CN106701880A - Method for improving Phaffia rhodozyma strain high-yield astaxanthin - Google Patents
Method for improving Phaffia rhodozyma strain high-yield astaxanthin Download PDFInfo
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- CN106701880A CN106701880A CN201710030949.XA CN201710030949A CN106701880A CN 106701880 A CN106701880 A CN 106701880A CN 201710030949 A CN201710030949 A CN 201710030949A CN 106701880 A CN106701880 A CN 106701880A
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- Prior art keywords
- astaxanthin
- parts
- phaffia rhodozyma
- fermentation
- yield
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- 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 62
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 62
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 62
- 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 62
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 62
- 241000081271 Phaffia rhodozyma Species 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 45
- 230000004151 fermentation Effects 0.000 claims abstract description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 13
- 239000001888 Peptone Substances 0.000 claims abstract description 12
- 108010080698 Peptones Proteins 0.000 claims abstract description 12
- 235000019319 peptone Nutrition 0.000 claims abstract description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 10
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 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 abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 239000007836 KH2PO4 Substances 0.000 claims abstract description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 5
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 5
- 239000012138 yeast extract Substances 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 14
- 239000012530 fluid Substances 0.000 claims description 14
- 239000001301 oxygen Substances 0.000 claims description 14
- 229910052760 oxygen Inorganic materials 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 9
- 239000006227 byproduct Substances 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 9
- 239000002243 precursor Substances 0.000 claims description 9
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 8
- JIHQDMXYYFUGFV-UHFFFAOYSA-N 1,3,5-triazine Chemical compound C1=NC=NC=N1 JIHQDMXYYFUGFV-UHFFFAOYSA-N 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 6
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 6
- 238000002386 leaching Methods 0.000 claims description 6
- BWDBEAQIHAEVLV-UHFFFAOYSA-N 6-methylheptan-1-ol Chemical compound CC(C)CCCCCO BWDBEAQIHAEVLV-UHFFFAOYSA-N 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 5
- 241000238557 Decapoda Species 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 150000001408 amides Chemical class 0.000 claims description 4
- 235000015278 beef Nutrition 0.000 claims description 4
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical group CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229940094933 n-dodecane Drugs 0.000 claims description 4
- 235000015193 tomato juice Nutrition 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 238000003811 acetone extraction Methods 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 3
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 3
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(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)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 claims description 2
- 235000013734 beta-carotene Nutrition 0.000 claims description 2
- 239000011648 beta-carotene Substances 0.000 claims description 2
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 claims description 2
- 229960002747 betacarotene Drugs 0.000 claims description 2
- 235000016709 nutrition Nutrition 0.000 claims description 2
- 230000035764 nutrition Effects 0.000 claims description 2
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(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)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 8
- 238000003786 synthesis reaction Methods 0.000 abstract description 8
- 235000019764 Soybean Meal Nutrition 0.000 abstract 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 abstract 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 abstract 1
- 235000019796 monopotassium phosphate Nutrition 0.000 abstract 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 239000004455 soybean meal Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 8
- 244000005700 microbiome Species 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- ZZVUWRFHKOJYTH-UHFFFAOYSA-N diphenhydramine Chemical compound C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1 ZZVUWRFHKOJYTH-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229940031098 ethanolamine Drugs 0.000 description 1
- 238000001914 filtration Methods 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
- 235000012907 honey Nutrition 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- -1 keto-acid carotenoid Chemical class 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 235000020995 raw meat Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for improving Phaffia rhodozyma strain high-yield astaxanthin. The method comprises the steps of multiplication culture and fermentation and separation, wherein a fermentation culture medium is prepared from, by weight, 75-85 parts of an agricultural and sideline product pretreatment solution, 20-30 parts of soybean meal, 8-14 parts of glucose, 4-8 parts of peptone, 4-8 parts of concentrated yeast extract powder, 0.2-0.4 part of CaCl2.2H2O, 1-4 parts of MgSO4.7H2O, 1-2 parts of KH2PO4, 1-5 parts of (NH4)2SO4, 1-3 parts of ethanol and 0.01-1 part of triethanolamine. According to the method, while the strain is stimulated to produce the astaxanthin, synthesis of the astaxanthin is promoted in the fermentation process, and therefore the astaxanthin yield is obviously increased, the fermentation cost is lowered, resources are fully utilized, triethanolamine promotes synthesis of the astaxanthin, and then the yield is obviously increased.
Description
Technical field
The invention belongs to fermentation arts, and in particular to the method for improving Phaffia rhodozyma strain high-yield astaxanthin.
Technical background
Astaxanthin (3,3 '-dihydroxy -4,4 ' diketo-β, β ' carrotene) is a kind of keto-acid carotenoid, naturally
Take on a red color.Astaxanthin is initially used as the feed addictive of aquatic products industry, and later pharmacology and Physiologic Studies show that astaxanthin has
Extremely strong function anti-oxidant, that free radical is quenched, and the generation of antibody can be promoted, strengthen the immunologic function of host, in food, doctor
The field such as medicine and cosmetics has broad application prospects, with economic worth very high.
Red phaffia rhodozyma（Phaffia rhodozyma）It is the only naturally production bacterial strain for producing astaxanthin, its trans shrimp
Blue or green element obtained FDA approvals in 2000, for food additives.It is mycota, Eumycota, Deuteromycotina, hidden ball ferment
Female section, red phaffia rhodozyma category unique kind.Used as the production bacterial strain of astaxanthin, red phaffia rhodozyma has many advantages：It is available
Various sugar carry out quick heterotrophism metabolism, and incubation time is short, are not required to illumination and can realize High Density Cultivation etc..Such as prior art Shen
Please number be the Chinese invention patent of CN103614444A, the invention belongs to biological technical field, there is provided one kind is using flammable
Astaxanthin contains in concentration of alcohol in the red phaffia rhodozyma zymotic fluid of gas sensor constant control, and then the red phaffia rhodozyma cell of raising
The method of amount, the method utilizes combustible gas sensor, and ethanol stream dosage, successfully constant control are controlled by switching peristaltic pump
Arbitrary value of the concentration of alcohol in red phaffia rhodozyma zymotic fluid in the range of 1 ~ 20g/L is made.In the red phaffia rhodozyma of constant control
In the case that concentration of alcohol is 10g/L in zymotic fluid, then by the culture of 1 day, the astaxanthin in red phaffia rhodozyma dry cell weight
Content can reach 145.7 μ g/g, comparison impinge upon do not flow plus ethanol under conditions of improve 140.4%, so as to reach red method
The purpose of husband's yeast high-yield astaxanthin, with important researching value and being widely applied prospect.The patent of invention is for equipment
It is very high with precise requirements, and inapplicable industrial production, such as Chinese invention of prior art Application No. CN105053519A
A kind of patent, method for improving red Phaffia Rhodozyma astaxanthin of the disclosure of the invention, first modulates pH=by red phaffia rhodozyma fermented liquid
9.0~11.5,35~60 DEG C are controlled, stir 80~150r/min, broken time 1.0~5.0；PH=6.0~7.5 are neutralized to again,
And mix with the water of 0.5~1 times of volume, by centrifugal concentrating, obtain concentrating the red Phaffia Rhodozyma slurry of broken wall；Then, with it is suitable
It is spray-dried after amount compound antioxidant is well mixed, 170~185 DEG C of EAT, 70~85 DEG C of leaving air temp obtains shrimp blue or green
Plain dry powder, dry powder moisture is less than 8%, and spray loss amount is less than 4.0%.The invention is simple and practical, can not only lift 10~12%
Total pigment, and broken time is short, low cost, suitable industrialized production, the astaxanthin dry powder storage rack time of production is longer, taste
Road raw meat is fragrant unique, and good palatability, power-product is widely used in animal feed industries.The prior art meets low cost, fits
Suitable industrialized production, but the yield of its astaxanthin need to be improved.
The content of the invention
The present invention provides a kind of astaxanthin yield raising Phaffia rhodozyma strain high yield high to solve above-mentioned technical problem
The method of astaxanthin.
The present invention is for the solution scheme taken of above-mentioned technical problem：Improve Phaffia rhodozyma strain high-yield astaxanthin
Method, including following steps：
Phaffia rhodozyma strain（Phaffia rhodozyma）CZ10 was preserved in positioned at Beijing Chaoyang on July 25th, 2012
In China Committee for Culture Collection of Microorganisms's common micro-organisms of the Institute of Microorganism, Academia Sinica of area North Star West Road 1
The heart, preserving number is CGMCC No.6355.
1）Activation Phaffia rhodozyma strain is inoculated in middle Shake flask medium at 20 ~ 27 DEG C for 5 ~ 12% by inoculum concentration, it is logical
Tolerance is 1.0 ~ 1.2vvm, stirs 100~200r/min, 20 ~ 35h of Amplification Culture；
2）Will be enlarged by the Phaffia rhodozyma strain after culture and be seeded to the fermentation tank equipped with fermentation medium by 6 ~ 10% inoculum concentration
In, ammonium sulfate is added as nitrogen source, make C/N ratios be 2；
3）The carrier of oxygen is added in fermentation tank, by volume, the addition of the carrier of oxygen is the 0.5 of fermentation medium percent by volume
~ 5%, 20 ~ 27 DEG C of cultivation temperature, the r/min of mixing speed 500 ~ 600, the vvm of throughput 1.2 ~ 1.5；
4）Fermentation process adds nutrient solution, while adding astaxanthin precursor substance every 12 ~ 24 h, 30 L is added every time, adds 2 ~ 5
Continue 20 ~ 30 h that ferment after secondary；
5）By 2 ~ 6 collects thallines of zymotic fluid centrifuge washing, thalline is suspended in 50 ~ 60 DEG C of dimethyl sulfoxide (DMSO)s, vibrated, acetone extraction
After taking, after vacuum concentration, dissolved again with dichloromethane, prepare the isolated astaxanthin component of HPLC chromatogram, be concentrated in vacuo
To astaxanthin dry powder；
Wherein, fermentation medium is made up of following composition and composition by weight：75 ~ 85 parts of agricultural byproducts pretreatment fluid, dregs of beans 20 ~ 30
Part, 8 ~ 14 parts of glucose, 4 ~ 8 parts of peptone, 4 ~ 8 parts of yeast extract powder of concentration, CaCl22H2O0.2 ~ 0.4 part, MgSO4
7H2O1 ~ 4 part, KH2PO41 ~ 2 part, (NH4) 2SO41 ~ 5 part, 1 ~ 3 part of ethanol, 0.01 ~ 1 part of triethanolamine, fermentation medium pH
Be worth is 5.5 ~ 6.5, hence it is evident that improve astaxanthin yield, while reducing fermentation costs, resource is fully used, wherein three ethanol
Amine promotes the synthesis of astaxanthin, so as to improve yield.Wherein Shake flask medium is made up of following composition and percentage：1 ~ 5% albumen
Peptone, 1 ~ 5% beef leaching thing, 4 ~ 10%NaCl, 0.01 ~ 0.11%MnSO4•H2O, 5 ~ 10% agar, 0.01 ~ 0.1% isooctanol and remaining
The sterilized water of amount, makes mycelial growth good, and bacterium colony is clearly demarcated, and wherein isooctanol stimulates bacterial strain to produce astaxanthin, so as to improve fermentation
During astaxanthin yield.The nutrient solution for adding during the fermentation is made up of following composition and percentage：1~4%(NH4)2·
The sterilized water of SO4,0.1 ~ 1% cellobiose, 0.1 ~ 1% KH2PO4,0.1 ~ 1% MnSO4,0.01 ~ 0.1% s-triazine and surplus,
Consumption and the shortage of carbon source in fermentation process are avoided, it is fully fermented, wherein s-triazine promotes fermentation abundant, so as to improve shrimp
Blue or green element yield, meanwhile, shorten its fermentation period.The carrier of oxygen added in fermentation process is n-dodecane and perfluocarbon；It is compound
Ratio is 1 ~ 2：1 ~ 2, promote oxygen transmission, dissolved oxygen amount in zymotic fluid is improved, so as to promote chemical activators, improve astaxanthin and produce
Amount, astaxanthin precursor substance described in astaxanthin precursor substance is tomato juice, beta carotene and amide blend, and compositely proportional is
5:4:1；The synthesis of astaxanthin can be promoted；Agricultural byproducts include blackstrap or brewer's yeast or microcrystalline cellulose or long-grained nonglutinous rice or corn
Core.
Compared with prior art, advantage for present invention is：While the present invention stimulates bacterial strain high-yield astaxanthin,
Promote the synthesis of astaxanthin simultaneously during the fermentation, so as to significantly improve astaxanthin yield, reduce fermentation costs, obtain resource
To making full use of, wherein triethanolamine promotes the synthesis of astaxanthin, so as to significantly improve yield.
Specific embodiment
It is described in further detail with reference to embodiments
Embodiment 1：
The method that Phaffia rhodozyma strain produces astaxanthin is improved, including following steps：
Phaffia rhodozyma strain（Phaffia rhodozyma）CZ10 was preserved in positioned at Beijing Chaoyang on July 25th, 2012
In China Committee for Culture Collection of Microorganisms's common micro-organisms of the Institute of Microorganism, Academia Sinica of area North Star West Road 1
The heart, preserving number is CGMCC No.6355.
1）Activation Phaffia rhodozyma strain is inoculated in middle Shake flask medium at 20 ~ 27 DEG C for 5 ~ 12% by inoculum concentration, it is logical
Tolerance is 1.0 ~ 1.2vvm, stirs 100~200r/min, 20 ~ 35h of Amplification Culture；
2）Will be enlarged by the Phaffia rhodozyma strain after culture and be seeded to the fermentation tank equipped with fermentation medium by 6 ~ 10% inoculum concentration
In, ammonium sulfate is added as nitrogen source, make C/N ratios be 2；
3）The carrier of oxygen is added in fermentation tank, by volume, the addition of the carrier of oxygen is the 0.5 of fermentation medium percent by volume
~ 5%, 20 ~ 27 DEG C of cultivation temperature, the r/min of mixing speed 500 ~ 600, the vvm of throughput 1.2 ~ 1.5；
4）Fermentation process adds nutrient solution, while adding astaxanthin precursor substance every 12 ~ 24 h, 30 L is added every time, adds 2 ~ 5
Continue 20 ~ 30 h that ferment after secondary；
5）By 2 ~ 6 collects thallines of zymotic fluid centrifuge washing, thalline is suspended in 50 ~ 60 DEG C of dimethyl sulfoxide (DMSO)s, vibrated, acetone extraction
After taking, after vacuum concentration, dissolved again with dichloromethane, prepare the isolated astaxanthin component of HPLC chromatogram, be concentrated in vacuo
To astaxanthin dry powder；
Wherein, fermentation medium is made up of following composition and composition by weight：75 ~ 85 parts of agricultural byproducts pretreatment fluid, dregs of beans 20 ~ 30
Part, 8 ~ 14 parts of glucose, 4 ~ 8 parts of peptone, 4 ~ 8 parts of yeast extract powder of concentration, CaCl2·2H2O0.2 ~ 0.4 part, MgSO4·
7H2O1 ~ 4 part, KH2PO41 ~ 2 part, (NH4)2SO41 ~ 5 part, 1 ~ 3 part of ethanol, 0.01 ~ 1 part of triethanolamine, fermentation medium pH value
It is 5.5 ~ 6.5, preferred ferment culture medium is made up of following composition and composition by weight：79 parts of agricultural byproducts pretreatment fluid, dregs of beans 26
Part, 13 parts of glucose, 6 parts of peptone, 6 parts of yeast extract powder of concentration, CaCl2·2H20.3 part of O, MgSO4·7H2O3 parts,
KH2PO41 part, (NH4)2SO43 parts, 1 part of ethanol, 0.01 part of triethanolamine, because the mechanism of action is still not clear, or because of synergy
Its astaxanthin yield is significantly improved, while reducing fermentation costs, resource is fully used, wherein triethanolamine promotes shrimp blue or green
The synthesis of element, so as to improve yield.Wherein Shake flask medium is made up of following composition and percentage：1 ~ 5% peptone, 1 ~ 5% N
Meat leaching thing, 4 ~ 10%NaCl, 0.01 ~ 0.11%MnSO4•H2O, 5 ~ 10% agar, 0.01 ~ 0.1% isooctanol and surplus it is aseptic
Water, it is preferred that Shake flask medium is made up of following composition and percentage：5% peptone, 5% beef leaching thing, 6%NaCl,
0.01%MnSO4•H2The sterilized water of O, 8% agar, 0.01% isooctanol and surplus；Make mycelial growth good, bacterium colony is clearly demarcated, wherein different
Octanol stimulates bacterial strain to produce astaxanthin, so as to improve astaxanthin yield in fermentation process.The nutrient solution for adding during the fermentation
It is made up of following composition and percentage：1~4%(NH4)2·SO4, 0.1 ~ 1% cellobiose, 0.1 ~ 1% KH2PO4、0.1~1%
MnSO4, 0.01 ~ 0.1% s-triazine and surplus sterilized water, it is preferred that the nutrient solution added in fermentation process by following composition and
Percentage is constituted：1%(NH4)2·SO4, 0.1% cellobiose, 0.3% KH2PO4、0.15% MnSO4, 0.01% s-triazine and surplus
Sterilized water, it is to avoid the consumption of carbon source and shortage in fermentation process, it is fully fermented, wherein s-triazine promotes fermentation abundant,
So as to improve astaxanthin yield, meanwhile, shorten its fermentation period.The carrier of oxygen added in fermentation process is n-dodecane and perfluor
Change carbon；Compositely proportional is 1 ~ 2：1 ~ 2, it is preferred that compositely proportional is 2：1, hence it is evident that promote oxygen transmission, improve dissolved oxygen in zymotic fluid
Amount, so as to promote chemical activators, hence it is evident that improve astaxanthin yield, astaxanthin precursor substance described in astaxanthin precursor substance for kind
Tomato juice, bata-carotene and amide blend, compositely proportional are 5：4:1；The synthesis of astaxanthin can be promoted；Agricultural byproducts include useless sugar
Honey or schlempe or microcrystalline cellulose or long-grained nonglutinous rice or corncob, reduce cost.
Embodiment 2：
Shake flask medium：5% peptone, 5% beef leaching thing, 6%NaCl, 0.01%MnSO4•H2It is O, 8% agar, 0.01% different pungent
The sterilized water of alcohol and surplus；79 parts of agricultural byproducts pretreatment fluid, 26 parts of dregs of beans, 13 parts of glucose, 6 parts of peptone, concentration yeast
Leach 6 parts of powder, CaCl2·2H20.3 part of O, MgSO4·7H2O3 parts, KH2PO41 part, (NH4)2SO43 parts, 1 part of ethanol, three second
0.01 part of hydramine；
Fermentation medium：79 parts of agricultural byproducts pretreatment fluid, 26 parts of dregs of beans, 13 parts of glucose, 6 parts of peptone, concentration yeast leaching
Go out 6 parts of powder, CaCl2·2H20.3 part of O, MgSO4·7H2O3 parts, KH2PO41 part, (NH4)2SO43 parts, 1 part of ethanol, three ethanol
0.01 part of amine；
Nutrient solution：：1%(NH4)2·SO4, 0.1% cellobiose, 0.3% KH2PO4、0.15% MnSO4, 0.01% s-triazine and remaining
The sterilized water of amount.
The good strain of activation culture is inoculated into Shake flask medium with 10% inoculum concentration, 21 DEG C, 170 rpm cultivate 18 h；
Finally it is inoculated with shaking flask, 6% inoculum concentration, 21 DEG C, 180 rpm cultures, 20 h；After corncob is crushed, with 4% sodium hydroxide solution
Treatment overnight, after filtering, is washed with water to neutrality, drying for standby.The sodium citrate buffer solution of 0.05 m of pH4.8 is subsequently adding,
115 DEG C of 20 min of sterilizing, add ammonium sulfate as nitrogen source, make C/N ratios be 2, are squeezed into culture medium with 700 L liquid amounts after mixing
In 1000 L fermentation tanks, inoculum concentration 6%, the carrier of oxygen is n-dodecane perfluocarbon, compositely proportional 2：1, the vvm of throughput 1.2, stir
Mix the r/min of speed 500,21 DEG C of fermentation temperature, pH is adjusted to 5.5 ~ 6.5, every 12 h addition one time of nutrition liquid adjust humidity to
30 ~ 40%, while every 24 h streams plus astaxanthin precursor substance tomato juice, bata-carotene and acid amides, being combined than row 5：4：1, every time
Plus 30 L, plus after 4 times, stop stream plus, continue 20 h that ferment.After fermentation ends, astaxanthin is extracted, astaxanthin is obtained after isolating and purifying
Yield is 81.56mg/L, refines total recovery and reaches 94.33%.
Although the present invention is disclosed as above with embodiment, it is not limited to protection scope of the present invention, any ripe
Those skilled in the art is known, in the change and retouching done without departing from the spirit and scope of the invention, this hair all should be belonged to
The protection domain of bright appended claims.
Claims (7)
1. the method for improving Phaffia rhodozyma strain high-yield astaxanthin, it is characterised in that following steps：
1）Activation Phaffia rhodozyma strain is inoculated in Shake flask medium at 20 ~ 27 DEG C for 5 ~ 12% by inoculum concentration, throughput is
1.0 ~ 1.2vvm, stirs 100~200r/min, 20 ~ 35h of Amplification Culture；
2）Will be enlarged by the Phaffia rhodozyma strain after culture to be seeded in fermentation tank by 6 ~ 10% inoculum concentration, add ammonium sulfate to make
It is nitrogen source, makes C/N ratios be 2；
3）The carrier of oxygen is added in fermentation tank, by volume, the addition of the carrier of oxygen is the 0.5 of fermentation medium percent by volume
~ 5%, 20 ~ 27 DEG C of cultivation temperature, the r/min of mixing speed 500 ~ 600, the vvm of throughput 1.2 ~ 1.5；
4）Fermentation process adds nutrient solution, while adding astaxanthin precursor substance every 12 ~ 24 h, 30 L is added every time, adds 2 ~ 5
Continue 20 ~ 30 h that ferment after secondary；
5）By 2 ~ 6 collects thallines of zymotic fluid centrifuge washing, thalline is suspended in 50 ~ 60 DEG C of dimethyl sulfoxide (DMSO)s, vibrated, acetone extraction
After taking, after vacuum concentration, dissolved again with dichloromethane, prepare the isolated astaxanthin component of HPLC chromatogram, be concentrated in vacuo
To astaxanthin dry powder；
Wherein, the fermentation medium is made up of following composition and composition by weight：75 ~ 85 parts of agricultural byproducts pretreatment fluid, dregs of beans
20 ~ 30 parts, 8 ~ 14 parts of glucose, 4 ~ 8 parts of peptone, concentration 4 ~ 8 parts of yeast extract powder, CaCl2·2H2O0.2 ~ 0.4 part,
MgSO4·7H2O1 ~ 4 part, KH2PO41 ~ 2 part, (NH4)2SO41 ~ 5 part, 1 ~ 3 part of ethanol, 0.01 ~ 1 part of triethanolamine.
It is 2. according to claim 1 to improve the method that Phaffia rhodozyma strain produces astaxanthin, it is characterised in that：The shaking flask
Culture medium is made up of following composition and percentage：1 ~ 5% peptone, 1 ~ 5% beef leaching thing, 4 ~ 10%NaCl, 0.01 ~ 0.11%
MnSO4•H2The sterilized water of O, 5 ~ 10% agar, 0.01 ~ 0.1% isooctanol and surplus.
It is 3. according to claim 1 to improve the method that Phaffia rhodozyma strain produces astaxanthin, it is characterised in that：The nutrition
Liquid is made up of following composition and percentage：1 ~ 4% (NH4) 2SO4,0.1 ~ 1% cellobiose, 0.1 ~ 1% KH2PO4,0.1 ~ 1%
The sterilized water of MnSO4,0.01 ~ 0.1% s-triazine and surplus.
It is 4. according to claim 1 to improve the method that Phaffia rhodozyma strain produces astaxanthin, it is characterised in that：The oxygen is carried
Body is n-dodecane and perfluocarbon；Compositely proportional is 1 ~ 2：1~2.
It is 5. according to claim 1 to improve the method that Phaffia rhodozyma strain produces astaxanthin, it is characterised in that：The shrimp is blue or green
Plain precursor substance is tomato juice, beta carotene and amide blend, and compositely proportional is 5：4:1.
6. it is according to claim 1 improve Phaffia rhodozyma strain high-yield astaxanthin method, it is characterised in that：The agriculture
Byproduct includes blackstrap or schlempe or microcrystalline cellulose or long-grained nonglutinous rice or corncob.
7. it is according to claim 1 improve Phaffia rhodozyma strain high-yield astaxanthin method, it is characterised in that：The hair
Ferment Medium's PH Value is 5.5 ~ 6.5.
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| CN108410939A (en) * | 2018-07-12 | 2018-08-17 | 烟台大学 | A method of improving Determination of Astaxanthin in Haematococcus Pluvialis content |
| CN108893517A (en) * | 2018-07-19 | 2018-11-27 | 威海利达生物科技有限公司 | A kind of fermentation medium and method of red phaffia rhodozyma fermenting and producing astaxanthin |
| CN108913746A (en) * | 2018-07-19 | 2018-11-30 | 威海利达生物科技有限公司 | By improving red phaffia rhodozyma biomass synthesizing astaxanthin and method for measuring |
| CN108931601A (en) * | 2018-08-14 | 2018-12-04 | 威海利达生物科技有限公司 | A method of efficiently preparing astaxanthin mark product |
| CN108977493A (en) * | 2018-08-17 | 2018-12-11 | 青岛中科潮生生物技术有限公司 | The method that astaxanthin is prepared using lignocellulosic |
| CN108998493A (en) * | 2018-06-25 | 2018-12-14 | 浙江皇冠科技有限公司 | A kind of formula technique of high-yield astaxanthin fermentation medium and application |
| CN112266945A (en) * | 2020-09-29 | 2021-01-26 | 自然资源部第三海洋研究所 | Method for extracting astaxanthin from phaffia rhodozyma |
| CN114561304A (en) * | 2022-01-20 | 2022-05-31 | 厦门昶科生物工程有限公司 | Fermentation process for improving astaxanthin production of phaffia rhodozyma strains |
| CN115418384A (en) * | 2022-09-23 | 2022-12-02 | 中国科学院过程工程研究所 | Method for producing astaxanthin by utilizing lignocellulose raw material |
| CN118497015A (en) * | 2024-07-18 | 2024-08-16 | 天津科技大学 | A thermotolerant red yeast and its application in producing astaxanthin |
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| CN108998493B (en) * | 2018-06-25 | 2020-11-06 | 浙江皇冠科技有限公司 | Formula technology and application of fermentation medium for high-yield astaxanthin |
| CN108410939A (en) * | 2018-07-12 | 2018-08-17 | 烟台大学 | A method of improving Determination of Astaxanthin in Haematococcus Pluvialis content |
| CN108893517A (en) * | 2018-07-19 | 2018-11-27 | 威海利达生物科技有限公司 | A kind of fermentation medium and method of red phaffia rhodozyma fermenting and producing astaxanthin |
| CN108913746A (en) * | 2018-07-19 | 2018-11-30 | 威海利达生物科技有限公司 | By improving red phaffia rhodozyma biomass synthesizing astaxanthin and method for measuring |
| CN108931601A (en) * | 2018-08-14 | 2018-12-04 | 威海利达生物科技有限公司 | A method of efficiently preparing astaxanthin mark product |
| CN108977493A (en) * | 2018-08-17 | 2018-12-11 | 青岛中科潮生生物技术有限公司 | The method that astaxanthin is prepared using lignocellulosic |
| CN112266945A (en) * | 2020-09-29 | 2021-01-26 | 自然资源部第三海洋研究所 | Method for extracting astaxanthin from phaffia rhodozyma |
| CN114561304A (en) * | 2022-01-20 | 2022-05-31 | 厦门昶科生物工程有限公司 | Fermentation process for improving astaxanthin production of phaffia rhodozyma strains |
| CN114561304B (en) * | 2022-01-20 | 2024-01-30 | 厦门昶科生物工程有限公司 | Fermentation process for improving astaxanthin production by rhodozyma strain |
| CN115418384A (en) * | 2022-09-23 | 2022-12-02 | 中国科学院过程工程研究所 | Method for producing astaxanthin by utilizing lignocellulose raw material |
| CN115418384B (en) * | 2022-09-23 | 2026-01-09 | 中国科学院过程工程研究所 | A method for producing astaxanthin using lignocellulose raw materials |
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