L of calcium, 1 g/L of yeast extract, 1.5 g/L of agar powder, remaining is water, and pH is natural.
(3) fermentation medium: 85% phosphoric acid, 26.7 ml/L, 0.93 g/L of calcium sulphate dihydrate, potassium sulfate 18.2 g/L, two
14.9 g/L of water magnesium sulfate, 4.13 g/L of potassium hydroxide, 30 g/L of glycerol, 4 ml/L of trace element solution, 1 L of water；
(4) 26.7 ml/L of fermentation medium 2:85% phosphoric acid, 0.93 g/L of calcium sulphate dihydrate, potassium sulfate 18.2 g/L, two
14.9 g/L of water magnesium sulfate, potassium hydroxide 4.13 g/L, methanol 20ml/L, 4 ml/L of trace element solution, 1 L of water, when culture
Per addition 20ml/L methanol for 24 hours.
The trace element solution are as follows: 6 g/L of cupric sulfate pentahydrate, 0.088 g/L of potassium iodide, 3 g/L of manganese sulfate monohydrate,
0.2 g/L of Sodium Molybdate Dihydrate, 0.02 g/L of boric acid, 0.5 g/L of CoCL2 6H2O, 20 g/L of zinc chloride, ferrous sulfate heptahydrate 65
G/L, 0.2 g/L of biotin, mass concentration are 5 ml/L of the concentrated sulfuric acid of 98 %, remaining is water.
Screening step is specific as follows:
(1) high concentration methanol plate primary dcreening operation:
Slide glass after mutagenesis in embodiment 1 is placed in the tool plug test tube equipped with 1-2ml physiological saline, is acutely shaken, it will
Bacterial strain elution on slide glass, is diluted to various concentration and is coated on the Selective agar medium plate of the methanol containing 500mM, 37 DEG C of culture 3-
4 days, pick out eugonic bacterium colony.
(2) it is rich in the screening of lysine single cell protein bacterial strain: choosing eugonic single colonie and be inoculated in respectively and include
In 200 ml fermentation mediums of 500 mM methanol, 37 DEG C, 200 rpm cultivate 48h, and thalline were collected by centrifugation albumen detects thallus
The content of middle albumen and the content of lysine select protein content and the higher bacterial strain of lysine content.
(3) domestication of resisting high-concentration methanol: the higher bacterial strain of protein content and lysine content in selecting step 2 after
It is continuous to be seeded on the Selective agar medium containing 500 mM methanol, after it grows to logarithmic phase, it is forwarded to the selection training of the methanol containing 1M
It supports on base；It is forwarded on the Selective agar medium containing 1.5 M, finally bacterium solution is transferred to containing 2 M methanol again in the same way later
Selective agar medium in, repeat domestication 2 times after, take 0.2 ml bacterium solution to be coated on the Selective agar medium plate comprising 2 M methanol,
37 DEG C of constant temperature incubations obtain multiple-shaped nuohan inferior yeastHansenula polymorphaAP23。
(4) shake flask fermentation screens:
Strains A P23 and original strain the access seed culture medium that step (3) are obtained, which expand, to be cultivated, and 37 DEG C of cultivation temperature,
100 mL triangular flask liquid amount 20 mL, 24 h of incubation time.Then it ferments in the fermentation medium, inoculum concentration 10%(v/v),
It 37 DEG C of fermentation temperature, 200 mL of 1L triangular flask liquid amount, after culture for 24 hours, stands overnight, replaces fermentation medium 2, fermentation time
Protein content and the lysine content that each bacterial strain is detected after 72 h are as shown in table 1:
Table 1
Embodiment 3
This example demonstrates that the genetic stability of the multiple-shaped nuohan inferior yeast AP23 of embodiment 2 is tested, strain passage fermentation is real
It tests as follows.
Using shake flask fermentation, the mitotic stability of mutant strain AP23 is detected.Strains A P23 passes on fermentation test result such as table 2
It is shown:
2 strains A P23 of table passes on fermentation test result
As can be seen from the table, it is passed on by 7 times, strains A P23 albumen and lysine content are stablized, and mitotic stability is good
It is good.
Embodiment 4
This example demonstrates that multiple-shaped nuohan inferior yeast AP23 efficiently utilizes methanol production answering rich in lysine single cell protein
With.
Wherein, used culture medium prescription:
(1) seed slant medium: 10 g/L of yeast powder, 20 g/L of peptone, 20 g/L of glucose, 20 g/ of agar powder
L, remaining is water.
(2) Selective agar medium: 500 mM of methanol, 1 g/L of ammonium sulfate, 1 g/L of potassium dihydrogen phosphate, 0.7 g/L of magnesium sulfate, chlorine
Change 0.4 g/L of calcium, 1 g/L of yeast extract, remaining is water, and pH is natural.
(3) fermentation medium: 85% phosphoric acid, 26.7 ml/L, 0.93 g/L of calcium sulphate dihydrate, potassium sulfate 18.2 g/L, two
14.9 g/L of water magnesium sulfate, 4.13 g/L of potassium hydroxide, 30 g/L of glycerol, 4 ml/L of trace element solution, 1 L of water；
The trace element solution are as follows: 6 g/L of cupric sulfate pentahydrate, 0.088 g/L of potassium iodide, 3 g/L of manganese sulfate monohydrate,
0.2 g/L of Sodium Molybdate Dihydrate, 0.02 g/L of boric acid, 0.5 g/L of CoCL2 6H2O, 20 g/L of zinc chloride, ferrous sulfate heptahydrate 65
G/L, 0.2 g/L of biotin, mass concentration are 5 ml/L of the concentrated sulfuric acid of 98 %, remaining is water.
Multiple-shaped nuohan inferior yeast AP23 bacterial strain is in the method for 5 L fermentor middle-high density fermenting and producing Methanol Proteins, and step is such as
Under:
(1) culture of seed: multiple-shaped nuohan inferior yeast AP23 bacterial strain is seeded on seed slant medium with method of scoring, 37
DEG C culture 48 hours, take 1 cultured inclined-plane seed, seed rinsed with 3 mL sterile waters, is seeded to comprising 200
In 1 L triangular flask of mL Selective agar medium, 37 DEG C, 220 rpm shaken cultivations about 24 hours to OD=9-10.
The fermentation of (2) 5 L fermentor middle-high densities: multiple-shaped nuohan inferior yeast AP23 bacterial strain is in 5 L fermentors using fermentation training
It supports base and carries out mycoprotein production, wherein fermentation medium liquid amount is 2L, 121 DEG C of 15 min of sterilizing, while to inoculated tube
Road, ammonium hydroxide and methanol feeding pipeline, sample tap pipeline, air filter sterilize, and are down to 37 DEG C or so to fermentation medium
When, pH to 5.0 is adjusted with ammonium hydroxide, seed liquor is added and ferments, fermentation temperature is 37 DEG C, controls dissolved oxygen 20%-60%, and stirring turns
Fast 250-500 rpm；After 24 hours, glycerol depletion in culture medium starts stream plus methanol, monitors methanol concentration with gas phase, makes it
Between 0.5%-2%, thallus OD is measured by sampling in every 8 hours for control600And weight in wet base, after 96-100 hours, fermentation ends are collected
Fermentation liquid.Fermentation liquid is concentrated through 5000 r/min continuous centrifugal of centrifuge then and obtains thallus, with claiming after clear water washing thalline
Measure its weight in wet base；The thallus of collection is dried to constant mass in 55 DEG C, collecting dry powder is single-cell methanol protein.
Final thallus 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 (4)
1. one plant of polymorpha for preparing high-lysine single cell protein using methanol, which is characterized in that classification naming
For multiple-shaped nuohan inferior yeast (Hansenulapolymorpha) AP23, Chinese Typical Representative culture is preserved on April 5th, 2016
Collection CCTCC, deposit number: CCTCC NO:M 2016173.
2. application of the polymorpha described in claim 1 in the preparation of high-lysine single cell protein.
3. application of the polymorpha in the preparation of high-lysine single cell protein according to claim 2, feature
It is, includes the following steps:
(1) seed culture: by multiple-shaped nuohan inferior yeast AP23 strain inoculated to seed slant medium, 37 DEG C are cultivated 48 hours
Afterwards, cultured inclined-plane seed is taken to be seeded in the triangular flask comprising Selective agar medium, 37 DEG C, 220 rpm shaken cultivations about 24
Hour to OD=9-10；
(2) fermented and cultured: multiple-shaped nuohan inferior yeast AP23 bacterial strain uses fermentation medium to carry out mycoprotein production in the fermenter,
121 DEG C of 15 min of sterilizing adjust pH to 5.0 when fermentation medium is down to 37 DEG C, and seed liquor is added and ferments, 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 starts to flow
Add methanol, monitor methanol concentration with gas phase, makes its control between 0.5%-2%, thallus OD is measured by sampling in every 8 hours600With it is wet
Weight, after 96-100 hours, fermentation ends collect fermentation liquid；
(3) it extracts albumen: fermentation liquid being concentrated through 5000 r/min continuous centrifugal of centrifuge and obtains thallus, with clear water washing thalline
After weigh its weight in wet base；The thallus of collection is dried to constant mass in 55 DEG C, collects dry powder.
4. application of the polymorpha in the preparation of high-lysine single cell protein according to claim 3, feature
It is, as follows using medium component:
Seed slant medium: 10 g/L of yeast powder, 20 g/L of peptone, 20 g/L of glucose, 20 g/L of agar powder, remaining
For water；
Selective agar medium: 500 mM of methanol, 1 g/L of ammonium sulfate, 1 g/L of potassium dihydrogen phosphate, 0.7 g/L of magnesium sulfate, calcium chloride 0.4
G/L, 1 g/L of yeast extract, remaining is water, and pH is natural；
Fermentation medium: 85% phosphoric acid, 26.7 ml/L, 0.93 g/L of calcium sulphate dihydrate, 18.2 g/L of potassium sulfate, sulfate dihydrate magnesium
14.9 g/L, 4.13 g/L of potassium hydroxide, 30 g/L of glycerol, 4 ml/L of trace element solution, 1 L of water；
The trace element solution are as follows: 6 g/L of cupric sulfate pentahydrate, 0.088 g/L of potassium iodide, 3 g/L of manganese sulfate monohydrate, two water
0.2 g/L of sodium molybdate, 0.02 g/L of boric acid, 0.5 g/L of CoCL2 6H2O, 20 g/L of zinc chloride, 65 g/L of ferrous sulfate heptahydrate,
0.2 g/L of biotin, mass concentration are 5 ml/L of the concentrated sulfuric acid of 98 %, remaining is water.
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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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| CN112646740B (en) * | 2020-11-27 | 2021-11-30 | 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 |
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| US4414329A (en) * | 1980-01-15 | 1983-11-08 | Phillips Petroleum Company | Biochemical conversions by yeast fermentation at high cell densities |
| CN101457206A (en) * | 2008-05-28 | 2009-06-17 | 中国农业科学院饲料研究所 | Acidic xylanase XYL10A and gene and application thereof |
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| US4242458A (en) * | 1978-10-25 | 1980-12-30 | Texaco Development Corporation | Biosynthesis of protein by fermentation of methanol obtained from the gasification of coal or residual oil |
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| CN101457206A (en) * | 2008-05-28 | 2009-06-17 | 中国农业科学院饲料研究所 | Acidic xylanase XYL10A and gene and application thereof |
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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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