CN113862210B - Method for promoting Phaffia rhodozyma to produce astaxanthin in high yield - Google Patents
Method for promoting Phaffia rhodozyma to produce astaxanthin in high yield Download PDFInfo
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- CN113862210B CN113862210B CN202110510672.7A CN202110510672A CN113862210B CN 113862210 B CN113862210 B CN 113862210B CN 202110510672 A CN202110510672 A CN 202110510672A CN 113862210 B CN113862210 B CN 113862210B
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- phaffia rhodozyma
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- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 99
- 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 98
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 98
- 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 98
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 98
- 241000081271 Phaffia rhodozyma Species 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 35
- 230000001737 promoting effect Effects 0.000 title claims abstract description 19
- 238000000855 fermentation Methods 0.000 claims abstract description 167
- 230000004151 fermentation Effects 0.000 claims abstract description 167
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000007788 liquid Substances 0.000 claims abstract description 41
- 229910010413 TiO 2 Inorganic materials 0.000 claims abstract description 29
- 229910052593 corundum Inorganic materials 0.000 claims abstract description 25
- 229910001845 yogo sapphire Inorganic materials 0.000 claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 claims abstract description 23
- 239000006185 dispersion Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- SOQBVABWOPYFQZ-UHFFFAOYSA-N oxygen(2-);titanium(4+) Chemical compound [O-2].[O-2].[Ti+4] SOQBVABWOPYFQZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 230000037353 metabolic pathway Effects 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 42
- 239000001963 growth medium Substances 0.000 claims description 30
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 28
- 239000002609 medium Substances 0.000 claims description 27
- 238000003756 stirring Methods 0.000 claims description 23
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 19
- 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 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 229940041514 candida albicans extract Drugs 0.000 claims description 18
- 239000008103 glucose Substances 0.000 claims description 18
- 239000012138 yeast extract Substances 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 17
- 238000011218 seed culture Methods 0.000 claims description 16
- 235000013970 phaffia yeast Nutrition 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
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 238000009423 ventilation Methods 0.000 claims description 12
- 239000010413 mother solution Substances 0.000 claims description 11
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 8
- 210000005253 yeast cell Anatomy 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 239000012452 mother liquor Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 241001542817 Phaffia Species 0.000 abstract description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 22
- 230000003834 intracellular effect Effects 0.000 description 17
- 239000004408 titanium dioxide Substances 0.000 description 10
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- YVLPJIGOMTXXLP-UHFFFAOYSA-N 15-cis-phytoene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-N 0.000 description 6
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- 241000251468 Actinopterygii Species 0.000 description 5
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 235000019688 fish Nutrition 0.000 description 5
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- 238000009835 boiling Methods 0.000 description 4
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- 238000001914 filtration Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
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- 235000019515 salmon Nutrition 0.000 description 4
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- 238000012360 testing method Methods 0.000 description 4
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 description 3
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- VWFJDQUYCIWHTN-UHFFFAOYSA-N farnesyl diphosphate Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCOP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-UHFFFAOYSA-N 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
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- 235000011765 phytoene Nutrition 0.000 description 3
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- 235000013594 poultry meat Nutrition 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 description 2
- OINNEUNVOZHBOX-QIRCYJPOSA-K 2-trans,6-trans,10-trans-geranylgeranyl diphosphate(3-) Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O OINNEUNVOZHBOX-QIRCYJPOSA-K 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 2
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 2
- OINNEUNVOZHBOX-XBQSVVNOSA-N Geranylgeranyl diphosphate Natural products [P@](=O)(OP(=O)(O)O)(OC/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)O OINNEUNVOZHBOX-XBQSVVNOSA-N 0.000 description 2
- 241000168517 Haematococcus lacustris Species 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
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- 230000003859 lipid peroxidation Effects 0.000 description 2
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- 238000007254 oxidation reaction Methods 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
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- XDIYNQZUNSSENW-UUBOPVPUSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O XDIYNQZUNSSENW-UUBOPVPUSA-N 0.000 description 1
- WFCGYZPOBJZGDF-UHFFFAOYSA-N 2-methylbuta-1,3-diene;phosphono dihydrogen phosphate Chemical compound CC(=C)C=C.OP(O)(=O)OP(O)(O)=O WFCGYZPOBJZGDF-UHFFFAOYSA-N 0.000 description 1
- 241000881711 Acipenser sturio Species 0.000 description 1
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- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
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- 241001282110 Pagrus major Species 0.000 description 1
- 241000287502 Phoenicopteriformes Species 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
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- 235000006708 antioxidants Nutrition 0.000 description 1
- MQZIGYBFDRPAKN-UWFIBFSHSA-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=C\C=C(/C)\C=C\C1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-UWFIBFSHSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001409 beta-carotene group Chemical group 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 150000001749 carotenones Chemical class 0.000 description 1
- 235000005472 carotenones Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 230000001276 controlling effect Effects 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 210000000514 hepatopancreas Anatomy 0.000 description 1
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- 125000000468 ketone group Chemical group 0.000 description 1
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- 210000003205 muscle Anatomy 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 229940005657 pyrophosphoric acid Drugs 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
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- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
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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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- Chemical & Material Sciences (AREA)
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- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for promoting Phaffia rhodozyma to produce astaxanthin in high yield, which is characterized by comprising the following steps of: the method comprises the following steps: inoculating Phaffia rhodozyma seed liquid into a sterile fermentation medium added with nano TiO 2/Al2O3 mother liquid for fermentation culture, wherein the nano TiO 2/Al2O3 mother liquid is nano titanium dioxide mixed liquid taking nano alumina aqueous dispersion liquid as a carrier, and the pH value imbalance inside and outside Phaffia rhodozyma cells is kept during fermentation, so that the stress reaction of Phaffia rhodozyma is excited, and the metabolic pathway in Phaffia rhodozyma body is carried out towards the direction of astaxanthin production. The invention has the advantages that: by using nano alumina water dispersion as carrier, nano titanium dioxide is treated and dissolved in fermentation medium, pH imbalance inside and outside Phaffia cells is maintained during fermentation, phaffia stress reaction is stimulated, metabolic pathway in Phaffia cells is conducted towards astaxanthin production direction, and astaxanthin content is increased.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for promoting Phaffia rhodozyma to produce astaxanthin in high yield.
Background
Astaxanthin (Astaxanthin) has chemical name of 3,3' -dihydroxy-4, 4' -diketo-beta, beta ' -carotene, molecular formula of C 40H52O4, relative molecular weight of 596.86, belongs to ketocarotenoid, and is a terpene unsaturated compound. Crystalline astaxanthin is pink, has a melting point of 215/216 ℃, is insoluble in water, has fat solubility, and is easily dissolved in most organic solvents such as chloroform, acetone, benzene and the like. The conjugated double bond chain in the astaxanthin molecular structure, and unsaturated ketone group and hydroxyl group at the tail end of the conjugated double bond chain can attract unpaired electrons of free radicals or provide electrons for the free radicals, so that the free radicals are removed, and the astaxanthin molecular structure plays an antioxidant role. The chemical structural formula of astaxanthin is shown in figure 1.
Astaxanthin exists mainly in both free and esterified forms. Astaxanthin in the free form is extremely unstable and is easily oxidized, and is usually chemically synthesized in the free form. The esterified astaxanthin is mainly formed by fatty acid and ester formed by one hydroxyl group in the end ring structure of astaxanthin, and astaxanthin on skin and shell of aquatic animals is mainly formed by lipidation, and free form on meat and viscera, wherein astaxanthin in rhodotorula and haematococcus pluvialis is mainly formed by esterification.
Astaxanthin has a strong oxidation resistance. Animal experiments show that the astaxanthin can remove NO 2, sulfide and disulfide, can reduce lipid peroxidation, and can effectively inhibit lipid peroxidation caused by free radicals. Meanwhile, the compound feed has anticancer activity, can obviously influence the immune function of animals, strengthen aerobic metabolism, obviously strengthen the muscle strength and tolerance of people, and has anti-infective activity. In the feed industry, astaxanthin is mainly used as a feed additive for crustaceans such as fish (salmon, sturgeon, rainbow trout, red sea bream, etc.), shrimp, crab, etc., and poultry. Astaxanthin can effectively protect the hepatopancreas and the like of aquatic animals, and enables the aquatic animals to present healthy and bright color. Astaxanthin has strong functions in preventing and treating diseases of fishes, shrimps, crabs and birds, can improve immunity and survival rate, and has important functions on normal growth and healthy cultivation, survival rate and reproductive rate. Astaxanthin also increases the flavor of fish, and can be used directly as a precursor compound for food flavor such as salmon and hilsa herring, or can promote the conversion of fatty acid or other lipid precursor into salmon flavor compound. The astaxanthin is added into the poultry feed to increase the pigment content of egg yolk, and can also improve the laying rate of hens and promote the health of laying hens.
At present, the production of astaxanthin mainly comprises two modes of chemical synthesis and microbial synthesis, and the chemically synthesized astaxanthin is high in price, and has obvious differences with natural astaxanthin in terms of molecular structure biological functions, application effects and biological safety performance. At present, methods for extracting astaxanthin by microbial fermentation synthesis are gradually dominant. Astaxanthin in nature is derived from algae, bacteria and phytoplankton. Some aquatic species, including crustaceans such as shrimp and crab, are predatory by fish such as salmon and forced fish, birds such as flamingos, chickens and ducks, and poultry, and pigments are stored in skin and adipose tissue to make their skin and feathers red in appearance due to long term consumption of these algae, bacteria and phytoplankton. The research shows that a plurality of algae such as snow algae, chlamydomonas, euglena, umbrella algae and the like contain astaxanthin, wherein the accumulation amount of the haematococcus pluvialis astaxanthin is higher than that of other green algae, and the algae are recognized as good biological sources for producing natural astaxanthin at present. The bacteria are affected by the factors of the bacteria, so that the bacteria have low utilization value. Phaffia is considered to be the most suitable source of astaxanthin in current fungal fermentation production. Extraction of astaxanthin from Phaffia rhodozyma is one of the main ways of producing astaxanthin.
Phaffia yeast is a natural astaxanthin-producing yeast, and trans-astaxanthin thereof has been approved by FDA in 2000 for use as a food additive. Phaffia yeast is mainly single cells, sometimes pseudohyphae are formed, the propagation mode is bud reproduction of asexual propagation, no asexual propagation exists, and cells not only contain abundant proteins, lipids and vitamins, but also contain a large amount of unsaturated fatty acids and precursors of various astaxanthin, and the growth temperature range is 0-27 ℃. The method for producing astaxanthin by using Phaffia rhodozyma is characterized in that: the Phaffia rhodozyma does not need illumination, can utilize various sugars as carbon sources to carry out quick heterotrophic metabolism, has short fermentation period and high production speed, can realize high-density culture in a fermentation tank, and can be used as bait, feed additive and the like after pigment extraction.
Microorganisms such as yeast, or E.coli, can synthesize astaxanthin in vivo using energy substances such as glucose or oil. A schematic diagram of the synthesis pathway of astaxanthin in Phaffia rhodozyma is shown in FIG. 2. After Glucose (Glucose) or other energy substances are ingested by the microorganism, the microorganism can be converted into a key metabolic intermediate Acetyl coenzyme A (Acetyl/CoA), isoprene pyrophosphoric acid (IPP) is synthesized through a mevalonate pathway (MVA), farnesyl pyrophosphoric acid (FPP) is generated through condensation reaction under the action of related enzymes, two molecules of FPP can be further condensed to generate ridge Niu Er of bovine pyrophosphoric acid (GGPP), two molecules of GGPP are further condensed to generate phytoene (Phytoene), and the phytoene is subjected to dehydrogenation and cyclization to generate beta/carotene, and is subjected to oxidation, ketonization and hydroxylation to generate the final product astaxanthin (Astaxanthin).
It is notable that the Phaffia rhodozyma strain cultivated under natural conditions has low astaxanthin yield and is easily degraded, and industrial production is impossible. Thus, the use of various mutagenesis methods to increase astaxanthin production is the first target of research. The methods adopted at present are ultraviolet mutagenesis, high-energy ray mutagenesis, chemical reagent mutagenesis, genetic engineering modification and the like. For example, Gong 2012(Gong,J.,Duan,N.,Zhao,X.,2012.Evolutionary engineering of Phaffia rhodozyma forastaxanthin/overproducing strain.Front.Chem.Sci.Eng.6(2),174–178.) discloses that the addition of micron-sized titanium dioxide (TiO 2) to Phaffia yeast medium can induce stress in the yeast, thereby over-expressing certain specific genes to yield astaxanthin. Although the literature reports that the addition of titanium dioxide can promote the study of the production of astaxanthin by Phaffia rhodozyma, the solid particle size of titanium dioxide mentioned in the literature is only on the order of microns, and the titanium dioxide on the order of microns is very low in solubility and almost insoluble in water because yeast fermentation is usually carried out in aqueous solution, so that the titanium dioxide on the order of microns cannot be efficiently delivered into yeast cells during fermentation, and therefore the astaxanthin content in Phaffia rhodozyma cultivated by the method reported in the literature is low.
Disclosure of Invention
The invention aims to provide a method for effectively promoting high-yield astaxanthin of Phaffia rhodozyma.
In order to achieve the above purpose, the invention adopts the following technical scheme: a method for promoting Phaffia rhodozyma to produce astaxanthin in high yield, which comprises the following steps: inoculating Phaffia rhodozyma seed liquid into a sterile fermentation medium added with nano TiO 2/Al2O3 mother liquid for fermentation culture, wherein the nano TiO 2/Al2O3 mother liquid is nano titanium dioxide mixed liquid taking nano alumina aqueous dispersion liquid as a carrier, and the pH value imbalance inside and outside Phaffia rhodozyma cells is kept during fermentation, so that the stress reaction of Phaffia rhodozyma is excited, and the metabolic pathway in Phaffia rhodozyma body is carried out towards the direction of astaxanthin production.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: 100-200 mg of nano TiO 2/Al2O3 mother liquor is added into each liter of sterile fermentation medium, the pH value of the sterile fermentation medium is kept to be 5.2-7.8, the pH value in Phaffia cells is lower than the pH value outside the cells during fermentation, and the pH difference value between the pH value in Phaffia cells and the pH value outside the cells during fermentation is kept to be-2.0 to-1.4.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: the preparation method of the nano TiO 2/Al2O3 mother solution comprises the following steps: mixing nano titanium dioxide into nano alumina aqueous dispersion, uniformly stirring to obtain nano TiO 2/Al2O3 mother liquor, wherein the nano TiO2/Al2O3 mother liquor contains 18-22 wt% of nano alumina particles, 1-2 wt% of nano titanium dioxide and 76-81 wt% of water, the nano titanium dioxide solid particles have a size of 40-80 nanometers, and the particle size of the nano alumina particles is 5-10 nanometers.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: 2-5 mol/L NaOH aqueous solution and 1-2 mol/L hydrochloric acid aqueous solution are used for controlling and adjusting the pH value of the sterile fermentation medium.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: the preparation method of the Phaffia rhodozyma seed liquid comprises the following steps: inoculating Phaffia rhodozyma into a seed culture medium for fermentation to prepare Phaffia rhodozyma seed solution, wherein the seed culture medium generally adopts a yeast extract peptone glucose culture medium, and the seed culture medium comprises the following components: 1-3% of glucose, 1-3% of yeast extract and 1-2% of peptone.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: the seed fermentation process is carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the Phaffia rhodozyma seed liquid is controlled at 15-25 ℃.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: the aseptic fermentation medium comprises the following components: 10-100 g/L glucose, 0.5-1 g/L yeast extract, 0.5-1 g/L magnesium sulfate, 2-6 g/L ammonium sulfate, 1-3 g/L monopotassium phosphate, 1-3 g/L dipotassium phosphate and 0.1-0.2 g/L calcium chloride.
Further, the method for promoting high-yield astaxanthin production by Phaffia rhodozyma comprises the following steps: inoculating Phaffia rhodozyma seed liquid into a sterile fermentation culture medium, and then fermenting and culturing in a fermentation tank for 48-72 hours; wherein the temperature of fermentation liquor in the fermentation tank is controlled at 20 ℃, the initial stirring rotation speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation amount in the fermentation tank is 0.5-2 vvm, and the dissolved oxygen in the fermentation liquor is ensured to be 20% -60% by setting the stirring rotation speed and ventilation amount in the whole fermentation culture process.
Through implementation of the technical prescription decide on a verdict, the invention has the following beneficial effects: according to the invention, the nano-alumina aqueous dispersion is used as a carrier to treat and dissolve nano-scale titanium dioxide into a fermentation medium, so that the nano-scale titanium dioxide can be efficiently delivered into yeast cells during fermentation, further, the pH value in the cells can be rapidly regulated, the imbalance of the pH value inside and outside the cells of Phaffia rhodozyma is kept during fermentation, namely, the pH environment inside and outside the cells of Phaffia rhodozyma is changed, so that the pH value gradient inside and outside the cells of Phaffia rhodozyma is caused, further, the stress reaction of Phaffia rhodozyma is stimulated, the metabolic pathway in the Phaffia rhodozyma is carried out towards the direction of producing astaxanthin, and the astaxanthin content in the Phaffia rhodozyma is effectively increased.
Drawings
FIG. 1 is a chemical structural formula of astaxanthin.
FIG. 2 is a schematic representation of the astaxanthin synthesis pathway in Phaffia rhodozyma.
Detailed Description
The technical scheme of the invention is further described in detail below in connection with the preferred embodiments.
Comparative example one
The fermentation method of the invention can be applied to batch fermentation occasions and continuous fermentation occasions. This example describes the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermenter.
Firstly, inoculating Phaffia rhodozyma Phaffia rhodozyma (ATCC 24202) into 100ml of seed culture medium for fermentation to prepare Phaffia rhodozyma seed liquid; the seed culture medium is usually yeast extract peptone glucose culture medium, and the composition of the seed culture medium generally comprises: 2% of glucose, 1% of yeast extract, 2% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the Phaffia rhodozyma seed liquid is controlled at 15-20 ℃;
2L of a carbon source-free sterile fermentation medium subjected to high-pressure sterilization at 121 ℃ is put into a 5L fermentation tank, the high-pressure sterilization time is 30 minutes, and the pH value of the fermentation medium is controlled and regulated to 7.2 by using 5mol/L NaOH aqueous solution and 2mol/L hydrochloric acid aqueous solution; the aseptic fermentation medium described in this example comprises the following components: 80g/L glucose, 1g/L yeast extract, 1g/L magnesium sulfate, 6g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L dipotassium phosphate and 0.2g/L calcium chloride; specifically, 500mg/L of titanium dioxide (Allatin, CAS 13463/67/7, SKU#T164497) having a solid particle size of the order of microns was additionally added to the medium;
Inoculating the obtained Phaffia rhodozyma seed liquid into the aseptic fermentation culture medium with the constant pH value of 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of fermentation liquor in the fermentation tank is controlled at 15-25 ℃, the initial stirring rotation speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation amount in the fermentation tank is 0.5-2 vvm, and the dissolved oxygen in the fermentation liquor is ensured to be 20-60% by setting the stirring rotation speed and ventilation amount in the whole fermentation culture process;
When sampling after 72 hours fermentation was completed, the intracellular pH was tested at this time point using the intracellular pH test kit (purchased from abcam, intracellular pH Assay kit ab 228552) at a pH of 5.8 on the inner wall of the cell membrane; since the pH of the fermenter was kept constant at 7.2, it was calculated that there was a pH gradient ΔpH (intracellular pH/extracellular pH) inside and outside the cell membrane of about-1.4, i.e., the pH difference between the intracellular pH and the extracellular pH of Phaffia rhodozyma during fermentation was kept at-1.4;
Cell dry weight determination was performed after the whole 72 hour fermentation was completed: after weighing the fermentation broth, centrifuging at 4000rpm for 5 minutes, washing the obtained precipitate once with water, centrifuging at 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃ and weighing, and measuring the dry cell weight concentration in the fermentation broth to be 39g/L.
1 G of strain sample after fermentation for 72 hours is taken, after centrifugal washing, an equal volume of 3mol/L hydrochloric acid aqueous solution is added and heated in a boiling water bath for 5 minutes to cause cell wall rupture. The cells after wall breaking are extracted by acetone to obtain an acetone solution of astaxanthin. All acetone residues were distilled off at 45 degrees under dark conditions. Then using High Performance Liquid Chromatography (HPLC), the astaxanthin content was determined to be about 3.2mg/g dry cell weight; the astaxanthin content obtained by the measurement is multiplied by 3.2mg/g to obtain the cell dry weight concentration 39g/L of the fermentation liquid, and the concentration of the astaxanthin obtained by the batch fermentation is 124.8mg/L.
Example two
The fermentation method of the invention can be applied to batch fermentation occasions and continuous fermentation occasions. This example describes the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermenter.
First, 200mg of nano-titania (Allatin, CAS 13463/67/7, SKU#T104936) having a solid particle size of 60 nm was weighed out; in addition, 10mL of a nano-alumina aqueous dispersion (Allatin, CAS 1344/28/1, SKU#A119404) containing 20wt% of nano-alumina particles having a particle size of 5 to 10 nm was prepared; mixing 200mg of nano titanium dioxide into 10mL of nano alumina aqueous dispersion, and uniformly stirring to obtain 10mL of nano TiO 2/Al2O3 mother solution containing 200mg of nano titanium dioxide; (since the amount of 200mg is small, the volume is hardly increased after 10mL of nano alumina aqueous dispersion is added, and the prepared nano TiO 2/Al2O3 mother solution is also 10 mL);
Inoculating Phaffia rhodozyma Phaffia rhodozyma (ATCC 24202) into 100ml of seed culture medium for fermentation to prepare Phaffia rhodozyma seed liquid; the seed culture medium usually adopts yeast extract peptone glucose culture medium, and the composition of the seed culture medium usually comprises the following components: 1% of glucose, 1% of yeast extract, 1% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the Phaffia rhodozyma seed liquid is controlled at 15-25 ℃;
2L of a carbon source-free sterile fermentation medium subjected to high-pressure sterilization at 121 ℃ is put into a 5L fermentation tank, the high-pressure sterilization time is 30 minutes, and the pH value of the fermentation medium is controlled and regulated to 7.2 by using 2mol/L of NaOH aqueous solution and 1mol/L of hydrochloric acid aqueous solution; the aseptic fermentation medium described in this example comprises the following components: glucose 60g/L, yeast extract 0.5g/L, magnesium sulfate 0.5g/L, ammonium sulfate 2g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, and calcium chloride 0.1g/L; specifically, 10mL of prepared nano TiO 2/Al2O3 mother solution containing 200mg of nano titanium dioxide is added into the culture medium, so that 100mg/L of nano TiO 2 is added into the fermentation liquid;
Inoculating the obtained Phaffia rhodozyma seed liquid into the aseptic fermentation culture medium with the constant pH value of 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of fermentation liquor in the fermentation tank is controlled at 15-25 ℃, the initial stirring rotation speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation amount in the fermentation tank is 0.5-2 vvm, and the dissolved oxygen in the fermentation liquor is ensured to be 20-60% by setting the stirring rotation speed and ventilation amount in the whole fermentation culture process;
When sampling after 72 hours fermentation was completed, the intracellular pH was tested at this time point using the intracellular pH test kit (purchased from abcam, intracellular pH Assay kit ab 228552) at a pH of 5.3 on the inner wall of the cell membrane. Since the pH of the fermenter was kept constant at 7.2, it was calculated that the pH gradient ΔpH (intracellular pH/extracellular pH) inside and outside the cell membrane was about-1.9, i.e., the pH difference between the pH in the Phaffia yeast cell and the pH outside the cell during fermentation was kept at-1.9;
Cell dry weight determination was performed after the whole 72 hour fermentation was completed: after weighing the fermentation broth, centrifuging at 4000rpm for 5 minutes, washing the obtained precipitate once with water, centrifuging at 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃ and weighing, and measuring the dry cell weight concentration in the fermentation broth to be 36g/L.
1 G of strain sample after fermentation for 72 hours is taken, after centrifugal washing, an equal volume of 3mol/L hydrochloric acid aqueous solution is added and heated in a boiling water bath for 5 minutes to cause cell wall rupture. The cells after wall breaking are extracted by acetone to obtain an acetone solution of astaxanthin. All acetone residues were distilled off at 45 degrees under dark conditions. Then using High Performance Liquid Chromatography (HPLC), the astaxanthin content is determined to be about 12.3mg/g dry cell weight; the astaxanthin content obtained by the measurement is multiplied by 12.3mg/g to obtain 36g/L of cell dry weight concentration of the fermentation liquid, and the concentration of the astaxanthin obtained by the batch fermentation is 442.8mg/L.
Example III
The fermentation method of the invention can be applied to batch fermentation occasions and continuous fermentation occasions. This example describes the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermenter.
First, 400mg of nano-titania (Allatin, CAS 13463/67/7, SKU#T104936) having a solid particle size of 60 nm was weighed out; in addition, 10mL of a nano-alumina aqueous dispersion (Allatin, CAS 1344/28/1, SKU#A119404) containing 20wt% of nano-alumina particles having a particle size of 5 to 10 nm was prepared; mixing 300mg of nano titanium dioxide into 10mL of nano aluminum oxide aqueous dispersion liquid, and uniformly stirring to obtain 10mL of nano TiO 2/Al2O3 mother solution containing 300mg of nano TiO 2; (since the volume of 300mg is small, the volume is hardly increased after 10mL of nano alumina aqueous dispersion is added, and the prepared nano TiO 2/Al2O3 mother solution is also 10 mL);
Inoculating Phaffia rhodozyma Phaffia rhodozyma (ATCC 24202) into 100ml of seed culture medium for fermentation to prepare Phaffia rhodozyma seed liquid; the seed medium is usually YPD medium (Yeast extract peptone glucose medium), and its composition generally comprises: 3% of glucose, 3% of yeast extract, 2% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the Phaffia rhodozyma seed liquid is controlled at 15-25 ℃;
2L of a carbon source-free sterile fermentation medium subjected to high-pressure sterilization at 121 ℃ is put into a 5L fermentation tank, the high-pressure sterilization time is 30 minutes, and the pH value of the fermentation medium is controlled and regulated to 7.2 by using 5mol/L NaOH aqueous solution and 2mol/L hydrochloric acid aqueous solution; the aseptic fermentation medium described in this example comprises the following components: glucose 100g/L, yeast extract 1g/L, magnesium sulfate 1g/L, ammonium sulfate 6g/L, potassium dihydrogen phosphate 3g/L, dipotassium hydrogen phosphate 3g/L, and calcium chloride 0.2g/L; specifically, 10mL of prepared nano TiO 2/Al2O3 mother solution containing 300mg of nano TiO 2 is added into a culture medium, so that 150mg/L of nano TiO 2 is added into the fermentation liquid;
Inoculating the obtained Phaffia rhodozyma seed liquid into the aseptic fermentation culture medium with the constant pH value of 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of fermentation liquor in the fermentation tank is controlled at 15-25 ℃, the initial stirring rotation speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation amount in the fermentation tank is 0.5-2 vvm, and the dissolved oxygen in the fermentation liquor is ensured to be 20-60% by setting the stirring rotation speed and ventilation amount in the whole fermentation culture process;
When sampling after 72 hours fermentation was completed, the intracellular pH was tested at this time point using the intracellular pH test kit (purchased from abcam, intracellular pH Assay kit ab 228552) at a pH of 5.5 on the inner wall of the cell membrane. Since the pH of the fermenter was kept constant at 7.2, it was calculated that the pH gradient ΔpH (intracellular pH/extracellular pH) inside and outside the cell membrane was about-1.7, i.e., the pH difference between the pH in the Phaffia yeast cell and the pH outside the cell during fermentation was kept at-1.7;
Cell dry weight determination was performed after the whole 72 hour fermentation was completed: after weighing the fermentation broth, centrifuging at 4000rpm for 5 minutes, washing the obtained precipitate once with water, centrifuging at 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃ and weighing, and measuring the dry cell weight concentration in the fermentation broth to be 34g/L.
1 G of strain sample after fermentation for 72 hours is taken, after centrifugal washing, an equal volume of 3mol/L hydrochloric acid aqueous solution is added and heated in a boiling water bath for 5 minutes to cause cell wall rupture. The cells after wall breaking are extracted by acetone to obtain an acetone solution of astaxanthin. All acetone residues were distilled off at 45 degrees under dark conditions. Then using High Performance Liquid Chromatography (HPLC), the astaxanthin content was determined to be about 11.8mg/g dry cell weight; the astaxanthin content obtained by the measurement is multiplied by 11.8mg/g to obtain the cell dry weight concentration 34g/L of the fermentation liquid, and the concentration of the fermented astaxanthin in the batch is 401.2mg/L.
Example IV
The fermentation method of the invention can be applied to batch fermentation occasions and continuous fermentation occasions. This example describes the production of astaxanthin by batch fermentation of Phaffia rhodozyma in a 5L fermenter.
First, 400mg of nano-titania (Allatin, CAS 13463/67/7, SKU#T104936) having a solid particle size of 60 nm was weighed out; in addition, 10mL of a nano-alumina aqueous dispersion (Allatin, CAS 1344/28/1, SKU#A119404) containing 20wt% of nano-alumina particles having a particle size of 5 to 10 nm was prepared; mixing 400mg of nano titanium dioxide into 10mL of nano aluminum oxide aqueous dispersion liquid, and uniformly stirring to obtain 10mL of nano TiO 2/Al2O3 mother solution containing 400mg of nano titanium dioxide; (since the amount of 400mg is small, the volume is hardly increased after 10mL of nano alumina aqueous dispersion is added, and the prepared nano TiO 2/Al2O3 mother solution is also 10 mL);
Inoculating Phaffia rhodozyma Phaffia rhodozyma (ATCC 24202) into 100ml of seed culture medium for fermentation to prepare Phaffia rhodozyma seed liquid; the seed culture medium usually adopts yeast extract peptone glucose culture medium, and the composition of the seed culture medium usually comprises the following components: 2% of glucose, 1% of yeast extract, 2% of peptone and the balance of deionized water; the seed fermentation process can be carried out in a shaking table, the rotating speed of the shaking table is usually 100-200 rpm, and the fermentation temperature of the Phaffia rhodozyma seed liquid is controlled at 15-25 ℃;
2L of a carbon source-free sterile fermentation medium subjected to high-pressure sterilization at 121 ℃ is put into a 5L fermentation tank, the high-pressure sterilization time is 30 minutes, and the pH value of the fermentation medium is controlled and regulated to 7.2 by using 5mol/L NaOH aqueous solution and 2mol/L hydrochloric acid aqueous solution; the aseptic fermentation medium described in this example comprises the following components: 80g/L glucose, 1g/L yeast extract, 1g/L magnesium sulfate, 6g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L dipotassium phosphate and 0.2g/L calcium chloride; specifically, 10ml of prepared nano TiO 2/Al2O3 mother solution containing 400mg of nano TiO 2 is added into a culture medium, so that 200mg/L of nano TiO 2 is added into the fermentation liquid;
Inoculating the obtained Phaffia rhodozyma seed liquid into the aseptic fermentation culture medium with the constant pH value of 7.2 according to the inoculation concentration of 5% for fermentation culture, wherein the culture period is 72 hours; wherein the temperature of fermentation liquor in the fermentation tank is controlled at 15-25 ℃, the initial stirring rotation speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation amount in the fermentation tank is 0.5-2 vvm, and the dissolved oxygen in the fermentation liquor is ensured to be 20-60% by setting the stirring rotation speed and ventilation amount in the whole fermentation culture process;
When sampling after 72 hours fermentation was completed, the intracellular pH was tested at this time point using the intracellular pH test kit (purchased from abcam, intracellular pH Assay kit ab 228552) at a pH of 5.2 on the inner wall of the cell membrane. Since the pH of the fermenter was kept constant at 7.2, it was calculated that the pH gradient ΔpH (intracellular pH/extracellular pH) inside and outside the cell membrane was about-2.0, i.e., the pH difference between the pH in the Phaffia yeast cell and the pH outside the cell during fermentation was kept at-2.0;
Cell dry weight determination was performed after the whole 72 hour fermentation was completed: after weighing the fermentation broth, centrifuging at 4000rpm for 5 minutes, washing the obtained precipitate once with water, centrifuging at 4000rpm for 5 minutes, filtering the obtained precipitate, drying at 80 ℃ and weighing, and measuring the dry cell weight concentration in the fermentation broth to be 35g/L.
1 G of strain sample after fermentation for 72 hours is taken, after centrifugal washing, an equal volume of 3mol/L hydrochloric acid aqueous solution is added and heated in a boiling water bath for 5 minutes to cause cell wall rupture. The cells after wall breaking are extracted by acetone to obtain an acetone solution of astaxanthin. All acetone residues were distilled off at 45 degrees under dark conditions. Then using High Performance Liquid Chromatography (HPLC), the astaxanthin content was determined to be about 16.8mg/g dry cell weight; the astaxanthin content obtained by the measurement was multiplied by 16.8mg/g to obtain a cell dry weight concentration of 35g/L, and the concentration of the astaxanthin obtained by the measurement was 588mg/L.
Examples one to four astaxanthin-producing data are compared as shown in the following table
The above table summarizes the effect of adding different nano-TiO 2/Al2O3 on astaxanthin production by phaffia fermentation. In the example of adding 500mg/L of common micron-sized titanium dioxide, the astaxanthin content is 3.2mg/g dry cell weight, and the total astaxanthin concentration is about 124.8mg/L. In the embodiment of adding 100mg/L and 200mg/L of nano TiO 2/Al2O3, the synthesis of astaxanthin reaches 442.9mg/L and 588mg/L, so that a good effect of exciting astaxanthin synthesis is obtained.
The invention has the advantages that: according to the invention, the nano-alumina aqueous dispersion is used as a carrier to treat and dissolve nano-scale titanium dioxide into a fermentation medium, so that the nano-scale titanium dioxide can be efficiently delivered into yeast cells during fermentation, further, the pH value in the cells can be rapidly regulated, the imbalance of the pH value inside and outside the cells of Phaffia rhodozyma is kept during fermentation, namely, the pH environment inside and outside the cells of Phaffia rhodozyma is changed, so that the pH value gradient inside and outside the cells of Phaffia rhodozyma is caused, further, the stress reaction of Phaffia rhodozyma is stimulated, the metabolic pathway in the Phaffia rhodozyma is carried out towards the direction of producing astaxanthin, and the astaxanthin content in the Phaffia rhodozyma is effectively increased.
Claims (6)
1. A method for promoting Phaffia rhodozyma to produce astaxanthin in high yield is characterized in that: the method comprises the following steps: inoculating Phaffia rhodozyma seed liquid into a sterile fermentation medium added with nano TiO 2/Al2O3 mother liquid for fermentation culture, wherein the nano TiO 2/Al2O3 mother liquid is nano titanium dioxide mixed liquid taking nano alumina aqueous dispersion liquid as a carrier, and the pH value inside and outside Phaffia rhodozyma cells is kept unbalanced during fermentation, so that the stress reaction of Phaffia rhodozyma is excited, and the metabolic pathway in Phaffia rhodozyma body is carried out towards the direction of astaxanthin production;
Adding 100-200 mg of nano TiO 2/Al2O3 mother liquor into each liter of sterile fermentation medium, keeping the pH value of the sterile fermentation medium to be 7.2, keeping the pH value in Phaffia yeast cells in the fermentation period to be lower than the pH value outside the cells, and keeping the pH difference value between the pH value in Phaffia yeast cells and the pH value outside the cells in the fermentation period to be-2.0 to-1.7;
The preparation method of the nano TiO 2/Al2O3 mother solution comprises the following steps: mixing nano titanium dioxide into nano alumina aqueous dispersion, and uniformly stirring to obtain nano TiO 2/Al2O3 mother liquor, wherein the nano TiO2/Al2O3 mother liquor contains 18-22 wt% of nano alumina particles, 1-2-wt% of nano titanium dioxide and 76-81 wt% of water, the nano titanium dioxide solid particles are 40-80 nanometers in size, and the particle size of the nano alumina particles is 5-10 nanometers.
2. The method for promoting high-yield astaxanthin by phaffia yeast according to claim 1, wherein: 2-5 mol/L NaOH aqueous solution and 1-2 mol/L hydrochloric acid aqueous solution are used for controlling and adjusting the pH value of the sterile fermentation medium.
3. The method for promoting high-yield astaxanthin by phaffia yeast according to claim 1, wherein: the preparation method of the Phaffia rhodozyma seed liquid comprises the following steps: inoculating Phaffia rhodozyma into a seed culture medium for fermentation to prepare Phaffia rhodozyma seed solution, wherein the seed culture medium generally adopts a yeast extract peptone glucose culture medium, and the seed culture medium comprises the following components: 1-3% of glucose, 1-3% of yeast extract and 1-2% of peptone.
4. A method for promoting high astaxanthin production by phaffia yeast according to claim 3, wherein: the seed fermentation process is carried out in a shaking table, the rotating speed of the shaking table is 100-200 rpm, and the fermentation temperature of the Phaffia rhodozyma seed liquid is controlled at 15-25 ℃.
5. The method for promoting high-yield astaxanthin by phaffia yeast according to claim 1, wherein: the aseptic fermentation medium comprises the following components: 10-100 g/L glucose, 0.5-1 g/L yeast extract, 0.5-1 g/L magnesium sulfate, 2-6 g/L ammonium sulfate, 1-3 g/L monopotassium phosphate, 1-3 g/L dipotassium phosphate and 0.1-0.2 g/L calcium chloride.
6. The method for promoting high-yield astaxanthin by phaffia yeast according to claim 5, wherein: inoculating Phaffia rhodozyma seed liquid into a sterile fermentation culture medium, and then fermenting and culturing in a fermentation tank for 48-72 hours; wherein the temperature of fermentation liquor in the fermentation tank is controlled at 20 ℃, the initial stirring rotation speed of stirring equipment in the fermentation tank is 100-200 rpm, the initial ventilation amount in the fermentation tank is 0.5-2 vvm, and the dissolved oxygen in the fermentation liquor is ensured to be 20% -60% by setting the stirring rotation speed and ventilation amount in the whole fermentation culture process.
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