CN106676019B - A method of utilizing Production of Astaxanthin from Fermentation by Microorganisms - Google Patents
A method of utilizing Production of Astaxanthin from Fermentation by Microorganisms Download PDFInfo
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- CN106676019B CN106676019B CN201611059924.4A CN201611059924A CN106676019B CN 106676019 B CN106676019 B CN 106676019B CN 201611059924 A CN201611059924 A CN 201611059924A CN 106676019 B CN106676019 B CN 106676019B
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- 238000000855 fermentation Methods 0.000 title claims abstract description 62
- 230000004151 fermentation Effects 0.000 title claims abstract description 62
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 56
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 56
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 56
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 56
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 244000005700 microbiome Species 0.000 title abstract description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 48
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- 239000007788 liquid Substances 0.000 claims description 23
- 238000011218 seed culture Methods 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 19
- 238000013019 agitation Methods 0.000 claims description 14
- 239000006052 feed supplement Substances 0.000 claims description 14
- 229940088594 vitamin Drugs 0.000 claims description 14
- 235000013343 vitamin Nutrition 0.000 claims description 14
- 239000011782 vitamin Substances 0.000 claims description 14
- 229930003231 vitamin Natural products 0.000 claims description 14
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 239000007836 KH2PO4 Substances 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 229910052751 metal Inorganic materials 0.000 claims description 7
- 239000002184 metal Substances 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
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000012531 culture fluid Substances 0.000 claims description 6
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 6
- 229960000367 inositol Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 241000235342 Saccharomycetes Species 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 4
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 3
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 3
- 241000235015 Yarrowia lipolytica Species 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- 229960000304 folic acid Drugs 0.000 claims description 3
- 235000019152 folic acid Nutrition 0.000 claims description 3
- 239000011724 folic acid Substances 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 3
- 235000008160 pyridoxine Nutrition 0.000 claims description 3
- 239000011677 pyridoxine Substances 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- 239000000908 ammonium hydroxide Substances 0.000 claims description 2
- 229910021645 metal ion Inorganic materials 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- 229910001868 water Inorganic materials 0.000 claims description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 claims 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 claims 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims 2
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims 2
- 239000001110 calcium chloride Substances 0.000 claims 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims 2
- 229940055726 pantothenic acid Drugs 0.000 claims 2
- 235000019161 pantothenic acid Nutrition 0.000 claims 2
- 239000011713 pantothenic acid Substances 0.000 claims 2
- 239000011684 sodium molybdate Substances 0.000 claims 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims 2
- 239000011686 zinc sulphate Substances 0.000 claims 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims 1
- 239000006071 cream Substances 0.000 claims 1
- 238000002386 leaching Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000005070 sampling Methods 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000235013 Yarrowia Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- RASZIXQTZOARSV-BDPUVYQTSA-N astacin Chemical compound CC=1C(=O)C(=O)CC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)C(=O)CC1(C)C RASZIXQTZOARSV-BDPUVYQTSA-N 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 102000034498 Astacin Human genes 0.000 description 1
- 108090000658 Astacin Proteins 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- FMKGDHLSXFDSOU-BDPUVYQTSA-N Dienon-Astacin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)C(=O)C(=CC1(C)C)O)C=CC=C(/C)C=CC2=C(C)C(=O)C(=CC2(C)C)O FMKGDHLSXFDSOU-BDPUVYQTSA-N 0.000 description 1
- 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 description 1
- 241000168517 Haematococcus lacustris Species 0.000 description 1
- 229910017906 NH3H2O Inorganic materials 0.000 description 1
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000081271 Phaffia rhodozyma Species 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 102220594896 Vasopressin-neurophysin 2-copeptin_M20A_mutation Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 235000003676 astacin Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 241000238565 lobster Species 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 150000003505 terpenes Chemical group 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 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
- C12N1/145—Fungal isolates
-
- 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
-
- 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
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods using Production of Astaxanthin from Fermentation by Microorganisms, belong to bioengineering fermentation technical field.The present invention utilizes Production of Astaxanthin from Fermentation by Microorganisms, and the yield of astaxanthin reaches 450mg/L, and the raw material utilized is less expensive, and production cost substantially reduces, and provides effective method for fast and efficient production natural astaxanthin.
Description
Technical field
The present invention relates to a kind of methods using Production of Astaxanthin from Fermentation by Microorganisms, belong to bioengineering fermentation technique neck
Domain.
Background technique
Astaxanthin (3,3 '-dihydroxy -4,4 '-diketo-β, β '-carrotene, also known as astacin), chemical molecular formula is
C40H52O4, it is terpenes unsaturated compounds, is a kind of natural carotenoid being widespread in nature, many crusts
Animal such as shrimp, lobster, crab etc. all contains astaxanthin in vivo, and astaxanthin sheet, can be with when it and protein combine as red
Show it is dark blue or green, and in process due to heat destroy astaxanthin and protein combination and restored shrimp blueness
The original color of element.A large number of studies show that, astaxanthin antioxidant activity with super strength in strengthen immunity, inhibits both at home and abroad
Tumour, inhibit diabetic nephropathy, remove free radical and active oxygen etc. have the function of it is very positive.It is served not only as
The additive of bait of precious aquaculture of aquatic animal and the food additives of the mankind, in drug, cosmetics and high-grade nutrient health care product
Equal fields also have great application potential.
Currently, the production technology of astaxanthin mainly have from processing of aquatic products waste extract astaxanthin, using algae such as
The production such as haematococcus pluvialis astaxanthin utilizes red phaffia rhodozyma fermenting and producing astaxanthin and chemical synthesis etc..Chemical synthesis
Astaxanthin with natural astaxanthin structure, function, application and in terms of have significant difference, U.S.'s food and drug
Management board has forbidden chemically synthesized astaxanthin to enter health food market.Currently, the production of astaxanthin generally tends to develop
The biological source of natural astaxanthin.
The main source of natural astaxanthin has aquatic products waste, phaffiafhodozyma and microalgae, due to waste of aquatic and
Content astaxanthin is lower in microalgae, and extraction is costly, is unsuitable for being mass produced.
Summary of the invention
Present invention firstly provides a kind of saccharomycete (Yarrowia lipolytica) that can be used for producing natural astaxanthin
As is preserved in China typical culture collection center on November 17th, 2016, and deposit number is CCTCC NO:M
2016644, preservation address is Wuhan, China Wuhan University.
The present invention also provides the methods that the application microbial fermentation produces astaxanthin, comprising the following steps:
(1) it prepares seed culture fluid: the bacterial strain of preservation is inoculated in liquid seed culture medium, at 28~30 DEG C, 180
Under the conditions of~200rpm/min, culture 20~for 24 hours, obtain seed culture fluid；
(2) fermentation produces astaxanthin: seed culture fluid is inoculated in fermentation medium in 1~10% ratio, tank temperature 28~
30 DEG C, ventilation quantity: 1.0-5.0VVM, pH: sodium hydroxide or ammonium hydroxide control pH are 4.0~6.0, incubation time: 55~75 hours.
In one embodiment of the invention, Do-stat mode feed supplement is begun to take after fermentation fermentation 20-30h, controlled
Dissolved oxygen is not less than 10~30%.Feed-batch culture based formulas are as follows: glycerol 500g/L, KH2PO45g/L、MgSO43g/L.Ferment 55-75h
Terminate, it is 400mg/L, purity 82.6% that content astaxanthin in fermentation liquid, which is measured by sampling,.
In one embodiment of the invention, fermentative medium formula are as follows: weigh glycerol 120g, yeast extract 10.5g,
KH2PO46.75g、MgSO42.1g、(NH4)2SO41500mL deionized water, 121 DEG C, 20mins are added in 22.8g, inositol 0.15g
Mixed vitamin solution 10mL/L, mixed metal solion 10mL/L is added in sterilizing.Mixed vitamin solution composition are as follows: general
Acid: 5.4g/L, pyridoxol: 1.4g/L, niacin: 6.1g/L, folic acid: 0.04g/L, biotin: 0.06g/L, filtration sterilization.Mixing
Metal ion solution composition are as follows: Fe3Cl3.6H2O:2.7g/L、ZnSO4.7H2O:1.4g/L、CaCl2.2H2O:2.0g/L、
Na2MoO4.2H2O:2.0g/L、CuSO4.2H2O:1.9g/L、H3Bo3:0.5g/L、MnSO4.H2O:2.0g/L, 121 DEG C, 20mins
Sterilizing.
In one embodiment of the invention, seed culture based formulas are as follows: weigh glycerol 2g, yeast extract 0.3g,
KH2PO40.45g、MgSO40.2g、(NH4)2SO42.4g, inositol 0.01g, are added 100mL deionized water, and 121 DEG C, 20mins goes out
Mixed vitamin solution 10mL/L, mixed metal solion 10mL/L is added in bacterium.
In one embodiment of the invention, inoculum concentration is 10% when fermentation.
In one embodiment of the invention, fermentation temperature is 30 DEG C.
In one embodiment of the invention, it is 4.0 that pH is controlled when fermentation.
In one embodiment of the invention, 28 DEG C of 3L fermentor initial temperature, agitation revolution 200rpm, ventilating ratio
It is cultivated under the conditions of 1L/min；Enter feed supplement period after fermentation 20-26h, agitation revolution is improved to 1200rpm, divulged information as 1-5L/
Min controls pH4.0 with NaOH, starts feed supplement.
In one embodiment of the invention, fermentation initial stage controls pH4.0 with NaOH and uses instead into feed supplement period
NH3H2O controls pH4.0.
The present invention is using Production of Astaxanthin from Fermentation by Microorganisms, and advantage is compared with prior art, the yield of astaxanthin
It is effectively improved, is 450mg/L, the raw material utilized is less expensive, and production cost substantially reduces, rapidly and efficiently to give birth to
It produces natural astaxanthin and effective method is provided.
Biomaterial preservation
A kind of saccharomycete (Yarrowia lipolytica) As can be used for producing natural astaxanthin, November 17 in 2016
Day, it is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2016644, and preservation address is that China is military
Chinese Wuhan University.
Detailed description of the invention
Fig. 1 is the HPLC map of astaxanthin.
Fig. 2 is the yield of astaxanthin under the conditions of different vaccination amount.
Fig. 3 is the yield of astaxanthin under condition of different pH.
Fig. 4 is the yield of astaxanthin under the conditions of control by stages pH
Fig. 5 is the yield of astaxanthin under the conditions of different passage numbers.
Specific embodiment
Analysis method: using the astaxanthin in high-efficient liquid phase chromatogram technique analysis fermentation liquid.Wherein, measurement uses Shimadzu SPD-
M20A diode array detector, Phenomenex luna 5u C18 chromatographic column (150mm × 4.6mm, 5 μ)；Mobile phase 85%
Acetonitrile: 15% isopropanol；Flow velocity 1.5ml/min；35 DEG C of column temperature；10 μ L of sample volume.The measurement of glycerol uses Composition distribution；
Phenomenex luna 5u NH2 chromatographic column (250mm × 4.6mm, 5 μ), 30 DEG C of column temperature；35 DEG C of RID temperature；Mobile phase 85%
Acetonitrile, flow velocity 1.0ml/min；10 μ L of sample volume.
The culture of 1 seed liquor of embodiment
(1) the preparation of fermenting microbe: going bail for -80 DEG C of presence of Yarrowia strain in solid medium, scribing line culture,
28 DEG C~30 DEG C of cultivation temperature, cultivate 24~48h.
The preparation method of solid medium: ingredient be yeast extract 1g, peptone 2g, glucose 2g, agar powder 1.5g, so
It is cooling that solid plate is made in 121 DEG C of temperature, 15~20min of high pressure steam sterilization afterwards with deionized water constant volume to 100ml.
(2) seed liquor culture: glycerol tube preservation bacterium is inoculated in seed liquor, in 30 DEG C of temperature, revolving speed 200r/min, training
Support 20~26h, culture to logarithmic phase.
The preparation method of seed culture medium: ingredient is glycerol 2g, yeast extract 0.3g, KH2PO40.45g、MgSO40.2g、
(NH4)2SO4Then 2.4g, inositol 0.01g are settled to 100ml with deionized water, in 121 DEG C of temperature, high pressure steam sterilization
20min is added mixed vitamin solution 1mL, mixed metal solion 1mL after cooling, seed culture medium is made.
2 shake flask fermentation culture of embodiment
Yarrowia seed liquor is inoculated in fermentation medium by inoculum concentration 10% (V/V) in Example 1, in temperature 28
DEG C~30 DEG C, 20~30h of revolving speed 200r/min training.
Fermentation medium preparation: ingredient is glycerol 120g, yeast extract 10.5g, KH2PO46.75g、MgSO42.1g、
(NH4)2SO422.8g, inositol 0.15g, then with deionized water constant volume to 1.5L, in 121 DEG C of temperature, high pressure steam sterilization
20min is added mixed vitamin solution 15mL, mixed metal solion 15mL after cooling, fermentation liquid is made.
3 3L fermentor of embodiment produces astaxanthin
(1) take glycerol tube to save bacterial strain, be inoculated in the 500ml triangular flask equipped with 50ml seed culture medium, 30 DEG C,
20~30h of 200rpm shaking table culture.Seed culture based formulas and embodiment 1 are identical.
(2) cultured seed liquor is inoculated in 3L fermentor by 10% inoculum concentration, starts to ferment, fermentative medium formula
Identical with embodiment 2,3L fermentor initial liquid amount 1.5L, 121 DEG C of sterilizing 20min, wherein vitamin mixture is filtered out with crossing
Bacterium mode is added.
(3) 28 DEG C of 3L fermentor initial temperature are cultivated under the conditions of agitation revolution 200rpm, ventilating ratio 1L/min, agitation revolution
It improves to 1200rpm, divulges information and begin to take Do-stat mode after the 20-30h that ferments with NaOH control pH4.0 for 1-5L/min
Feed supplement, feed-batch culture based formulas are as follows: glycerol 500g/L, KH2PO45g/L、MgSO43g/L.Fermentation 55-75h terminates, and is measured by sampling
Content astaxanthin is 400mg/L, purity 82.6% in fermentation liquid.
Astaxanthin is produced under the conditions of 4 3L fermentor different vaccination amount of embodiment
(1) take glycerol tube to save bacterial strain, be inoculated in the 500ml triangular flask equipped with 50ml seed culture medium, 30 DEG C,
20~30h of 200rpm shaking table culture.Seed culture based formulas and embodiment 1 are identical.
(2) cultured seed liquor is inoculated in 3L fermentor, starts to ferment, 2 phase of fermentative medium formula and embodiment
Together, the initial liquid amount 1.5L of 3L fermentor, 121 DEG C of sterilizing 20min, wherein vitamin mixture is added in a manner of filtration sterilization.
(3) 28 DEG C of 3L fermentor initial temperature are cultivated under the conditions of agitation revolution 200rpm, ventilating ratio 1L/min, agitation revolution
It improves to 1200rpm, divulges information and begin to take Do-stat mode after the 20-26h that ferments with NaOH control pH4.0 for 1-5L/min
Feed supplement.Fermentation 55-75h terminates.
Inoculum concentration 1%, fermentation to 56h are measured by sampling content astaxanthin in fermentation liquid and reach up to 252mg/L, purity
It is 62.7%.
Inoculum concentration 5%, fermentation to 56h are measured by sampling content astaxanthin in fermentation liquid and reach up to 317mg/L, purity
It is 52.2%.
Inoculum concentration 10%, fermentation to 56h are measured by sampling content astaxanthin in fermentation liquid and reach up to 335mg/L, purity
It is 62.7%.
Astaxanthin is produced under 5 3L fermentor condition of different pH of embodiment
(1) take glycerol tube to save bacterial strain, be inoculated in the 500ml triangular flask equipped with 50ml seed culture medium, 30 DEG C,
20~30h of 200rpm shaking table culture.Seed culture based formulas and embodiment 1 are identical.
(2) cultured seed liquor is inoculated in 3L fermentor by 10% inoculum concentration, starts to ferment, fermentative medium formula
Identical with embodiment 2,3L fermentor initial liquid amount 1.5L, 121 DEG C of sterilizing 20min, wherein vitamin mixture is filtered out with crossing
Bacterium mode is added.
(3) 28 DEG C of 3L fermentor initial temperature are cultivated under the conditions of agitation revolution 200rpm, ventilating ratio 1L/min, agitation revolution
It improves to 1200rpm, divulges information as 1-5L/min, begin to take Do-stat mode feed supplement after the 20-26h that ferments.Ferment 65-75h knot
Beam.
Fermentation process NaOH controls pH4.0, and fermentation to 65h is measured by sampling content astaxanthin in fermentation liquid and reaches
183.3mg/L, purity 83.7%.
Fermentation process NaOH controls pH5.0, and fermentation to 65h is measured by sampling content astaxanthin in fermentation liquid and reaches highest
For 164.8mg/L, purity 83.3%.
Fermentation process NaOH controls pH6.0, and fermentation to 65h is measured by sampling content astaxanthin in fermentation liquid and reaches highest
For 89.9mg/L, purity 73.4%.
6 3L fermentor control by stages pH of embodiment produces astaxanthin
(1) take glycerol tube to save bacterial strain, be inoculated in the 500ml triangular flask equipped with 50ml seed culture medium, 30 DEG C,
20~30h of 200rpm shaking table culture.Seed culture based formulas and embodiment 1 are identical.
(2) cultured seed liquor is inoculated in 3L fermentor, starts to ferment, 2 phase of fermentative medium formula and embodiment
Together, the initial liquid amount 1.5L of 3L fermentor, 121 DEG C of sterilizing 20min, wherein vitamin mixture is added in a manner of filtration sterilization.
(3) 28 DEG C of 3L fermentor initial temperature are cultivated under the conditions of agitation revolution 200rpm, ventilating ratio 1L/min, agitation revolution
It improves to 1200rpm, divulges information as 1-5L/min, begin to take Do-stat mode feed supplement after the 20-26h that ferments.Ferment 75-80h knot
Beam.
(4) fermentation initial stage controls pH4.0 with NaOH and uses NH instead into feed supplement period3H2O controls pH4.0.After fermentation,
It is 450mg/L, purity 78% that content astaxanthin in fermentation liquid, which is measured by sampling,.
Astaxanthin is produced under the conditions of embodiment 73L fermentor difference passage number
(1) take glycerol tube to save bacterial strain, be inoculated in the 500ml triangular flask equipped with 50ml seed culture medium, 30 DEG C,
20~30h of 200rpm shaking table culture.Seed culture based formulas and embodiment 1 are identical.
(2) cultured seed liquor is inoculated in 3L fermentor, starts to ferment, 2 phase of fermentative medium formula and embodiment
Together, the initial liquid amount 1.5L of 3L fermentor, 121 DEG C of sterilizing 20min, wherein vitamin mixture is added in a manner of filtration sterilization.
(3) 28 DEG C of 3L fermentor initial temperature are cultivated under the conditions of agitation revolution 200rpm, ventilating ratio 1L/min, agitation revolution
It improves to 1200rpm, divulges information and begin to take Do-stat mode after the 20-26h that ferments with NaOH control pH4.0 for 1-5L/min
Feed supplement.Fermentation 75-80h terminates.
(4) generation seed, after fermentation, it is 330mg/L that content astaxanthin in fermentation liquid, which is measured by sampling, and purity is
85.3%.
(5) five generation seed, after fermentation, it is 320mg/L that content astaxanthin in fermentation liquid, which is measured by sampling, and purity is
82.6%.
The extraction of astaxanthin and HPLC detection in 8 fermentation liquid of embodiment
Fermentation liquid 20 μ L-200 μ L, 12000r/min are taken, centrifugation 5min removes supernatant, 0.5mL methanol and 0.5mL chlorine is added
Imitative, whirlpool shakes 10mins, is centrifuged 10mins, takes supernatant, and organic membrane filtration carries out HPLC detection.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (9)
1. a kind of saccharomycete (Yarrowia lipolytica) As that can be used for producing natural astaxanthin, on November 17th, 2016,
It is preserved in China typical culture collection center, deposit number is CCTCC NO:M 2016644, and preservation address is Wuhan, China,
Wuhan University.
2. a kind of method for producing astaxanthin using the fermentation of saccharomycete As described in claim 1, which is characterized in that including following step
It is rapid:
(1) it prepares seed culture fluid: saccharomycete As is inoculated in liquid seed culture medium, at 28~30 DEG C, 180~200rpm
Under the conditions of, culture 20~for 24 hours, obtain seed culture fluid；
(2) fermentation produces astaxanthin: seed culture fluid is inoculated in fermentation medium in 1~10% ratio, 28~30 DEG C of tank temperature,
Ventilation quantity 1.0-5.0vvm, with sodium hydroxide or ammonium hydroxide control pH 4.0~6.0, fermentation time is 55~75 hours.
3. according to the method described in claim 2, it is characterized in that, fermentation 20-30h after begin to take Do-stat mode feed supplement,
It controls dissolved oxygen and is not less than 10%；Feed-batch culture based formulas are as follows: glycerol 500g/L, KH2PO4 5g/L、MgSO4 3g/L。
4. according to the method described in claim 2, it is characterized in that, fermentative medium formula are as follows: weigh glycerol 120g, yeast leaching
Cream 10.5g, KH2PO4 6.75g、MgSO4 2.1g、(NH4)2SO41500mL deionized water is added in 22.8g, inositol 0.15g,
121 DEG C, 20min sterilizing, are added mixed vitamin solution 10mL/L, mixed metal solion 10mL/L, mixed vitamin is molten
Liquid composition are as follows: pantothenic acid 5.4g/L, pyridoxol 1.4g/L, niacin 6.1g/L, folic acid 0.04g/L, biotin 0.06g/L are crossed and filtered out
Bacterium；Mixed metal solion composition are as follows: FeCl3·6H2O 2.7g/L、ZnSO4·7H2O1.4g/L、CaCl2·2H2O
2.0g/L、Na2MoO4·2H2O 2.0g/L、CuSO4·2H2O1.9g/L、H3BO3 0.5g/L、MnSO4·H2O2.0g/L, 121
DEG C, 20min sterilizing.
5. according to the method described in claim 2, it is characterized in that, seed culture based formulas are as follows: weigh glycerol 2g, yeast extract
0.3g、KH2PO4 0.45g、MgSO4 0.2g、(NH4)2SO42.4g, inositol 0.01g, addition 100mL deionized water, 121 DEG C,
20min sterilizing, is added mixed vitamin solution 10mL/L, mixed metal solion 10mL/L, mixed vitamin solution composition
Are as follows: pantothenic acid 5.4g/L, pyridoxol 1.4g/L, niacin 6.1g/L, folic acid 0.04g/L, biotin 0.06g/L, filtration sterilization；Mixing
Metal ion solution composition are as follows: FeCl3·6H2O 2.7g/L、ZnSO4·7H2O1.4g/L、CaCl2·2H2O2.0g/L、
Na2MoO4·2H2O 2.0g/L、CuSO4·2H2O1.9g/L、H3BO3 0.5g/L、MnSO4·H2O 2.0g/L, 121 DEG C,
20min sterilizing.
6. according to the method described in claim 3, it is characterized in that, 28 DEG C, agitation revolution 200rpm of 3L fermentor initial temperature,
It ferments under the conditions of ventilating ratio 1L/min；Enter feed supplement period after fermentation 20-26h, agitation revolution is improved to 1200rpm, and divulging information is
1-5L/min is 4.0 with NaOH control pH, starts feed supplement.
7. according to the method described in claim 6, it is characterized in that, it is 4.0 that fermentation initial stage, which controls pH with NaOH, into when feed supplement
Phase uses NH instead3H2It is 4.0 that O, which controls pH,.
8. according to the method in claim 2 or 3, which is characterized in that inoculum concentration is 10% when fermentation, and control pH is when fermentation
4.0。
9. according to the method in claim 2 or 3, which is characterized in that fermentation temperature is 30 DEG C or 28 DEG C.
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| CN103589650A (en) * | 2005-03-18 | 2014-02-19 | 米克罗比亚公司 | Production of carotenoids in oleaginous yeast and fungi |
| WO2008073367A1 (en) * | 2006-12-12 | 2008-06-19 | E. I. Du Pont De Nemours And Company | Carotenoid production in a recombinant oleaginous yeast |
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