CN107400693B - Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells - Google Patents
Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells Download PDFInfo
- Publication number
- CN107400693B CN107400693B CN201710883642.4A CN201710883642A CN107400693B CN 107400693 B CN107400693 B CN 107400693B CN 201710883642 A CN201710883642 A CN 201710883642A CN 107400693 B CN107400693 B CN 107400693B
- Authority
- CN
- China
- Prior art keywords
- aqueous solution
- phaffia rhodozyma
- concentration
- protoplast
- chromatographic column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 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 38
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 38
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 38
- 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 38
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 38
- 239000012528 membrane Substances 0.000 title claims abstract description 36
- 238000010041 electrostatic spinning Methods 0.000 title claims abstract description 18
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 210000001822 immobilized cell Anatomy 0.000 title claims abstract description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 72
- 241000081271 Phaffia rhodozyma Species 0.000 claims abstract description 55
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 50
- 210000001938 protoplast Anatomy 0.000 claims abstract description 41
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 25
- 239000002121 nanofiber Substances 0.000 claims abstract description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 20
- 150000002632 lipids Chemical class 0.000 claims abstract description 17
- 229920001661 Chitosan Polymers 0.000 claims abstract description 14
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims abstract description 14
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 14
- 239000002131 composite material Substances 0.000 claims abstract description 14
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 14
- 238000001914 filtration Methods 0.000 claims abstract description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 10
- 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 10
- 240000008042 Zea mays Species 0.000 claims abstract description 10
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 10
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 10
- 239000001110 calcium chloride Substances 0.000 claims abstract description 10
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 10
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 10
- 235000005822 corn Nutrition 0.000 claims abstract description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 10
- 239000008103 glucose Substances 0.000 claims abstract description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 10
- 239000012138 yeast extract Substances 0.000 claims abstract description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 9
- -1 polyoxyethylene Polymers 0.000 claims abstract description 8
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims description 43
- 239000000725 suspension Substances 0.000 claims description 23
- 239000002502 liposome Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000005303 weighing Methods 0.000 claims description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 17
- 239000002244 precipitate Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 10
- 229930195725 Mannitol Natural products 0.000 claims description 10
- 239000000594 mannitol Substances 0.000 claims description 10
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 238000011049 filling Methods 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 210000000805 cytoplasm Anatomy 0.000 claims description 8
- 229940088598 enzyme Drugs 0.000 claims description 8
- 230000006196 deacetylation Effects 0.000 claims description 7
- 238000003381 deacetylation reaction Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 108010059892 Cellulase Proteins 0.000 claims description 6
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 6
- 229910052782 aluminium Inorganic materials 0.000 claims description 6
- 229940106157 cellulase Drugs 0.000 claims description 6
- 239000011888 foil Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 239000004417 polycarbonate Substances 0.000 claims description 5
- 229920000515 polycarbonate Polymers 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 108060004795 Methyltransferase Proteins 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 11
- 238000011068 loading method Methods 0.000 abstract description 2
- 238000001338 self-assembly Methods 0.000 abstract description 2
- 230000002924 anti-infective effect Effects 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 238000000707 layer-by-layer assembly Methods 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 230000001954 sterilising effect Effects 0.000 abstract 1
- 229960001701 chloroform Drugs 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 239000010410 layer Substances 0.000 description 5
- 210000002706 plastid Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000004005 microsphere Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000002390 rotary evaporation Methods 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 229940051921 muramidase Drugs 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000168517 Haematococcus lacustris Species 0.000 description 1
- 241001532508 Mycobacterium lactis Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910018540 Si C Inorganic materials 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000000819 hypertonic solution Substances 0.000 description 1
- 229940021223 hypertonic solution Drugs 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052909 inorganic silicate Inorganic materials 0.000 description 1
- 235000012680 lutein Nutrition 0.000 description 1
- 239000001656 lutein Substances 0.000 description 1
- KBPHJBAIARWVSC-RGZFRNHPSA-N lutein Chemical compound C([C@H](O)CC=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\[C@H]1C(C)=C[C@H](O)CC1(C)C KBPHJBAIARWVSC-RGZFRNHPSA-N 0.000 description 1
- 229960005375 lutein Drugs 0.000 description 1
- ORAKUVXRZWMARG-WZLJTJAWSA-N lutein 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=CC2C(=CC(O)CC2(C)C)C ORAKUVXRZWMARG-WZLJTJAWSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 150000001282 organosilanes Chemical class 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000003904 phospholipids Chemical group 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- FJHBOVDFOQMZRV-XQIHNALSSA-N xanthophyll Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C=C(C)C(O)CC2(C)C FJHBOVDFOQMZRV-XQIHNALSSA-N 0.000 description 1
Images
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
-
- 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
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for producing astaxanthin by self-assembly silica-body electrostatic spinning fibrous membrane immobilized cells, which comprises the steps of wrapping phaffia rhodozyma protoplasts in stable silica bodies formed by organic-inorganic composite lipids by using an electrostatic self-assembly method; completely dissolving chitosan, polyvinyl alcohol and polyoxyethylene in an acetic acid solution, adding glycerol and a silica body, preparing a nano-fiber membrane by electrostatic spinning, and loading the nano-fiber membrane into a chromatographic column in a coiled manner; preparing a mixed solution of glucose, yeast extract, corn steep liquor, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride and magnesium sulfate, filtering and sterilizing, flowing through a chromatographic column at a certain flow rate, ventilating the lower part of the chromatographic column, and detecting that effluent contains astaxanthin. The method for immobilizing the cells has the characteristics of high efficiency, anti-infection, high yield and high product concentration, and is convenient for subsequent product separation and purification.
Description
Technical Field
The invention relates to a method for producing astaxanthin by self-assembled silica body electrostatic spinning fibrous membrane immobilized cells, belonging to the field of biochemical engineering.
Background
Astaxanthin is a fat-soluble natural carotenoid with extremely strong physiological functions, is lutein which is most widely distributed in nature, has unique antioxidant function and coloring function, can resist tumors and enhance immunity, is safe, non-toxic, environment-friendly and high in biological value, so that the preparation and the application of the astaxanthin are favored by people. Natural astaxanthin is generally derived from: the method comprises extracting astaxanthin from aquatic product processing waste, producing astaxanthin from algae such as Haematococcus pluvialis, producing astaxanthin from bacteria such as Mycobacterium lactis, producing astaxanthin by fermentation with Phaffia rhodozyma, etc. Phaffia rhodozyma serves as an important natural astaxanthin source, and the biosynthesis of Phaffia rhodozyma is an important way for commercial production and development of astaxanthin.
The silica liposome is a liposome-like body, and is a novel lipid molecule formed from a molecule connecting two hydrophobic carbon chains and a hydrophilic organosilane molecule, said molecule can be self-assembled in water to form vesicle, the surface of the vesicle is covered with inorganic silicate shell layer with nano-grade thickness, and the stable Si-C bond is used for connecting inorganic layer with organic layer bipartite body together. Compared with the traditional liposome vesicle, the liposome is a very stable vesicle structure and is an organic-inorganic hybrid material. Compared with the traditional conventional liposome, the siloxane network on the surface of the liposome obviously improves the stability of the liposome and immobilized cells, and has good tolerance to a surfactant and acid-base. The silica body is used as a carrier for immobilized cells, compared with other conventional carriers, one cell can be loaded on one silica body, and the cell is bound in the silica body, so that the silica body has great mechanical strength, great specific surface area and pollution resistance.
Compared with the conventional immobilized carrier, the electrostatic spinning nanofiber membrane has the characteristics of high porosity, large specific surface area and good pore connectivity, can reduce the diffusion resistance of a substrate, and is favorable for improving the reactivity of immobilized cells.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects of the prior art, the invention provides an immobilized cell method which has the advantages of high efficiency, antibacterium, high yield and high product concentration for producing astaxanthin and is convenient for separating and purifying subsequent products.
In order to solve the technical problem, the invention discloses a method for producing astaxanthin by self-assembled silica body electrostatic spinning fibrous membrane immobilized cells, which comprises the following steps:
the method comprises the following steps: preparing a phaffia rhodozyma protoplast: weighing organic-inorganic composite lipid and phaffia rhodozyma protoplast in a mass ratio of 0.1-0.2: 1-3, preferably 0.15: 2; dissolving organic-inorganic composite lipid in chloroform, and evaporating to remove chloroform to obtain lipid film; suspending the phaffia rhodozyma protoplast in a mannitol aqueous solution, adding the phaffia rhodozyma protoplast into the obtained lipid-like film, stirring and uniformly mixing, rapidly heating to 45-50 ℃, preferably 48 ℃, thermally shocking for 10-30 s, simultaneously performing water bath ultrasound, cooling to 4 ℃, standing, and extruding by a liposome extruder to obtain a phaffia rhodozyma protoplast suspension with uniform particle size;
step two: preparing a nanofiber membrane: dissolving chitosan, polyvinyl alcohol and polyoxyethylene in an acetic acid aqueous solution, adding glycerol, adding the phaffia rhodozyma protoplasm liposome suspension prepared in the step one, uniformly stirring, preparing a nanofiber membrane through electrostatic spinning, filling the nanofiber membrane into a chromatographic column in a coiled manner, and leaving a space in the center of the chromatographic column for sterile air to pass through;
step three: preparing a mixed solution of glucose, yeast extract, corn steep liquor, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride and magnesium sulfate, filtering bacteria, passing through the chromatographic column prepared in the second step, and collecting the effluent.
In the first step, the phaffia rhodozyma protoplast is obtained through the following steps:
step a: taking the phaffia rhodozyma, fully washing with sterile water, centrifuging and taking a precipitate;
step b: preparing an enzymolysis liquid: respectively taking lywallzyme, helicase, cellulase and collapse enzyme with the mass of 2-4% of the precipitate obtained in the step a, and adding the lywallzyme, the helicase, the cellulase and the collapse enzyme into a mixture containing 0.3-0.5 mol/L KCl and 0.2-0.4 mol/LMgSO in a mass-volume ratio of 0.1: 5-10 g/mL
4Is dissolved in waterStirring for dissolving, and filtering to obtain enzymolysis solution;
step c: and (c) adding the precipitate obtained in the step (a) into the enzymolysis liquid obtained in the step (b), uniformly mixing in a vortex manner, carrying out water bath enzymolysis at the temperature of 25-30 ℃ for 3-8 h, and centrifuging to obtain the precipitate, thus obtaining the phaffia rhodozyma protoplast.
In the first step, the structural formula of the organic-inorganic composite lipid is as follows:
the structure forms a stable, extremely thin and rigid compact reticular membrane in the silicon plastid, and can restrain the protoplast, so that the protoplast cannot be broken due to the change of environmental osmotic pressure, and the action time of the protoplast is greatly prolonged; in addition, because the organic-inorganic composite lipid has similar phospholipid structure, the silica liposome combines the dual advantages of the liposome and the silica nanoparticles, avoids the respective defects of the liposome and the silica nanoparticles, can reduce the toxicity to the protoplast to the maximum extent, ensures the normal physiological functions of protein and enzyme in the protoplast, and simultaneously can not obstruct the entering and exiting of micromolecular substances.
In the first step, the mixing mass-volume ratio of the organic-inorganic composite lipid to the trichloromethane is 100: 200:10mg/mL, preferably 150:10 mg/mL; the concentration of the mannitol aqueous solution is 100-180g/L, and the mixing mass volume ratio of the phaffia rhodozyma protoplast to the mannitol aqueous solution is 1-3: 10g/mL, preferably 2:10 g/mL; the liposome extruder adopts a polycarbonate filter membrane, and the aperture of the filter membrane is 0.5-1.0 μm, preferably 1.0 μm.
In the second step, the viscosity average molecular weight of the chitosan is 5.0 multiplied by 10
5The deacetylation degree is 80-85%, and preferably 85%; the weight average molecular weight of the polyvinyl alcohol is 180000-200000, and is preferably 200000; the polyoxyethylene has a weight average molecular weight of 1 × 10
6～1×10
7Preferably 1X 10
7(ii) a The volume concentration of the acetic acid aqueous solution is 1-2%; the mass ratio of the chitosan to the polyvinyl alcohol to the polyoxyethylene is 1-22 to 4:7 to 9, preferably 2:3:8, chitosan, polyvinyl alcohol and polyethylene oxide are dissolved in an acetic acid aqueous solution at a total mass concentration of 1.5 to 2%, preferably 1.8%; the mass volume ratio of the added amount of the glycerol to the acetic acid water solution is 1-2 g/L, and 1.5g/L is preferred; the volume ratio of the addition amount of the phaffia rhodozyma protoplast suspension to the acetic acid aqueous solution is 1: 100-500, and preferably 1: 400.
In the second step, the receiving device of the electrostatic spinning adopts an aluminum foil, and the distance from the spray head to the receiving device is 5-30 cm, preferably 20 cm; the voltage is 3-30 KV, and preferably 20 KV; the spraying flow rate is 1-5 mL/h, preferably 1 mL/h.
In the third step, the concentration of glucose in the mixed aqueous solution is 40-60 g/L, preferably 50 g/L; the concentration of the yeast extract is 3-5 g/L, preferably 4 g/L; the concentration of the corn steep liquor is 2-3 g/L, preferably 2.5 g/L; the concentration of the ammonium sulfate is 4-6 g/L, preferably 5 g/L; the concentration of the monopotassium phosphate is 0.2-0.8 g/L, preferably 0.5 g/L; the concentration of the dipotassium phosphate is 1.5-3 g/L, preferably 2 g/L; the concentration of calcium chloride is 0.005-0.02 g/L, preferably 0.01 g/L; the concentration of magnesium sulfate is 0.5-0.8 g/L, preferably 0.6 g/L; the pH value of the mixed aqueous solution is adjusted to 5.0-5.8, preferably 5.5.
In the third step, the flow rate of the mixed aqueous solution is 0.2-0.5 mL/h, preferably 0.2 mL/h; the ventilation quantity of the sterile air in the chromatographic column is 1.0-2.0V/V.min, preferably 2.0V/V.min; the temperature of the chromatography column jacket is controlled to be 22-27 ℃, and preferably 25 ℃.
Has the advantages that:
1. the method adopts continuous operation, is simple to operate, has high raw material conversion efficiency and less time consumption, and can continuously biosynthesize the astaxanthin.
2. This application adopts the silica plastid as the direct carrier of cell, and the effectual constraint holds the cell to it reduces the cell volume to prepare protoplast through hypertonic solution, and this is favorable to the loading rate of cell in the silica plastid and keeps the stability of silica, and the activity of cell can be lastingly kept to the phaffia rhodozyma protoplast silica plastid of self-assembling, improves production efficiency, reduces the loss rate of cell.
3. The phaffia rhodozyma protoplast and the silicon plastid can enhance the permeability of cells to a greater extent, are beneficial to the secretion and discharge of astaxanthin, reduce the concentration of astaxanthin in cells, and effectively relieve the low yield of astaxanthin caused by product inhibition.
4. The method adopts electrostatic spinning to prepare a reaction medium, and utilizes the advantages of large porosity and small diffusion resistance and the combination of the silica body, so that the maximum reaction contact area can be obtained, and the maximum reaction efficiency can be exerted.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a schematic side view of the apparatus of the present invention.
FIG. 2 is a schematic diagram of the top view of the apparatus of the present invention.
Detailed Description
The invention will be better understood from the following examples. The description of the embodiments is intended to be illustrative of the invention and should not, nor should it be taken to limit the invention to the details set forth in the claims.
In the following examples, the HPLC detection method for astaxanthin is as follows:
the column used was Shim-pack CLC-ODS (6X 150mm, 5 μm) Shimadzu, Japan, and the mobile phase was methanol, acetonitrile and tetrahydrofuran in a flow ratio of 85:10:5 at a flow rate of 1mL/min, a column temperature of 30 ℃ and a detection wavelength of 480 nm.
And (3) porosity testing: taking a sample, accurately weighing the dry weight W of the sample
0Adding a certain amount of absolute ethyl alcohol into a weighing bottle, putting a sample into the weighing bottle, weighing the total weight W of the weighing bottle and the sample after the ethyl alcohol is fully immersed
1Then taking out the wet sample, and weighing the wet sample to obtain the weight W of the weighing bottle
2(ii) a Filling the pycnometer with absolute ethyl alcohol, weighing W
3The wet sample is put into a pycnometer, filled with ethanol to a specified scale and weighed as W
4。
The porosity was calculated according to the following formula.
Wherein P is porosity,%.
Example 1
The preparation method of the phaffia rhodozyma protoplast comprises the following steps:
(1) 3g of the Phaffia rhodozyma is fully washed by sterile water and centrifuged to obtain a precipitate.
(2) Preparing an enzymolysis liquid: adding muramidase 0.1g, snailase 0.1g, cellulase 0.1g, and collapse enzyme 0.1g into a solution containing KCl 0.3mol/L and MgSO 0.4mol/L
4Fully dissolving in 10mL of aqueous solution, and filtering to remove bacteria to obtain the enzymatic hydrolysate.
(3) Adding the precipitate into the enzymolysis liquid, mixing uniformly by vortex, carrying out enzymolysis for 8h in water bath at 30 ℃, centrifuging, and obtaining the precipitate as the phaffia rhodozyma protoplast.
Example 2
The preparation method of the phaffia rhodozyma protoplast comprises the following steps:
(1) 4g of the phaffia rhodozyma is fully washed by sterile water and centrifuged to take the sediment.
(2) Preparing an enzymolysis liquid: adding muramidase 0.1g, snailase 0.1g, cellulase 0.1g, and collapse enzyme 0.1g into a solution containing KCl 0.5mol/L and MgSO 0.2mol/L
4Fully dissolving in 5mL of aqueous solution, and filtering to remove bacteria to obtain enzymatic hydrolysate.
(3) Adding the precipitate into the enzymolysis liquid, mixing uniformly by vortex, carrying out enzymolysis for 3h in water bath at 25 ℃, centrifuging, and obtaining the precipitate as the phaffia rhodozyma protoplast.
Example 3
(1) Weighing 150mg of organic-inorganic composite lipid, dissolving the organic-inorganic composite lipid in 10mL of trichloromethane, removing the trichloromethane by rotary evaporation, and forming a layer of transparent lipid-like film at the bottom of a round-bottom flask; suspending 2g of phaffia rhodozyma protoplast with 10mL of 180g/L mannitol aqueous solution, adding the suspended phaffia rhodozyma protoplast into the obtained lipid-like film, fully stirring and uniformly mixing, rapidly heating to 48 ℃, thermally shocking for 20s and simultaneously performing water bath ultrasound; rapidly cooling to 4 ℃, standing for 12h, and extruding by a liposome extruder (the aperture of a polycarbonate filter membrane is 1.0 mu m) to obtain the phaffia rhodozyma protoplasm body suspension with uniform particle size.
(2) Weighing chitosan (viscosity average molecular weight of 5.0 × 10) according to the mass ratio of 2:3:8
5Degree of deacetylation 85%), polyvinyl alcohol (weight average molecular weight 200000) and polyethylene oxide (weight average molecular weight 1X 10)
7) Completely dissolving 1.8 percent of total mass concentration in an acetic acid aqueous solution with volume solubility of 2 percent, adding glycerol with the mass-to-volume ratio of the acetic acid aqueous solution of 1.5g/L, adding the silica suspension prepared in the step (1) according to the volume ratio of the phaffia rhodozyma protoplast silica suspension to the acetic acid aqueous solution of 1:400, stirring at 4 ℃ until the mixture is completely mixed, adopting an electrostatic spinning method, using an aluminum foil as a receiving device, setting the receiving distance to be 20cm, setting the voltage to be 20KV, preparing a nanofiber membrane with the jet flow of 1mL/h, measuring the porosity of the nanofiber membrane to be 97.1 percent, and filling the nanofiber membrane into a chromatographic column in a coiled manner, wherein the figure is 2;
(3) preparing a mixed aqueous solution according to the concentrations of 50g/L glucose, 4g/L yeast extract, 2.5g/L corn steep liquor, 5g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 2g/L dipotassium hydrogen phosphate, 0.01g/L calcium chloride and 0.6g/L magnesium sulfate, adjusting the pH value of the solution to 5.5, filtering out bacteria, flowing through the chromatographic column in the step (2) at the flow rate of 0.2mL/h, and simultaneously introducing sterile air into the chromatographic column at the flow rate of 2.0V/V.min, controlling the temperature of a jacket at 25 ℃, and collecting an effluent to determine the astaxanthin concentration by using HPLC.
The average astaxanthin concentration reached 1.72g/L after 30 days of continuous operation, i.e. the productivity of the process was 1.72 g/(L.h).
Example 4
(1) Weighing 100mg of organic-inorganic composite lipid, dissolving in 10mL of trichloromethane, removing the trichloromethane by rotary evaporation, and forming a layer of transparent lipid-like film at the bottom of a round-bottom flask; suspending 3g of phaffia rhodozyma protoplast with 10mL of 100g/L mannitol aqueous solution, adding into the obtained lipid-like film, fully stirring and uniformly mixing, rapidly heating to 45 ℃, thermally exciting for 10s and simultaneously performing water bath ultrasound; rapidly cooling to 4 deg.C, standing for 12 hr, and extruding with liposome extruder (with polycarbonate filter membrane aperture of 0.5 μm) to obtain phaffia rhodozyma protoplasm body suspension with uniform particle size;
(2) weighing chitosan (viscosity average molecular weight 5.0 × 10) at a mass ratio of 1:2:7
5Degree of deacetylation 80%), polyvinyl alcohol (weight average molecular weight of 180000) and polyethylene oxide (weight average molecular weight of 1 × 10)
6) Completely dissolving 1.5 percent of total mass concentration into 2 percent of acetic acid aqueous solution, adding 1g/L of glycerol in the mass-to-volume ratio of the acetic acid aqueous solution, adding the silica suspension prepared in the step (1) according to the volume ratio of 1:100 of the Phaffia rhodozyma protoplasm silica suspension to the acetic acid aqueous solution, stirring at 4 ℃ until the mixture is completely mixed, adopting an electrostatic spinning method, using an aluminum foil as a receiving device, setting the receiving distance to be 5cm and the voltage to be 3KV, preparing a nanofiber membrane with the jet flow of 1mL/h, measuring the porosity of the nanofiber membrane to be 96.3 percent, and filling the nanofiber membrane into a chromatographic column in a coiled manner;
(3) preparing a mixed solution according to the concentrations of 40g/L glucose, 3g/L yeast extract, 2g/L corn steep liquor, 4g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 1.5g/L dipotassium hydrogen phosphate, 0.005g/L calcium chloride and 0.5g/L magnesium sulfate, adjusting the pH value of the solution to 5.0, filtering out bacteria, flowing through the chromatographic column in the step (2) at the flow rate of 0.2mL/h, simultaneously introducing sterile air into the chromatographic column at the flow rate of 1.0V/V.min, controlling the temperature of a jacket water to be 22 ℃, collecting an effluent liquid, and measuring the astaxanthin concentration by using HPLC.
The average astaxanthin concentration reached 1.22g/L, i.e. the productivity of the process was 1.22 g/(L.h), operating continuously for 30 days.
Example 5
(1) Weighing 200mg of organic-inorganic composite lipid, dissolving in 10mL of trichloromethane, removing the trichloromethane by rotary evaporation, and forming a layer of transparent lipid-like film at the bottom of a round-bottom flask; suspending 1g of phaffia rhodozyma protoplast with 10mL of 180g/L mannitol aqueous solution, adding the suspended phaffia rhodozyma protoplast into the obtained lipid-like film, fully stirring and uniformly mixing, rapidly heating to 50 ℃, thermally shocking for 30s and simultaneously performing water bath ultrasound; rapidly cooling to 4 deg.C, standing for 12 hr, and extruding with liposome extruder (with polycarbonate filter membrane aperture of 0.8 μm) to obtain phaffia rhodozyma protoplasm body suspension with uniform particle size;
(2) weighing chitosan (viscosity average molecular weight 5.0 × 10) according to the mass ratio of 2:4:9
5Degree of deacetylation 80%), polyvinyl alcohol (weight average molecular weight of 180000) and polyethylene oxide (weight average molecular weight of 1 × 10)
6) Completely dissolving 2% of total mass concentration into 2% of acetic acid aqueous solution with volume solubility, adding glycerol with the mass-to-volume ratio of the acetic acid aqueous solution of 2g/L, adding the silica suspension prepared in the step (1) according to the volume ratio of the phaffia rhodozyma protoplasm silica suspension to the acetic acid aqueous solution of 1:500, stirring at 4 ℃ until the mixture is completely mixed, adopting an electrostatic spinning method, using an aluminum foil as a receiving device, setting the receiving distance to be 30cm and the voltage to be 30KV, preparing a nanofiber membrane with the jet flow of 5mL/h, measuring the porosity of the nanofiber membrane to be 93.9%, and filling the nanofiber membrane into a chromatographic column in a coiled manner;
(3) preparing a mixed solution according to the concentrations of 60g/L glucose, 5g/L yeast extract, 3g/L corn steep liquor, 6g/L ammonium sulfate, 0.8g/L potassium dihydrogen phosphate, 3g/L dipotassium hydrogen phosphate, 0.02g/L calcium chloride and 0.8g/L magnesium sulfate, adjusting the pH value of the solution to 5.8, filtering out bacteria, flowing through the chromatographic column in the step (2) at the flow rate of 0.5mL/h, simultaneously introducing sterile air into the chromatographic column at the flow rate of 1.5V/V.min, controlling the temperature of a jacket water to be 27 ℃, and collecting an effluent liquid to measure the astaxanthin concentration by utilizing HPLC.
The average astaxanthin concentration reached 1.21g/L, i.e. the productivity of the process was 1.21 g/(L.h), operating continuously for 30 days.
Comparative example 1
(1) Suspending 3g of phaffia rhodozyma protoplast by using 5mL of 180g/L mannitol aqueous solution to prepare phaffia rhodozyma protoplast suspension;
(2) weighing chitosan (viscosity average molecular weight 5.0 × 10) at a mass ratio of 1:2:7
5Degree of deacetylation 85%), polyvinyl alcohol (molecular weight 200000) and polyethylene oxide (molecular weight 1X 10)
7) Completely dissolving 1.8% of total mass concentration in 2% of volume solubility acetic acid aqueous solution, adding glycerol with the mass-to-volume ratio of the acetic acid aqueous solution of 2g/L, adding the phaffia rhodozyma protoplast suspension prepared in the step (1) according to the volume ratio of the phaffia rhodozyma protoplast suspension to the acetic acid aqueous solution of 1:400, stirring at 4 ℃ until the phaffia rhodozyma protoplast suspension is completely and uniformly mixed, adopting an electrostatic spinning method, using an aluminum foil as a receiving device, setting the receiving distance to be 20cm and the voltage to be 20KV, preparing a nanofiber membrane with the jet flow of 1mL/h, measuring the porosity of the nanofiber membrane to be 96.2%, and filling the nanofiber membrane into a chromatographic column in a coiled manner;
(3) preparing a mixed solution according to the concentrations of 50g/L glucose, 4g/L yeast extract, 2.5g/L corn steep liquor, 5g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 2g/L dipotassium hydrogen phosphate, 0.01g/L calcium chloride and 0.6g/L magnesium sulfate, adjusting the pH value of the solution to 5.5, filtering out bacteria, flowing through the chromatographic column in the step (2) at the flow rate of 0.2mL/h, simultaneously introducing sterile air into the chromatographic column at the flow rate of 2.0V/V.min, controlling the temperature of a jacket water to be 25 ℃, collecting effluent and measuring the astaxanthin concentration by using HPLC.
The average concentration of astaxanthin was 0.06g/L, i.e.the productivity of the process was 0.06 g/(L.h), operating continuously for 30 days.
Comparative example 2
(1) Suspending 3g of phaffia rhodozyma with 5mL of aqueous solution to prepare phaffia rhodozyma thallus suspension;
(2) weighing chitosan (viscosity average molecular weight 5.0 × 10) at a mass ratio of 1:2:7
5Degree of deacetylation 85%), polyvinyl alcohol (molecular weight 200000) and polyethylene oxide (molecular weight 1X 10)
7) Completely dissolving 1.8 percent of total mass concentration in 2 percent of volume solubility acetic acid aqueous solution, adding glycerol with the mass-volume ratio of the acetic acid aqueous solution of 2g/L, adding the phaffia rhodozyma thallus suspension prepared in the step (1) according to the volume ratio of the phaffia rhodozyma thallus suspension to the acetic acid solution of 1:400, stirring at 4 ℃ until the phaffia rhodozyma thallus suspension is completely and uniformly mixed, filling the mixture into an injector, dripping 1mol/L of NaOH solution to form microspheres, washing the microspheres to be neutral by using water, measuring the porosity of the microspheres to be 81.5 percent, and filling the microspheres into a chromatographic column;
(3) preparing a mixed solution according to the concentrations of 50g/L glucose, 4g/L yeast extract, 2.5g/L corn steep liquor, 5g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 2g/L dipotassium hydrogen phosphate, 0.01g/L calcium chloride and 0.6g/L magnesium sulfate, adjusting the pH value of the solution to 5.5, filtering out bacteria, flowing through the chromatographic column in the step (2) at the flow rate of 0.2mL/h, simultaneously introducing sterile air into the chromatographic column at the flow rate of 2.0V/V.min, controlling the temperature of a jacket water to be 25 ℃, collecting effluent and measuring the astaxanthin concentration by using HPLC.
The average concentration of astaxanthin was 0.09g/L, i.e. the productivity of the process was 0.09 g/(L.h), for 30 days of continuous operation.
The invention provides a method for producing astaxanthin by self-assembly silica body electrostatic spinning fiber membrane immobilized cells, and a method and a way for realizing the method are numerous, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and the improvements and decorations should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (6)
1. A method for producing astaxanthin by self-assembled silica body electrostatic spinning fibrous membrane immobilized cells is characterized by comprising the following steps:
the method comprises the following steps: preparing a phaffia rhodozyma protoplast: weighing organic-inorganic composite lipid and phaffia rhodozyma protoplast in a mass ratio of 0.1-0.2: 1-3; dissolving organic-inorganic composite lipid in chloroform, and evaporating to remove chloroform to obtain lipid film; suspending the phaffia rhodozyma protoplast in a mannitol aqueous solution, adding the phaffia rhodozyma protoplast into the obtained lipid-like film, stirring and uniformly mixing, rapidly heating to 45-50 ℃, thermally exciting for 10-30 s, simultaneously performing water bath ultrasound, cooling to 4 ℃, standing, and extruding by a liposome extruder to obtain a phaffia rhodozyma protoplast suspension;
step two: preparing a nanofiber membrane: dissolving chitosan, polyvinyl alcohol and polyoxyethylene in an acetic acid aqueous solution, adding glycerol, adding the phaffia rhodozyma protoplasm liposome suspension prepared in the step one, uniformly stirring, preparing a nanofiber membrane through electrostatic spinning, filling the nanofiber membrane into a chromatographic column in a coiled manner, and leaving a space in the center of the chromatographic column;
step three: preparing a mixed solution of glucose, yeast extract, corn steep liquor, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride and magnesium sulfate, filtering bacteria, passing through the chromatographic column prepared in the second step, and collecting an effluent liquid to obtain the compound enzyme preparation;
in the first step, the phaffia rhodozyma protoplast is obtained through the following steps:
step a: washing Phaffia rhodozyma with sterile water, centrifuging and taking precipitate;
step b: preparing an enzymolysis liquid: respectively taking lywallzyme, helicase, cellulase and collapse enzyme with the mass of 2-4% of the precipitate obtained in the step a, and adding the lywallzyme, the helicase, the cellulase and the collapse enzyme into a mixture containing 0.3-0.5 mol/L KCl and 0.2-0.4 mol/L MgSO (MgSO) according to the mass-volume ratio of 0.1: 5-10 g/mL
4Stirring and dissolving the mixture in the aqueous solution, and filtering bacteria to obtain an enzymolysis solution;
step c: adding the precipitate obtained in the step a into the enzymolysis liquid obtained in the step b, uniformly mixing in a vortex manner, carrying out water bath enzymolysis at the temperature of 25-30 ℃ for 3-8 h, and centrifuging to obtain the precipitate, namely the phaffia rhodozyma protoplast;
in the first step, the structural formula of the organic-inorganic composite lipid is as follows:
in the first step, the mass-volume ratio of the organic-inorganic composite lipid to the chloroform is 100-200:10 mg/mL; the concentration of the mannitol aqueous solution is 100-180g/L, and the mass volume ratio of the phaffia rhodozyma protoplast to the mannitol aqueous solution is 1-3: 10 g/mL;
the liposome extruder adopts a polycarbonate filter membrane, and the aperture of the filter membrane is 0.5-1.0 μm.
2. The method of producing astaxanthin according to claim 1, wherein in the second step, the viscosity average molecular weight of the chitosan is 5.0X 10
5The deacetylation degree is 80-85%; the weight average molecular weight of the polyvinyl alcohol is 180000-200000; the polyoxyethylene has a weight average molecular weight of 1 × 10
6～1×10
7(ii) a The volume concentration of the acetic acid aqueous solution is 1-2%; the mass ratio of the chitosan to the polyvinyl alcohol to the polyoxyethylene is 1-2: 2-4: 7-9, and the chitosan, the polyvinyl alcohol and the polyoxyethylene are dissolved in an acetic acid water solution with the total mass concentration of 1.5-2%.
3. The method for producing astaxanthin according to claim 1, wherein in the second step, the mass-to-volume ratio of the glycerol to the acetic acid aqueous solution is 1-2 g/L; the volume ratio of the addition amount of the phaffia rhodozyma protoplasm liposome suspension to the acetic acid aqueous solution is 1: 100-500.
4. The method for producing astaxanthin according to claim 1, wherein in the second step, aluminum foil is used as the receiving device for electrostatic spinning, the distance from the spray head to the receiving device is 5-30 cm, the voltage is 3-30 KV, and the spray flow rate is 1-5 mL/h.
5. The method for producing astaxanthin according to claim 1, wherein in the third step, the mixed aqueous solution has a glucose concentration of 40 to 60g/L, a yeast extract concentration of 3 to 5g/L, a corn steep liquor concentration of 2 to 3g/L, an ammonium sulfate concentration of 4 to 6g/L, a potassium dihydrogen phosphate concentration of 0.2 to 0.8g/L, a dipotassium hydrogen phosphate concentration of 1.5 to 3g/L, a calcium chloride concentration of 0.005 to 0.02g/L, and a magnesium sulfate concentration of 0.5 to 0.8 g/L; and adjusting the pH value of the mixed aqueous solution to 5.0-5.8.
6. The method for producing astaxanthin according to claim 1, wherein in the third step, the flow rate of the mixed aqueous solution is 0.2-0.5 mL/h; the ventilation quantity of sterile air in the chromatographic column is 1.0-2.0V/V.min, and the temperature of a chromatographic column jacket is controlled at 22-27 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710883642.4A CN107400693B (en) | 2017-09-26 | 2017-09-26 | Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710883642.4A CN107400693B (en) | 2017-09-26 | 2017-09-26 | Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107400693A CN107400693A (en) | 2017-11-28 |
| CN107400693B true CN107400693B (en) | 2020-02-11 |
Family
ID=60388792
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710883642.4A Active CN107400693B (en) | 2017-09-26 | 2017-09-26 | Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107400693B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109432021B (en) * | 2018-11-21 | 2021-05-25 | 江苏大学 | Astaxanthin nano preparation prepared by electrostatic spraying method and preparation method thereof |
| CN110373897B (en) * | 2019-07-15 | 2021-09-10 | 南通大学 | Natural washable anti-hypha and wool fabric finishing method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705274A (en) * | 2009-12-07 | 2010-05-12 | 中国农业大学 | Phaffia rhodozyma culture method for improving content of astaxanthin in phaffia rhodozyma cells |
| CN104450665A (en) * | 2014-11-27 | 2015-03-25 | 华南理工大学 | Method for immobilizing phaffia rhodozyma and preparing neokestose and application of method |
| CN105802950A (en) * | 2016-05-24 | 2016-07-27 | 天津工业大学 | Beaded electrospun nanofiber immobilized saccharomycete and preparation method thereof |
-
2017
- 2017-09-26 CN CN201710883642.4A patent/CN107400693B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705274A (en) * | 2009-12-07 | 2010-05-12 | 中国农业大学 | Phaffia rhodozyma culture method for improving content of astaxanthin in phaffia rhodozyma cells |
| CN104450665A (en) * | 2014-11-27 | 2015-03-25 | 华南理工大学 | Method for immobilizing phaffia rhodozyma and preparing neokestose and application of method |
| CN105802950A (en) * | 2016-05-24 | 2016-07-27 | 天津工业大学 | Beaded electrospun nanofiber immobilized saccharomycete and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| 新型有机-无机杂化陶瓷型脂质体"Cerasome"的研究与应用;李纪龙 等;《材料导报》;20090731;第23卷(第7期);第51页第1.1-1.2节 * |
| 红发夫酵母Phaffia rhodozyma培养生产虾青素的研究;朱明军;《中国优秀博硕士学位论文全文数据库 (博士) 工程科技Ⅰ辑》;20061215(第12（2016）期);摘要 * |
| 红发夫酵母催化合成新科思糖的研究;别晓颖;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20151215(第12（2015）期);摘要，第21页第5段 * |
| 红发夫酵母的生物学特性;裘娟萍 等;《工业微生物》;20010930;第31卷(第3期);第6-8页 * |
| 脂质体制备方法的里程碑-挤出法;蒋宫平 等;《沈阳药科大学学报》;20130831;第30 卷(第8 期);第646-652页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107400693A (en) | 2017-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107400689B (en) | Method for producing nisin by self-assembled silica body electrostatic spinning fibrous membrane immobilized cells | |
| Xi et al. | Drug loading techniques for exosome-based drug delivery systems | |
| CN107400693B (en) | Method for producing astaxanthin by self-assembled siliceous body electrostatic spinning fibrous membrane immobilized cells | |
| CN108714222A (en) | A kind of preparation and application of novel and multifunctional supermolecule vesica | |
| CN106974897B (en) | Targeting stimulation-responsive multifunctional cerium dioxide nano drug-carrying system | |
| CN103756002B (en) | A kind of porous magnetic spherex and preparation method thereof | |
| CN107686836B (en) | Method for producing cordycepin by self-assembled silica body electrostatic spinning fibrous membrane immobilized cells | |
| CN107475345B (en) | Method for producing ganoderic acid by using self-assembled silica body electrostatic spinning fibrous membrane immobilized cells | |
| CN102329340A (en) | Method for preparing D-mannose | |
| CN101744789B (en) | Biological medicine carrying microcapsule which can degrade nanometer porous polymer L- glutamic acid/ chitosan and preparing method thereof | |
| CN105435658B (en) | A kind of preparation method of supermolecule composite separating film | |
| CN101265448B (en) | Grease catalysis separation biphasic enzyme-film bioreactor and its preparation and application | |
| CN113081965B (en) | Sensitivity and H based on ROS 2 S-responsive multifunctional liposome and preparation method and application thereof | |
| CN101624607A (en) | Method for preparing hydroxytyrosol | |
| CN112646055A (en) | Preparation method of low-molecular-weight hyaluronic acid | |
| CN101429530A (en) | Process for producing reduction type cozymase Q10 based on redex enzyme | |
| CN103539669A (en) | Method for synthesizing triethyl citrate by molecular sieve supported ionic liquid catalysis | |
| CN103194857A (en) | Preparation method of electrostatic spinning glucose ether/acrylonitrile copolymer nanofiber membrane | |
| CN107653282A (en) | A kind of method of substrate and the double immobilization production glucoside compounds of catalyst | |
| CN107304413A (en) | A kind of excretion body quick separating and the kit of purifying | |
| CN102871985B (en) | Hemoglobin microcapsule blood substitute and its preparation method | |
| CN104059114A (en) | Glucoside containing azobenzene group, and preparation method and application thereof | |
| WO2017051882A1 (en) | Amphipathic block copolymer, molecule assembly and method for producing same, and protein-included agent | |
| KR101529709B1 (en) | Method for production of soybean isoflavone aglycone | |
| CN110859965B (en) | Multifunctional nanoparticle with AIE characteristics and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210618 Address after: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee after: Center for technology transfer, Nantong University Address before: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee before: NANTONG University |
|
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20171128 Assignee: NANTONG HAOYOU FOOD ADDITIVES Co.,Ltd. Assignor: Center for technology transfer, Nantong University Contract record no.: X2021980016476 Denomination of invention: A method for producing astaxanthin by immobilizing cells with self-assembled silica electrospinning fiber membrane Granted publication date: 20200211 License type: Common License Record date: 20211228 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee after: Nantong University Technology Transfer Center Co.,Ltd. Address before: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee before: Center for technology transfer, Nantong University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221128 Address after: Room 801, 85 Kefeng Road, Huangpu District, Guangzhou City, Guangdong Province Patentee after: Yami Technology (Guangzhou) Co.,Ltd. Address before: 226019 Jiangsu city of Nantong province sik Road No. 9 Patentee before: Nantong University Technology Transfer Center Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240301 Address after: Unit 3, No. 264 Xizhongjiawa, Shibei District, Qingdao City, Shandong Province, 266011 Patentee after: Wang Guisen Country or region after: China Patentee after: Qingdao Hengnuo Shijia Trading Co.,Ltd. Address before: Room 801, 85 Kefeng Road, Huangpu District, Guangzhou City, Guangdong Province Patentee before: Yami Technology (Guangzhou) Co.,Ltd. Country or region before: China |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240326 Address after: Building 608, No. 6 Fengjin Road, Chengyang District, Qingdao City, Shandong Province, 266000 Patentee after: Qingdao Hengnuo Shijia Brand management Co.,Ltd. Country or region after: China Address before: Unit 3, No. 264 Xizhongjiawa, Shibei District, Qingdao City, Shandong Province, 266011 Patentee before: Wang Guisen Country or region before: China Patentee before: Qingdao Hengnuo Shijia Trading Co.,Ltd. |