CN117867041A - Method for preparing flavor ester by ultraviolet/visible light response interface enzyme catalysis - Google Patents
Method for preparing flavor ester by ultraviolet/visible light response interface enzyme catalysis Download PDFInfo
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- CN117867041A CN117867041A CN202311696639.3A CN202311696639A CN117867041A CN 117867041 A CN117867041 A CN 117867041A CN 202311696639 A CN202311696639 A CN 202311696639A CN 117867041 A CN117867041 A CN 117867041A
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- hmss
- light response
- ultraviolet
- enzyme
- visible light
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 64
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 64
- 230000004298 light response Effects 0.000 title claims abstract description 63
- 150000002148 esters Chemical class 0.000 title claims abstract description 34
- 239000000796 flavoring agent Substances 0.000 title claims abstract description 33
- 235000019634 flavors Nutrition 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 64
- 238000006243 chemical reaction Methods 0.000 claims abstract description 48
- 239000000839 emulsion Substances 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 102000004882 Lipase Human genes 0.000 claims abstract description 28
- 108090001060 Lipase Proteins 0.000 claims abstract description 28
- 239000004367 Lipase Substances 0.000 claims abstract description 27
- 235000019421 lipase Nutrition 0.000 claims abstract description 27
- 238000003756 stirring Methods 0.000 claims abstract description 23
- 238000005886 esterification reaction Methods 0.000 claims abstract description 19
- 239000012074 organic phase Substances 0.000 claims abstract description 14
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 7
- 229930195729 fatty acid Natural products 0.000 claims abstract description 7
- 239000000194 fatty acid Substances 0.000 claims abstract description 7
- 150000004666 short chain fatty acids Chemical group 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract 4
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 37
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 25
- 235000019441 ethanol Nutrition 0.000 claims description 24
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 claims description 18
- 239000012071 phase Substances 0.000 claims description 17
- NCDCLPBOMHPFCV-UHFFFAOYSA-N hexyl hexanoate Chemical compound CCCCCCOC(=O)CCCCC NCDCLPBOMHPFCV-UHFFFAOYSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- YPQSPODHFDGVAC-UHFFFAOYSA-N Butyl heptanoate Chemical compound CCCCCCC(=O)OCCCC YPQSPODHFDGVAC-UHFFFAOYSA-N 0.000 claims description 8
- -1 N-carboxyl indolium iodide Chemical compound 0.000 claims description 8
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 claims description 8
- PQLMXFQTAMDXIZ-UHFFFAOYSA-N isoamyl butyrate Chemical compound CCCC(=O)OCCC(C)C PQLMXFQTAMDXIZ-UHFFFAOYSA-N 0.000 claims description 8
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 8
- RPRPDTXKGSIXMD-UHFFFAOYSA-N butyl hexanoate Chemical compound CCCCCC(=O)OCCCC RPRPDTXKGSIXMD-UHFFFAOYSA-N 0.000 claims description 7
- 239000003995 emulsifying agent Substances 0.000 claims description 7
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 238000000967 suction filtration Methods 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- MNWFXJYAOYHMED-UHFFFAOYSA-N heptanoic acid Chemical compound CCCCCCC(O)=O MNWFXJYAOYHMED-UHFFFAOYSA-N 0.000 claims description 6
- PBGWNXWNCSSXCO-UHFFFAOYSA-N hexyl octanoate Chemical compound CCCCCCCC(=O)OCCCCCC PBGWNXWNCSSXCO-UHFFFAOYSA-N 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 5
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 claims description 4
- BBMCTIGTTCKYKF-UHFFFAOYSA-N 1-heptanol Chemical compound CCCCCCCO BBMCTIGTTCKYKF-UHFFFAOYSA-N 0.000 claims description 4
- XVSZRAWFCDHCBP-UHFFFAOYSA-N 3-methylbutyl hexanoate Chemical compound CCCCCC(=O)OCCC(C)C XVSZRAWFCDHCBP-UHFFFAOYSA-N 0.000 claims description 4
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 claims description 4
- RGXWDWUGBIJHDO-UHFFFAOYSA-N ethyl decanoate Chemical compound CCCCCCCCCC(=O)OCC RGXWDWUGBIJHDO-UHFFFAOYSA-N 0.000 claims description 4
- TVQGDYNRXLTQAP-UHFFFAOYSA-N ethyl heptanoate Chemical compound CCCCCCC(=O)OCC TVQGDYNRXLTQAP-UHFFFAOYSA-N 0.000 claims description 4
- BYEVBITUADOIGY-UHFFFAOYSA-N ethyl nonanoate Chemical compound CCCCCCCCC(=O)OCC BYEVBITUADOIGY-UHFFFAOYSA-N 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 229940094941 isoamyl butyrate Drugs 0.000 claims description 4
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 4
- KQNPFQTWMSNSAP-UHFFFAOYSA-N isobutyric acid Chemical compound CC(C)C(O)=O KQNPFQTWMSNSAP-UHFFFAOYSA-N 0.000 claims description 4
- FBUKVWPVBMHYJY-UHFFFAOYSA-N nonanoic acid Chemical compound CCCCCCCCC(O)=O FBUKVWPVBMHYJY-UHFFFAOYSA-N 0.000 claims description 4
- PSXNDMJWRZYVTM-UHFFFAOYSA-N octanoic acid butyl ester Natural products CCCCCCCC(=O)OCCCC PSXNDMJWRZYVTM-UHFFFAOYSA-N 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- CFNJLPHOBMVMNS-UHFFFAOYSA-N pentyl butyrate Chemical compound CCCCCOC(=O)CCC CFNJLPHOBMVMNS-UHFFFAOYSA-N 0.000 claims description 4
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims description 4
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 3
- FLHJIAFUWHPJRT-UHFFFAOYSA-N 2,3,3-trimethylindole Chemical compound C1=CC=C2C(C)(C)C(C)=NC2=C1 FLHJIAFUWHPJRT-UHFFFAOYSA-N 0.000 claims description 3
- IHFRMUGEILMHNU-UHFFFAOYSA-N 2-hydroxy-5-nitrobenzaldehyde Chemical compound OC1=CC=C([N+]([O-])=O)C=C1C=O IHFRMUGEILMHNU-UHFFFAOYSA-N 0.000 claims description 3
- KMRNTNDWADEIIX-UHFFFAOYSA-N 3-Iodopropanoic acid Chemical compound OC(=O)CCI KMRNTNDWADEIIX-UHFFFAOYSA-N 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 241001661345 Moesziomyces antarcticus Species 0.000 claims description 3
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 229960002446 octanoic acid Drugs 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 230000004043 responsiveness Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- WJTCHBVEUFDSIK-NWDGAFQWSA-N (2r,5s)-1-benzyl-2,5-dimethylpiperazine Chemical compound C[C@@H]1CN[C@@H](C)CN1CC1=CC=CC=C1 WJTCHBVEUFDSIK-NWDGAFQWSA-N 0.000 claims description 2
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 claims description 2
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 claims description 2
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 claims description 2
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 claims description 2
- 241000222175 Diutina rugosa Species 0.000 claims description 2
- ICMAFTSLXCXHRK-UHFFFAOYSA-N Ethyl pentanoate Chemical compound CCCCC(=O)OCC ICMAFTSLXCXHRK-UHFFFAOYSA-N 0.000 claims description 2
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 claims description 2
- 239000005643 Pelargonic acid Substances 0.000 claims description 2
- 241000223258 Thermomyces lanuginosus Species 0.000 claims description 2
- 241000235015 Yarrowia lipolytica Species 0.000 claims description 2
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 238000004945 emulsification Methods 0.000 claims description 2
- YYZUSRORWSJGET-UHFFFAOYSA-N octanoic acid ethyl ester Natural products CCCCCCCC(=O)OCC YYZUSRORWSJGET-UHFFFAOYSA-N 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 229940005605 valeric acid Drugs 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- 238000001354 calcination Methods 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 abstract description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 6
- 239000000178 monomer Substances 0.000 abstract description 6
- 238000002360 preparation method Methods 0.000 abstract description 6
- 230000002255 enzymatic effect Effects 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 230000032050 esterification Effects 0.000 abstract description 3
- 239000000377 silicon dioxide Substances 0.000 abstract description 3
- 150000002632 lipids Chemical class 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract 2
- 150000001412 amines Chemical class 0.000 abstract 1
- 125000003277 amino group Chemical group 0.000 abstract 1
- 238000006482 condensation reaction Methods 0.000 abstract 1
- 238000006911 enzymatic reaction Methods 0.000 abstract 1
- 238000003980 solgel method Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 31
- 230000000052 comparative effect Effects 0.000 description 7
- 108010093096 Immobilized Enzymes Proteins 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 4
- 230000002210 biocatalytic effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000001678 irradiating effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000011942 biocatalyst Substances 0.000 description 3
- 230000002051 biphasic effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- MGADZUXDNSDTHW-UHFFFAOYSA-N 2H-pyran Chemical compound C1OC=CC=C1 MGADZUXDNSDTHW-UHFFFAOYSA-N 0.000 description 1
- AWQSAIIDOMEEOD-UHFFFAOYSA-N 5,5-Dimethyl-4-(3-oxobutyl)dihydro-2(3H)-furanone Chemical compound CC(=O)CCC1CC(=O)OC1(C)C AWQSAIIDOMEEOD-UHFFFAOYSA-N 0.000 description 1
- 102100021851 Calbindin Human genes 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 101000898082 Homo sapiens Calbindin Proteins 0.000 description 1
- 101001021643 Pseudozyma antarctica Lipase B Proteins 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000002159 adsorption--desorption isotherm Methods 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- GONOPSZTUGRENK-UHFFFAOYSA-N benzyl(trichloro)silane Chemical compound Cl[Si](Cl)(Cl)CC1=CC=CC=C1 GONOPSZTUGRENK-UHFFFAOYSA-N 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000005543 nano-size silicon particle Substances 0.000 description 1
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- RUOJZAUFBMNUDX-UHFFFAOYSA-N propylene carbonate Chemical compound CC1COC(=O)O1 RUOJZAUFBMNUDX-UHFFFAOYSA-N 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 125000003003 spiro group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to the field of functional lipid enzymatic methods, and particularly relates to a method for preparing flavor ester by ultraviolet/visible light response interface enzyme catalysis. Firstly, synthesizing a light response monomer spiropyran SP-COOH, preparing a hollow mesoporous silica sphere HMSS by a sol-gel method, modifying an amino group to prepare HMSS-N, and then carrying out an amine condensation reaction on the SP-COOH and the HMSS-N to obtain a light response carrier (HMSS-SP); fixing free enzyme on a photoresponsive carrier through a multiple adsorption method to prepare photoresponsive immobilized lipase; mixing short/medium chain fatty acid and short/medium chain alcohol raw materials, adding light-responsive immobilized lipase, stirring or homogenizing to form a Pickering interfacial enzyme catalytic system (PIB), performing enzymatic esterification under a constant-temperature water bath, demulsifying by ultraviolet irradiation, and separating an upper-layer product organic phase; then adding new substrate, stirring again with visible light irradiation or homogenizing to form Pickering emulsion, and starting to catalyze the preparation of flavor ester by a new round of enzyme. The method has the advantages of wide raw material adaptability, high catalytic efficiency, high conversion rate of flavor ester, light response regulation and control, simple operation, environmental protection and the like.
Description
Technical Field
The invention belongs to the field of functional lipid enzymatic preparation, relates to a method for preparing flavor ester by ultraviolet/visible light response interface enzyme catalysis, and in particular relates to a method for synthesizing flavor ester by constructing an ultraviolet/visible light response interface biological enzyme catalysis system through efficient enzymatic esterification.
Background
Pickering Emulsion (PE) is stabilized by solid particles at the oil/water interface, and is a highly stable, biocompatible and environmentally friendly biocatalytic reaction platform. Compared with the traditional biphasic reaction system, the interfacial contact between the catalyst and the reactant is obviously enhanced; the lipase is adsorbed on the oil-water interface to improve the catalytic activity, and the immobilized enzyme is used as an emulsifying agent and a catalyst to stabilize emulsion. In addition, the byproduct water generated by the reaction is automatically transferred from the oil phase to the water phase, and the traditional laborious water removal step is not needed, so that the reaction is promoted to move forward. However, the emulsion breaking of Pickering emulsion is mainly carried out by means of centrifugation, filtration and the like, and has the problems of filter blockage, time consumption and the like. Second, there are problems of leaching, denaturation deactivation and decrease in biocatalytic activity due to the inherent brittleness of the free biocatalyst. The immobilized biocatalyst can promote the catalytic stability and promote the reutilization of the biocatalyst.
Stimulus responsive Pickering interface biocatalytic systems (PIB) may be the best choice to address these issues. Theoretically, by adjusting the environment's response to in situ stimuli, various morphological states of the emulsion, such as emulsion inversion or demulsification, can be achieved. On the other hand, the emulsifier can be recycled to support more environmentally friendly operation and adhere to green chemistry principles. So far, among external environmental factors, thermal triggers require more energy to overcome the high energy barrier of absorbed energy; CO 2 /N 2 The flip-flop requires a long time to invert; intentional introduction of pH or ionic strength changesThe incorporation of new ingredients may lead to chemical residual contamination and side effects on the enzymatic activity. In contrast, light can change the properties of the emulsion in a non-contact and non-invasive manner in the surrounding environment. Furthermore, light is a clean stimulus that triggers in an accurate and simple and easy to control manner compared to other external stimuli. To date, there has been no report of light response to pickering emulsion enzyme catalysis.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a method for preparing flavor ester by catalyzing with ultraviolet/visible light responsive interfacial enzyme, wherein Pickering emulsion can remarkably improve the catalytic interfacial area, the mass transfer performance of reactants and the catalytic activity of enzyme. However, the high stability of Pickering emulsions is detrimental to product separation and catalyst recovery and recycling. Stimulus responsive Pickering interface biocatalytic systems (PIB) may be the best choice to address these issues. Inversion or breaking of the emulsion can be achieved by adjusting the environmental response to the in situ stimulus. The emulsifier and the catalyst can be recycled to support more environment-friendly operation and adhere to the principles of green chemistry and sustainable development. For this purpose, the present invention has developed surface active particles having stimulus-induced properties and functional switching. Light is triggered in an accurate and simple and easily controllable manner, and Spiropyrans (SPs) can be reversibly switched between two different conformations (colorless SPs) and charge-type (purple cyanines MC) under uv and visible light irradiation. The O/W Pickering emulsion breaking and the reversible switching of the emulsion are realized by alternately irradiating ultraviolet light and visible light. The photoresponsive PIB system is successfully used for continuous green preparation of flavor ester, shows good substrate applicability and high stability, the conversion rate of the reaction is higher than 94% after 10 times of circulation, and the catalytic efficiency of enzyme is as high as 80.9mmol g -1 h -1 。
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
1) 1.0-12.0g of 2, 3-trimethyl indolenine, 1.25-15.0g of 3-iodopropionic acid and 7.5-90.0mL of toluene are weighed, reflux is carried out for 4-8 hours under the protection of nitrogen, and after the reaction is finished, dichloromethane is used for washing to obtain yellow N-carboxyl indolium iodide; 1.0-12.0g of N-carboxyl indolium iodide, 0.56-6.72g of 5-nitro salicylaldehyde and 0.5-6.0mL of piperidine are weighed into a 250mL round bottom flask containing 7.5-90.0mL of absolute ethyl alcohol, reflux is carried out for 4-8h under the protection of nitrogen, after the reaction is finished, the black solid is obtained through reduced pressure distillation, and methanol is used for washing and column chromatography to obtain yellow solid SP-COOH;
2) 0.16g CTAB was dissolved in 106mL of an aqueous ethanol solution containing ammonia (V) Water and its preparation method ：V Ethanol ：V Ammonia water =75: 30: 1) During stirring for 1h at 35 ℃, adding 0.25mL BTSE and 0.25mL TEOS, stirring for 24h at 35 ℃, suction-filtering to collect white nanoparticles, washing with ethanol three times, and drying the obtained HMSS at 60 ℃ overnight. The HMSS was sonicated in ultrapure water, incubated in an oven at 70 ℃ for 20h, collected by suction filtration and calcined in a tube furnace at 550 ℃ for 5h to remove the template CTAB. The mixed solution obtained by dispersing 1.0g of HMSS and 9mmol of APTES in 10mL of toluene is subjected to heat treatment in a 120 ℃ high-pressure reaction kettle for 20 hours, and the mixed solution is subjected to suction filtration, collected and washed 3 times by ethanol, and dried in a 60 ℃ oven to obtain HMSS-N. 200mg,0.526mmol SP-COOH was added to 30mL of a mixture of dimethylformamide containing 500mg of EDC and 400mg of NHS, stirred for 5min, then 50mg of HMSS-N was added, stirred in the dark at 25℃for 48h, and the resultant was washed 3 times with DMF and ethanol to give a photoresponsive carrier HMSS-SP;
3) Adding free enzyme into phosphate buffer solution to prepare enzyme solution; then mixing the enzyme solution with a photoresponsive carrier HMSS-SP, and drying to prepare immobilized lipase; wherein the light response carrier HMSS-SP is an ultraviolet light/visible light response carrier with the size of 270nm, the mesoporous aperture of 4.0nm and the water contact angle of 80-95 degrees;
4) Adding a light response carrier HMSS-SP/CL@HMSS-SP into an oil/water two-phase, forming a light response Pickering emulsion through stirring or homogenizing, realizing demulsification under the irradiation of ultraviolet light for 15-25min, and forming the Pickering emulsion through stirring or homogenizing again under the irradiation of visible light for 1-5min, wherein the number of alternating irradiation cycles is more than 10;
5) Adding short/medium chain fatty acid and short/medium chain alcohol into n-hexane/water containing light response carriers HMSS-SP and CL@HMSS-SP according to a molar ratio, stirring or homogenizing to form Pickering emulsion, then carrying out esterification reaction at 25-55 ℃ to prepare flavor ester, after the reaction is finished, carrying out ultraviolet irradiation demulsification, separating an upper product organic phase, continuously adding a new substrate, and stirring or homogenizing again after visible light irradiation to form Pickering emulsion to start a new round of enzyme catalysis to prepare the flavor ester.
Preferably, in step (3), the free enzyme is one or more of candida antarctica lipase, candida rugosa lipase, NS40086 lipase, thermomyces lanuginosus, candida lipolytica lipase; the pH of the enzyme solution is 6.0-8.0, and the concentration is 10-50mg/mL; the ratio of the carrier mass to the enzyme solution volume is 1:100-5:100 (m/v, g/mL); the immobilization time is 10-50min, and the temperature is 20-40 ℃; the pH of the phosphate buffer is 6.0-8.0.
Preferably, in the step (4), the oil-water ratio in the solution is 3:7-7:3, the oil phase is n-hexane, n-heptane or cyclohexane, the water phase is pure water or PBS buffer (pH=7.0), and the addition amount of the photoresponsive carrier is 1.0-3.0%.
Preferably, the short/medium chain fatty acid is one or more of butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, heptanoic acid, caprylic acid, pelargonic acid and capric acid; the short/medium chain alcohol is one or more of ethanol, propanol, butanol, isobutanol, amyl alcohol, isoamyl alcohol, hexanol, heptanol and octanol.
Preferably, in the step (4) and the step (5), ultraviolet light irradiates for 15-25min, and visible light irradiates for 1-5min; the number of irradiation cycles may be 10 or more alternately.
Preferably, the flavor esters produced are one or more of ethyl valerate, ethyl caproate, ethyl heptanoate, ethyl caprylate, ethyl pelargonate, ethyl caprate, amyl butyrate, isoamyl butyrate, butyl caproate, hexyl caproate, isoamyl caproate, butyl heptanoate, and hexyl caprylate.
Preferably, in step (5), the molar ratio of the short/medium chain fatty acid to the short/medium chain alcohol is 1:1-1:4, and the immobilized lipase with light responsiveness is in windThe addition amount of the flavoring ester in the synthesis is 0.1-1.0% and the addition amount of the HMSS-SP emulsifier is 0.8-2.9%, the esterification reaction is carried out in a constant temperature water bath at 25-55deg.C for 5-120min, the emulsion particle size is 30-110 μm, and the contact area is 1000-2000cm 2 /mL。
By the scheme, the conversion rate of the immobilized enzyme stabilized light response Pickering emulsion for preparing the flavor ester is up to more than 95.0%, and the conversion rate of the free enzyme and the emulsifier HMSS-SP stabilized light response Pickering emulsion for preparing the flavor ester is up to more than 82.0%; and the photoresponsive PIB system has high catalytic efficiency, good stability and reusability, and the conversion rate is still more than 94.0% after 10 cycles.
Compared with the prior art, the invention has the beneficial effects that:
1) Aiming at the problems that the high stability of Pickering emulsion is not beneficial to separation of products and recovery and recycling of catalysts, the invention designs and prepares a PIB system with ultraviolet/visible light response, hollow mesoporous silica spheres are grafted with light response monomer spiropyran on the surfaces, and meanwhile, the PIB system is used as an enzyme carrier and an emulsifier to establish a green and efficient responsive Pickering emulsion interface catalytic system, and the light response PIB system is successfully used for continuous green preparation of flavor esters.
2) According to the invention, through alternating irradiation of ultraviolet light and visible light, reversible switching between O/WPickering emulsion breaking and emulsification is realized; the emulsion breaking is realized by irradiating with ultraviolet light for 15-25min, and the Pickering emulsion can be formed after stirring or homogenizing with visible light for 1-5min, so that the circulation is realized for more than 10 times easily.
3) The method can be widely used for generating flavor ester through the esterification reaction of short/medium chain acid and short/medium chain alcohol, and the obtained product is controllable and various and meets the requirements of different application scenes;
4) The reaction process adopts an emulsion enzyme catalytic system, the reaction condition is mild, the reaction time is short, and the separation is simple; compared with the traditional chemical catalysis method, the method does not generate byproducts and wastes; compared with a nonaqueous enzyme catalysis method, the method has high product yield;
5) The invention also investigates the influence of ultraviolet/visible light on lipase structure and activity. Ultraviolet irradiation, wherein the spiropyran modified on the surface of the carrier is subjected to ring opening, so that the carrier becomes hydrophilic, and the lipase enzyme activity is reduced; after irradiation of visible light, the spiropyran modified on the surface of the carrier has ring-closing action, so that the carrier becomes hydrophobic and the lipase activity is recovered.
6) The photoresponsive PIB system of the invention exhibits good substrate suitability and stability, and Catalytic Efficiency (CE) of up to 80.9mmol g -1 h -1 Is 12.6 times of the free enzyme single-phase system. After 10 times of circulation, the reaction yield is still higher than 94 percent, and the invention provides a new idea for green and sustainable interfacial biocatalysis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a spiropyran (SP-COOH) monomer 1 HNMR(400MHz,CDCl 3 ,25℃,TMS)。
FIG. 2 shows the ultraviolet absorption spectrum of a spiropyran (SP-COOH) monomer, irradiated with ultraviolet light (a) and visible light (b).
FIG. 3 is an SEM (a) of hollow mesoporous silica spheres (HMSS-N) modified with a silane coupling agent, N of HMSS, HMSS-N, HMSS-SP and CL@HMSS-SP 2 Pore size distribution plot (b) of adsorption-desorption isotherms.
FIG. 4 is an absorption spectrum of SP-COOH at various concentrations (1.5-80. Mu.M) in absolute ethanol versus (inset: linear relationship of absorbance at 546nm to SP-COOH concentration) and an ultraviolet absorption spectrum of HMSS-SP at absolute ethanol (inset: picture of supernatant irradiated with ultraviolet lamp after HMSS-SP ultrasound for 1 h) before HMSS-SP ultrasonication for 1h and CL@HMSS-SP.
FIG. 5 is a FTIR spectrum of HMSS, HMSS-N, HMSS-SP, CL and CL@HMSS-SP.
FIG. 6 is a fluorescence confocal microscopy image of HMSS-SP or CL@HMSS-SP stabilized O/W Pickering emulsion (nile red labeled oil phase, FITC labeled lipase).
FIG. 7 is a photograph of example 6 UV/visible light irradiation O/W Pickering emulsion breaking and 10 alternating cycles of emulsion.
FIG. 8 is a graph of the reuse of the photo-responsive PIB system of example 6 to catalyze the esterification of hexanoic acid and hexanol to produce hexyl hexanoate.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The word "embodiment" as used herein does not necessarily mean that any embodiment described as "exemplary" is preferred or advantageous over other embodiments. Performance index testing in the examples herein, unless otherwise indicated, was performed using conventional testing methods in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; other test methods and techniques not specifically identified herein are those commonly employed by those of ordinary skill in the art.
Numerous specific details are set forth in the following examples in order to provide a better understanding of the present application. It will be understood by those skilled in the art that the present application may be practiced without some of these specific details. In the examples, some methods, means, instruments, devices, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present application. On the premise of no conflict, the technical features disclosed in the embodiments of the present application may be combined arbitrarily, and the obtained technical solution belongs to the disclosure of the embodiments of the present application.
The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.
In the following examples, the process for preparing HMSS-SP according to the invention is as follows:
(1) 1.0-12.0g of 2, 3-trimethyl indolenine, 1.25-15.0g of 3-iodopropionic acid and 7.5-90.0mL of toluene are weighed, reflux is carried out for 4-8 hours under the protection of nitrogen, and after the reaction is finished, dichloromethane is used for washing to obtain yellow N-carboxyl indolium iodide; 1.0-12.0g of N-carboxyl indolium iodide, 0.56-6.72g of 5-nitro salicylaldehyde and 0.5-6.0mL of piperidine are weighed into a 250mL round bottom flask containing 7.5-90.0mL of absolute ethyl alcohol, reflux is carried out for 4-8h under the protection of nitrogen, after the reaction is finished, the black solid is obtained through reduced pressure distillation, and methanol is used for washing and column chromatography to obtain yellow solid SP-COOH;
2) 0.16g CTAB was dissolved in 106mL of an aqueous ethanol solution containing ammonia (V) Water and its preparation method ：V Ethanol ：V Ammonia water =75: 30: 1) During stirring for 1h at 35 ℃, adding 0.25mL BTSE and 0.25mL TEOS, stirring for 24h at 35 ℃, suction-filtering to collect white nanoparticles, washing with ethanol three times, and drying the obtained HMSS at 60 ℃ overnight. The HMSS was sonicated in ultrapure water, incubated in an oven at 70 ℃ for 20h, collected by suction filtration and calcined in a tube furnace at 550 ℃ for 5h to remove the template CTAB. The mixed solution obtained by dispersing 1.0g of HMSS and 9mmol of APTES in 10mL of toluene is subjected to heat treatment in a 120 ℃ high-pressure reaction kettle for 20 hours, and the mixed solution is subjected to suction filtration, collected and washed 3 times by ethanol, and dried in a 60 ℃ oven to obtain HMSS-N. 200mg,0.526mmol SP-COOH was added to 30mL of a mixture of dimethylformamide containing 500mg of EDC and 400mg of NHS, stirred for 5min, then 50mg of HMSS-N was added, stirred for 48h in the dark at 25℃to give a product which was washed 3 times with DMF and ethanol to give HMSS-SP.
The successful synthesis of the above-described spiropyran (SP-COOH) monomer with light responsiveness, the results are shown in FIG. 1, respectively 1 HNMR spectra and figure 2 ultraviolet absorption spectra. The amino modified hollow mesoporous silicon sphere has a size of about 270nm, a core-shell mesoporous structure and a size of 4.0nm, and is shown in figure 3. According to the linear relation between the SP-COOH concentration and the absorbance of FIG. 4, the grafting rate of the spiro pyran (SP-COOH) subjected to ultrasonic treatment for 1h on the covalent coupling (HMSS-SP) of the amino-modified hollow mesoporous nano silicon spheres (HMSS-N) was 7.6%. In addition, there was little change in absorbance after lipase immobilization, indicating successful grafting of the photoresponsive SP-COOH monomer onto the surface of HMSS-N.
In the following examples, the specific procedure for preparing immobilized lipase by adsorption method is as follows:
3g of free Candida Antarctica Lipase (CALB) is dissolved in 50mL of phosphate buffer solution (50 mM) with pH of 7.0, HMSS-SP and enzyme solution are mixed according to the solid-to-liquid ratio of 10mg/mL, and the mixture is subjected to shaking table incubation for 40min and centrifugation; and freeze-drying the precipitate to obtain the immobilized lipase.
As a result of obtaining the immobilized lipase (CL@HMSS-SP) having a light-responsive property, the result is shown in FIG. 5, which shows stretching of aryl nitro group (1338, 1524cm -1 ) And vibration of acyl amide (1650 cm) -1 ) Indicating successful grafting of SP-COOH to HMSS-N, the lipase immobilization is characterized by the grafting of SP-COOH on HMSS-N at 1564, 1649 and 2930cm -1 At the peak, which is a characteristic signal of lipase CL.
Example 1
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol of caproic acid was added in molar ratio to caproic acid: butanol 1:1 is added into n-hexane/water (total volume 3mL, v: v=5:5) containing 1.0% of light response carrier (0.2% of light response CL@HMSS-SP and 0.8% of light response HMSS-SP), emulsion is formed by stirring and homogenizing, then esterification reaction is carried out at 30 ℃ for 2h to prepare butyl caproate, after the reaction is finished, ultraviolet light is irradiated for 15-25min to demulsify, and an upper product organic phase is separated to obtain the product.
The conversion of butyl hexanoate was determined to be 94.0%.
Example 2
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol of octanoic acid is used in molar ratio: hexanol 1:1 is added into n-hexane/water (total volume 3mL, v: v=7:3) containing 3.0% of light response carrier (0.3% of light response CL@HMSS-SP and 2.7% of light response HMSS-SP), emulsion is formed by stirring and homogenizing, then esterification reaction is carried out at 55 ℃ for 1h to prepare hexyl octanoate, after the reaction is finished, ultraviolet light is irradiated for 15-25min to demulsify, and an upper product organic phase is separated to obtain the product.
The conversion of hexyl octanoate was determined to be 96.4%.
Example 3
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol of caproic acid was added in molar ratio to caproic acid: isoamyl alcohol 1:2 is added into cyclohexane/water (total volume 3mL, v: v=6:4) containing 2.3% of light response carrier (0.5% of light response CL@HMSS-SP and 1.7% of light response HMSS-SP), emulsion is formed by stirring and homogenizing, then esterification reaction is carried out at 40 ℃ for 2 hours to prepare isoamyl caproate, after the reaction is finished, ultraviolet light is irradiated for 15-25 minutes to demulsify, and an upper product organic phase is separated to obtain the product.
The conversion of isoamyl hexanoate was determined to be 97.2%.
Example 4
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol of caproic acid was added in molar ratio to caproic acid: ethanol 1:2 is added into n-heptane/water (total volume 3mL, v: v=4:6) containing 1.7% of light response carrier (0.4% light response CL@HMSS-SP and 1.3% light response HMSS-SP), and the mixture is stirred and homogenized to form emulsion, then esterification reaction is carried out at 45 ℃ for 1h to prepare ethyl caproate, after the reaction is finished, ultraviolet light is irradiated for 15-25min to demulsify, and an upper product organic phase is separated to obtain the product.
The conversion of ethyl hexanoate was determined to be 98.8%.
Example 5
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol heptanoic acid was added in molar ratio heptanoic acid: butanol 1:1 is added into n-hexane/water (total volume 3mL, v: v=7:3) containing 3.0% of light response carrier (0.6% of light response CL@HMSS-SP and 2.4% of light response HMSS-SP), emulsion is formed by stirring and homogenizing, then esterification reaction is carried out at 50 ℃ for 1h to prepare butyl heptanoate, after the reaction is finished, ultraviolet light is irradiated for 15-25min to demulsify, and an upper product organic phase is separated to obtain the product.
The conversion of butyl heptanoate was determined to be 95.5%.
Example 6
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol of caproic acid was added in molar ratio to caproic acid: hexanol 1:1 is added into n-hexane/water (total volume 3mL, v: v=7:3) containing 2.3% of light response carrier (0.8% of light response CL@HMSS-SP and 1.5% of light response HMSS-SP), and is stirred and homogenized to form emulsion, then esterification reaction is carried out at 35 ℃ for 2h to prepare hexyl caproate, after the reaction is finished, ultraviolet light is irradiated for 15-25min to demulsify, and an upper product organic phase is separated to obtain the product. Adding new substrate, irradiating with visible light for 1-5min, stirring or homogenizing to form Pickering emulsion, and preparing flavor ester by enzyme catalysis.
The conversion of hexyl caproate was determined to be 97.0%, the Catalytic Efficiency (CE) was determined to be 80.9mmol g -1 h -1 The conversion was still greater than 94.0% over 10 cycles.
Example 7
A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis specifically comprises the following steps:
0.63mmol of butyric acid was reacted in molar ratio: isoamyl alcohol 1:2 is added into n-hexane/water (total volume 3mL, v: v=7:3) containing 2.3% of light response carrier (1.0% light response CL@HMSS-SP and 1.3% light response HMSS-SP), emulsion is formed by stirring or homogenizing, then esterification reaction is carried out at 25 ℃ for 2 hours to prepare isoamyl butyrate, after the reaction is finished, ultraviolet light is irradiated for 15-25 minutes to demulsify, and the organic phase of the upper product is separated to obtain the product.
The conversion of isoamyl butyrate was determined to be 94.3%.
Table 1 shows the conversion of the products of examples 1 to 7.
In order to further demonstrate the beneficial effects of the present invention for a better understanding of the present invention, the technical features disclosed herein are further illustrated by the following comparative examples, which are not to be construed as limiting the present invention. Other modifications of the invention which do not involve the inventive work, as would occur to those skilled in the art in light of the foregoing teachings, are also considered to be within the scope of the invention.
Comparative example 1
Free enzyme single phase system: 0.63mmol of caproic acid was added in molar ratio to caproic acid: hexanol 1:1 is added into 3mL of normal hexane containing 0.5% free enzyme, then the mixture is stirred in a constant temperature water bath at 35 ℃ for esterification reaction for 2 hours to prepare hexyl caproate, and after the reaction is finished, the organic phase product is separated to obtain the product.
The conversion of hexyl caproate was determined to be 6.4%, the Catalytic Efficiency (CE) 6.4mmol g -1 h -1 . The CE value of the photoresponsive Pickering interfacial enzyme catalytic system is 12.6 times that of the free enzyme single-phase system, and the total specific surface area of the photoresponsive PIB system is 1060cm 2 The method is improved by 84 times compared with a two-phase system.
Comparative example 2
Free enzyme biphasic system: 0.63mmol of caproic acid was added in molar ratio to caproic acid: hexanol 1:1 to n-hexane/water (total volume 3ml, v: v=7:3) containing 0.5% free enzyme, then stirring in a constant temperature water bath at 35 ℃ to carry out esterification reaction for 2 hours to prepare hexyl caproate, and separating the organic phase product after the reaction is finished to obtain the product.
The conversion of hexyl hexanoate was determined to be 23.9%, catalytic Efficiency (CE) 21.8mmol g -1 h -1 . The CE value of the photoresponsive Pickering interfacial enzyme catalytic system is 3.7 times that of the free enzyme biphasic system. In contrast to PIB systems, lipases cannot be recovered and are requiredThe product is separated by the means of cost consumption such as suction filtration, centrifugation and the like.
Comparative example 3
Immobilized enzyme single-phase system: 0.63mmol of caproic acid was added in molar ratio to caproic acid: hexanol 1:1 is added into 3mL of n-hexane containing 2.3% of light response HMSS-SP (containing 0.5% of light response CL@HMSS-SP), then the mixture is stirred in a constant temperature water bath at 35 ℃ for esterification reaction for 2 hours to prepare hexyl caproate, and after the reaction is finished, the organic phase product is separated to obtain the product.
The conversion of hexyl hexanoate was determined to be 81.9%, catalytic Efficiency (CE) 70.8mmol g -1 h -1 The CE value of the photoresponsive Pickering interfacial enzyme catalytic system is 1.1 times that of the immobilized enzyme single-phase system. Compared with the free enzyme single-phase system of comparative example 1, the lipase immobilization significantly improves the stability and enzyme activity of the lipase.
Comparative example 4
Pickering system of free enzyme: 0.63mmol of caproic acid was added in molar ratio to caproic acid: hexanol 1:1 is added into n-hexane/water (total volume 3mL, v: v=7:3) containing 2.3 percent of light response HMSS-SP and 0.5 percent of free enzyme, then the mixture is stirred in a constant temperature water bath at 35 ℃ for esterification reaction for 2 hours to prepare hexyl caproate, after the reaction is finished, ultraviolet light is irradiated for 15-25 minutes to demulsify, and an upper product organic phase is separated to obtain the product.
The conversion of hexyl caproate was determined to be 90.5%, catalytic Efficiency (CE) 73.3mmol g -1 h -1 The CE value of the photoresponsive Pickering interfacial enzyme catalytic system is 1.1 times that of the immobilized enzyme single-phase system. Compared with the free enzyme biphase system of comparative example 2, the Pickering emulsion system significantly improves the oil-water interface area and the mass transfer performance of the reactants. In addition, the emulsion breaking and the product separation can be realized only by irradiating ultraviolet light for 15-25 min.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis, which is characterized by comprising the following steps:
1) 1.0-12.0g of 2, 3-trimethyl indolenine, 1.25-15.0g of 3-iodopropionic acid and 7.5-90.0mL of toluene are weighed, reflux is carried out for 4-8 hours under the protection of nitrogen, and after the reaction is finished, dichloromethane is used for washing to obtain yellow N-carboxyl indolium iodide; 1.0-12.0g of N-carboxyl indolium iodide, 0.56-6.72g of 5-nitro salicylaldehyde and 0.5-6.0mL of piperidine are weighed into a 250mL round bottom flask containing 7.5-90.0mL of absolute ethyl alcohol, reflux is carried out for 4-8h under the protection of nitrogen, after the reaction is finished, the black solid is obtained through reduced pressure distillation, and methanol is used for washing and column chromatography to obtain yellow solid SP-COOH;
2) Dissolving 0.16g CTAB in 106mL of an aqueous solution of ammonia-containing aqueous ethanol, stirring for 1h at 35 ℃, adding 0.25mL of BTSE and 0.25mL of TEOS, stirring for 24h at 35 ℃, filtering to collect white nano particles, washing with ethanol three times, and drying the obtained HMSS at 60 ℃ overnight; dispersing HMSS in ultrapure water by ultrasonic, incubating for 20 hours in a baking oven at 70 ℃, collecting HMSS by suction filtration, and calcining for 5 hours in a tubular furnace at 550 ℃; dispersing 1.0g of HMSS and 9mmol of APTES into 10mL of toluene to obtain a mixed solution, carrying out heat treatment on the mixed solution for 20 hours in a high-pressure reaction kettle at 120 ℃, carrying out suction filtration, collecting, washing 3 times with ethanol, and drying in a baking oven at 60 ℃ to obtain HMSS-N; 200mg,0.526mmol SP-COOH was added to 30mL of a mixture of dimethylformamide containing 500mg of EDC and 400mg of NHS, stirred for 5min, then 50mg of HMSS-N was added, stirred in the dark at 25℃for 48h, and washed 3 times with DMF and ethanol to give a photoresponsive carrier HMSS-SP;
3) Adding free enzyme into phosphate buffer solution to prepare enzyme solution; mixing the enzyme solution with a light response carrier HMSS-SP, and drying to obtain immobilized lipase CL@HMSS-SP;
4) Adding a light response carrier HMSS-SP/CL@HMSS-SP into an oil/water two-phase, forming a light response Pickering emulsion through stirring or homogenizing, realizing demulsification under ultraviolet light irradiation, and forming the Pickering emulsion through stirring or homogenizing again under visible light irradiation;
5) Adding short/medium chain fatty acid and short/medium chain alcohol into n-hexane/water containing light response carriers HMSS-SP and CL@HMSS-SP according to a molar ratio, forming Pickering emulsion by stirring or homogenizing, and then carrying out esterification reaction at 25-55 ℃ to prepare flavor ester; after the reaction is finished, demulsification is realized under the irradiation of ultraviolet light, an organic phase of an upper product is separated, a new substrate is continuously added, and after the irradiation of visible light, the mixture is stirred or homogenized again to form Pickering emulsion, and a new round of enzyme catalytic reaction is started.
2. The method for preparing flavor ester by enzyme catalysis of an ultraviolet/visible light response interface according to claim 1, wherein in the step (3), the light response carrier HMSS-SP is an ultraviolet/visible light response carrier with a size of 270nm, a mesoporous pore diameter of 4.0nm and a water contact angle of 80 ° -95 °; the free enzyme is one or more of candida antarctica lipase, candida rugosa lipase, NS40086 lipase, thermomyces lanuginosus and candida lipolytica lipase; the pH of the enzyme solution is 6.0-8.0, and the concentration is 10-50mg/mL; the ratio relation between the carrier mass and the enzyme solution volume is 1:100-5:100 (m/v, g/mL); the immobilization time is 10-50min, and the temperature is 20-40 ℃; the pH of the phosphate buffer is 6.0-8.0.
3. The method of claim 1, wherein the short/medium chain fatty acid is one or more of butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid and capric acid; the short/medium chain alcohol is one or more of ethanol, propanol, butanol, isobutanol, amyl alcohol, isoamyl alcohol, hexanol, heptanol and octanol.
4. The method for preparing flavor ester by using ultraviolet/visible light response interfacial enzyme catalysis according to claim 1, wherein in the step (4), the oil-water ratio in the solution is 3:7-7:3, the oil phase is n-hexane, n-heptane or cyclohexane, the water phase is pure water or PBS buffer with pH=7.0, and the addition amount of the light response carrier HMSS-SP is 1.0-3.0%.
5. The method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis according to claim 1, wherein the ultraviolet irradiation demulsification time is 15-25min, the visible light irradiation emulsification time is 1-5min, and the alternating irradiation cycle times are more than 10 times.
6. The method of claim 1, wherein the flavor ester produced in step (5) is one or more of ethyl valerate, ethyl caproate, ethyl heptanoate, ethyl caprylate, ethyl pelargonate, ethyl caprate, amyl butyrate, isoamyl butyrate, butyl caproate, hexyl caproate, isoamyl caproate, butyl heptanoate, and hexyl caprylate.
7. The method for preparing flavor ester by ultraviolet/visible light response interfacial enzyme catalysis according to claim 1, wherein in the step (5), the molar ratio of short/medium chain fatty acid to short/medium chain alcohol is 1:1-1:4, the addition amount of immobilized lipase with light responsiveness in flavor ester synthesis is 0.1-1.0% and the addition amount of HMSS-SP emulsifier is 0.8-2.9%, the esterification reaction is carried out in a constant temperature water bath at 25-55 ℃, the reaction time is 5-120min, the emulsion particle size is 30-50 μm, and the contact area is 1000-2000cm 2 /mL。
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