CN110373442A - A kind of preparation method and application with high anti-oxidation activity fine jade disaccharides - Google Patents
A kind of preparation method and application with high anti-oxidation activity fine jade disaccharides Download PDFInfo
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- CN110373442A CN110373442A CN201910736279.2A CN201910736279A CN110373442A CN 110373442 A CN110373442 A CN 110373442A CN 201910736279 A CN201910736279 A CN 201910736279A CN 110373442 A CN110373442 A CN 110373442A
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- fine jade
- jade disaccharides
- disaccharides
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- 239000010977 jade Substances 0.000 title claims abstract description 104
- 150000002016 disaccharides Chemical class 0.000 title claims abstract description 86
- 230000000694 effects Effects 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 230000003064 anti-oxidating effect Effects 0.000 title claims abstract description 21
- 229920001817 Agar Polymers 0.000 claims abstract description 30
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 29
- 238000004321 preservation Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 63
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 19
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 17
- 238000000855 fermentation Methods 0.000 claims description 14
- 230000004151 fermentation Effects 0.000 claims description 14
- 238000004090 dissolution Methods 0.000 claims description 12
- 230000002779 inactivation Effects 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 11
- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 241000206672 Gelidium Species 0.000 claims description 9
- 235000010419 agar Nutrition 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 8
- 230000000593 degrading effect Effects 0.000 claims description 8
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 7
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 6
- 239000008236 heating water Substances 0.000 claims description 6
- 238000000703 high-speed centrifugation Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 4
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 241000927735 Penaeus Species 0.000 abstract description 18
- 235000013372 meat Nutrition 0.000 abstract description 16
- 241000238557 Decapoda Species 0.000 abstract description 15
- 150000003254 radicals Chemical class 0.000 abstract description 15
- 238000005259 measurement Methods 0.000 abstract description 8
- 230000003647 oxidation Effects 0.000 abstract description 8
- 238000007254 oxidation reaction Methods 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- -1 DPPH free radical Chemical class 0.000 abstract description 6
- 230000015556 catabolic process Effects 0.000 abstract description 6
- 238000006731 degradation reaction Methods 0.000 abstract description 6
- 230000002000 scavenging effect Effects 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 3
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 25
- ZTOJFFHGPLIVKC-YAFCTCPESA-N (2e)-3-ethyl-2-[(z)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound S\1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C/1=N/N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-YAFCTCPESA-N 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 8
- 230000007760 free radical scavenging Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 230000003078 antioxidant effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 150000002482 oligosaccharides Chemical class 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 244000276331 Citrus maxima Species 0.000 description 3
- 235000001759 Citrus maxima Nutrition 0.000 description 3
- 229910002567 K2S2O8 Inorganic materials 0.000 description 3
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 235000009508 confectionery Nutrition 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 229960004756 ethanol Drugs 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 229960004889 salicylic acid Drugs 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000530454 Litopenaeus schmitti Species 0.000 description 2
- CPLXHLVBOLITMK-UHFFFAOYSA-N Magnesium oxide Chemical compound [Mg]=O CPLXHLVBOLITMK-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000012055 fruits and vegetables Nutrition 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 230000003859 lipid peroxidation Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000238553 Litopenaeus vannamei Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 235000009727 food gelling agent Nutrition 0.000 description 1
- 235000003086 food stabiliser Nutrition 0.000 description 1
- 235000003132 food thickener Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000395 magnesium oxide Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- QGVNJRROSLYGKF-UHFFFAOYSA-N thiobarbital Chemical compound CCC1(CC)C(=O)NC(=S)NC1=O QGVNJRROSLYGKF-UHFFFAOYSA-N 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses a kind of preparation method and applications with high anti-oxidation activity fine jade disaccharides, belong to field of marine biotechnology.Fine jade disaccharides is prepared using agaropectin oligose as raw material, using enzyme edman degradation Edman in the present invention, by measurement fine jade disaccharides to DPPH free radical, ABTS+The Scavenging activity of free radical, OH free radical, realize the homogeneity preparation of high anti-oxidation activity fine jade disaccharides, and fine jade disaccharides is used for the fresh-keeping of Penaeus Vannmei meat, the effect that protein corruption in Penaeus Vannmei shrimp can effectively be slowed down, reduce fat oxidation rate, to improve freshness date, the shelf life for extending Penaeus Vannmei, realizes efficient utilization of the high anti-oxidation activity fine jade disaccharides in preservation of fishery field.
Description
Technical field
The present invention relates to a kind of preparation method with high anti-oxidation activity fine jade disaccharides more particularly to it is a kind of utilize enzymatic isolation method
The method with high anti-oxidation activity fine jade disaccharides and its application in preservation of fishery field are prepared, marine biotechnology is belonged to
Field.
Background technique
Agar-agar is a kind of water-soluble polysaccharide, is widely used in food, medicine and daily-use chemical industry industry.In food, agar-agar
It can be used as food gelling agents, stabilizer and thickener, play and increase food viscosity, make food is glutinous to slide, mouthfeel is rich in malleable；?
In medicine, agar-agar is mainly used as microbiological culture media.Catabolite of the agaropectin oligose as agar-agar, it is water-soluble preferable.Agar-agar
Oligosaccharides preparation method mainly has 3 kinds, respectively chemical method (including acid degradation method, oxidation degradation method), physical method (including microwave drop
Solution, irradiation-induced degradation method etc.) and enzyme process.Compared with chemical method and physical method, enzyme process, which prepares agaropectin oligose, has that reaction is mild, bottom
The advantages that object specificity is strong, yield is higher, by-product is few.Therefore, enzyme process prepares agaropectin oligose and its property research and becomes in recent years
Research hotspot.
Correlative study shows that agaropectin oligose has preferable antioxidant activity.Wherein fine jade disaccharides has preferable anti-oxidant work
Property, to ABTS+Three kinds of free radical, OH free radical, diphenyl bitter taste hydrazides (DPPH) free radicals all have stronger removing and make
With.In addition, fine jade disaccharides can also be used in the fresh-keeping of fruits and vegetables, sweet shaddock such as is handled with fine jade disaccharides, sweet shaddock fruit organic acid, total can be reduced
Sugar, soluble solid, lignin and VCLoss extend its freshness date and shelf life to improve the hardness of sweet shaddock fruit.
Protein rich in and a variety of unsaturated fatty acids in aquatic products, but because its water content is higher, microorganism is held
It easily breeds, is fatty oxidizable so as to cause putrid and deteriorated.Fine jade disaccharides is as a kind of nontoxic, efficient, tasteless, lower-cost natural
Antistaling agent has been applied in fruits and vegetables class plant is fresh-keeping, and it is fewer that fine jade disaccharides is applied to the research on preservation of fishery.
The application of low polymerization degree, high anti-oxidation ability agaropectin oligose in preservation of fishery field becomes the key technology of the art
Problem needs the integrated and application of key technology.
Summary of the invention
To overcome the deficiencies in the prior art described above, the present invention provides a kind of preparation with high anti-oxidation activity fine jade disaccharides
Fine jade disaccharides is prepared using enzyme edman degradation Edman using agaropectin oligose as raw material in method and application, by measurement fine jade disaccharides to DPPH from
By base, ABTS+The Scavenging activity of free radical, OH free radical realizes the homogeneity preparation of high anti-oxidation activity fine jade disaccharides, and will
Fine jade disaccharides is fresh-keeping for Penaeus Vannmei meat, realizes efficient benefit of the high anti-oxidation activity fine jade disaccharides in preservation of fishery field
With.
The present invention is achieved through the following technical solutions above-mentioned technical effect:
A kind of preparation method with high anti-oxidation activity fine jade disaccharides, specifically includes the following steps:
(1) preparation of fine jade disaccharides oligomer
1) dissolution of agaropectin oligose: the solution that mass concentration is 1% is configured by agaropectin oligose with deionized water, in 30 DEG C of water-baths
Heating stirring is allowed to be completely dissolved；
2) enzymatic hydrolysis of agaropectin oligose: crude enzyme liquid is mixed with the agaropectin oligose of dissolution according to the volume ratio of 1:5~10, in 30 DEG C of water
Bathe 5~10min of heating stirring；
3) enzymolysis liquid inactivates: by the liquid after enzymatic hydrolysis in 100 DEG C of 10~20min of inactivation；
4) enzymolysis liquid is centrifuged: by the product of enzymatic hydrolysis inactivation in 6000~8000r/min, 10~15min of high speed centrifugation；
5) enzymolysis liquid is lyophilized: enzymolysis liquid is centrifuged object vacuum freeze drying, obtains fine jade disaccharides oligomer powder；
(2) preparation of fine jade disaccharides
1) dissolution of fine jade disaccharides oligomer: configuring the solution that mass concentration is 1% for fine jade disaccharides oligomer with deionized water,
30 DEG C of heating water bath stirrings are allowed to be completely dissolved；
2) enzymatic hydrolysis of fine jade disaccharides oligomer: crude enzyme liquid is mixed with the fine jade disaccharides oligomer of dissolution according to the volume ratio of 1:5~10,
5~10min is stirred in 30 DEG C of heating water baths；
3) enzymolysis liquid inactivates: by the liquid after enzymatic hydrolysis in 100 DEG C of 10~20min of inactivation；
4) enzymolysis liquid is centrifuged: by the product of enzymatic hydrolysis inactivation in 6000~8000r/min, 10~15min of high speed centrifugation；
5) enzymolysis liquid is lyophilized: enzymolysis liquid is centrifuged object vacuum freeze drying, obtains fine jade disaccharides powder.
Above-mentioned two sugar degrading enzyme crude enzyme liquid of fine jade is made by the steps:
1) willAgarivorans albusStrain is inoculated in activation medium, in 25~35 DEG C, 150-250rpm/min shaking table
40~50h of middle culture, carries out culture activation, obtains seed liquor；
2) seed liquor is inoculated into fermentation medium by 5~10% inoculum concentration, is shaken in 25~35 DEG C, 150~250rpm/min
40~50h is cultivated in bed, obtains fermentation liquid；
3) by fermentation liquid under the conditions of 4 DEG C, in 4000~5000r/min, 15~30min of refrigerated centrifuge, supernatant is taken after centrifugation,
Obtain two sugar degrading enzyme crude enzyme liquid of fine jade.
It is above-mentionedAgarivorans albusThe autonomous bacterium of Chinese Marine University, strain system, can be by seeking advice from, buying
Etc. modes obtain.
Preferably, above-mentioned activation medium at being grouped as are as follows: agar-agar 5%, NaCl 3.0%, urea 0.1%, yeast powder
0.1%、MgSO4 0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
Preferably, above-mentioned fermentation medium at being grouped as are as follows: glucose 1%, agar-agar 5%, NaCl 3.0%, urea 0.1%,
Yeast powder 0.1%, MgSO4 0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
The present invention also provides application of the fine jade disaccharides in preservation of fishery.Fine jade disaccharides of the present invention is applied to South America
White shrimp shrimp is fresh-keeping, the effect that can effectively slow down protein corruption in Penaeus Vannmei shrimp, reduce fat oxidation rate, from
And freshness date is improved, extend the shelf life of Penaeus Vannmei.
The present invention provides a kind of preparation method and application with the active fine jade disaccharides of high anti-oxidation, with prior art phase
Than having the advantage that
First is that fine jade disaccharides is prepared using agaropectin oligose as raw material, using enzyme edman degradation Edman in the application, two sugared content of fine jade is high in product,
With stronger oxidation resistance, to DPPH free radical, ABTS+The Scavenging activity of free radical, OH free radical realizes high antioxygen
Change the homogeneity preparation of active fine jade disaccharides；
Second is that agaropectin oligose prepared by the present invention has effects that preferable aquatic products is fresh-keeping, fine jade disaccharides is applied to white leg Shrimp
Meat is fresh-keeping, the effect that can effectively slow down protein corruption in Penaeus Vannmei shrimp, reduce fat oxidation rate, to improve guarantor
The fresh time limit extends the shelf life of Penaeus Vannmei；
Third is that test result shows that adding glucose in two sugar degrading enzyme fermentation medium of fine jade has certain suppression to enzymatic production
Production is used, the research report of this and Xie Hui are consistent；But result of study shows that adding glucose in the fermentation medium has
Conducive to the preservation of fishery ability for improving the fine jade disaccharides prepared by crude enzyme liquid；Glucose fermentation culture is not added under the same terms
Crude enzyme liquid enzymatic hydrolysis after fine jade disaccharides crude enzyme liquid fresh-keeping effect compared with embodiment 4 decline 25% or more.
Detailed description of the invention:
Fig. 1: the HPLC chromatogram of fine jade disaccharides；
Fig. 2: the ESI-MS mass spectrogram of fine jade disaccharides；
Fig. 3: pH value obtains situation of change in Penaeus Vannmei meat storage；
Fig. 4: the situation of change of TVB-N value in Penaeus Vannmei meat storage；
Fig. 5: the situation of change of TBA value in Penaeus Vannmei meat storage.
Specific embodiment
The preparation of 1 high anti-oxidation activity fine jade disaccharides of embodiment
A kind of preparation method with high anti-oxidation activity fine jade disaccharides, specifically includes the following steps:
(1) prepared by two sugar degrading enzyme crude enzyme liquid of fine jade:
1) willAgarivorans albusStrain is inoculated in activation medium, is trained in 25~35 DEG C, 200rpm/min shaking table
45h is supported, culture activation is carried out, obtains seed liquor；
2) seed liquor is inoculated into fermentation medium by 7.5% inoculum concentration, is trained in 25~35 DEG C, 200rpm/min shaking table
40~50h is supported, fermentation liquid is obtained；
3) by fermentation liquid under the conditions of 4 DEG C, in 5000r/min refrigerated centrifuge 20min, supernatant is taken after centrifugation, obtains fine jade disaccharides
Degrading enzyme crude enzyme liquid；
(2) preparation of fine jade disaccharides oligomer
1) dissolution of agaropectin oligose: the solution that mass concentration is 1% is configured by agaropectin oligose with deionized water, in 30 DEG C of water-baths
Heating stirring is allowed to be completely dissolved；
2) enzymatic hydrolysis of agaropectin oligose: crude enzyme liquid is mixed with the agaropectin oligose of dissolution according to the volume ratio of 1:8, is added in 30 DEG C of water-baths
Thermal agitation 8min；
3) enzymolysis liquid inactivates: by the liquid after enzymatic hydrolysis in 100 DEG C of inactivation 20min；
4) enzymolysis liquid is centrifuged: by the product of enzymatic hydrolysis inactivation in 8000r/min, high speed centrifugation 12min；
5) enzymolysis liquid is lyophilized: enzymolysis liquid is centrifuged object vacuum freeze drying, obtains fine jade disaccharides oligomer powder；
(3) preparation of fine jade disaccharides
1) dissolution of fine jade disaccharides oligomer: configuring the solution that mass concentration is 1% for fine jade disaccharides oligomer with deionized water,
30 DEG C of heating water bath stirrings are allowed to be completely dissolved；
2) enzymatic hydrolysis of fine jade disaccharides oligomer: crude enzyme liquid is mixed with the fine jade disaccharides oligomer of dissolution according to the volume ratio of 1:7, in 30
DEG C heating water bath stirs 7min；
3) enzymolysis liquid inactivates: by the liquid after enzymatic hydrolysis in 100 DEG C of inactivation 20min；
4) enzymolysis liquid is centrifuged: by the product of enzymatic hydrolysis inactivation in 8000r/min, high speed centrifugation 15min；
5) enzymolysis liquid is lyophilized: enzymolysis liquid is centrifuged object vacuum freeze drying, obtains fine jade disaccharides powder.
2 fine jade of embodiment, two sugar detection
1, column front derivation high performance liquid chromatography (HPLC) is analyzed
Detecting instrument: peace 3000 liquid chromatograph of Dionex UltiMate is worn in the U.S.；Chromatographic condition: chromatographic column Kromasil
C18 (4.6mm × 150mm, 5 μm)；Mobile phase: phosphate buffer/acetonitrile (83:17, V/V)；Flow velocity: 0.8 mL/min；Column
Incubator: 30 DEG C；Sample volume: 10 μ L；Detector: diode array UV detector (DAD)；As a result as shown in Figure 1；
2, electrospray ionization mass spectrum (ESI-MS) is analyzed
Detecting instrument: U.S.'s Micromass Q-TOF Ultima Global Quadrupole-time of flight mass spectrometry instrument；Mass spectrum inspection
Survey condition: anionic textiles mode, capillary voltage 3 kV, orifice potential 40-80V, flow by 80 DEG C of ion source temperature, carrier gas N2
Fast 400L/h, 200 DEG C of temperature, syringe pump direct injected, 10 μ L/min of sample flow rate；As a result as shown in Figure 2.
The Scavenging ability of the active fine jade disaccharides of 3 high anti-oxidation of embodiment measures
1, scavenging ability of DPPH free radical measures
Two sugar juice of fine jade of various concentration is prepared, concentration series 1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL take 0.4 respectively
Two sugar juice of fine jade of mL various concentration sequentially adds 0.6 mL, 0.15 mmol/L DPPH solution, shakes up；It is protected from light and puts at room temperature
30 min are set, the absorbance value A of each group mixed solution is measured respectively at 517nm, is gone with 2.0mL dehydrated alcohol and 1.0 mL
Ionized water replaces sample to survey absorbance value A0 as blank group, replaces sample as positive control using ascorbic acid；It is measured in parallel 3
It is secondary.The DPPH free radical scavenging activity of antioxidant calculates as follows:
DPPH free radical scavenging activity (%)=[(A0-A)/A0] × 100
The preparation of DPPH solution: DPPH 1mg is taken to be dissolved in about 20mL dehydrated alcohol, ultrasonic 5min；
Shake well, each section up and down of make suring are uniform；
1 fine jade disaccharides of table is to DPPH free radical scavenging activity
By table 1 as it can be seen that as fine jade disaccharides concentration gradually increases, the ability for removing DPPH free radical is gradually increased, when fine jade disaccharides
When for 8mg/ml, the ability for removing DPPH free radical is maximum, reaches 27 times of positive control Vc.
2, ABTS is removed+The measurement of free radical ability
Two sugar juice of fine jade of preparation various concentration, concentration series 1mg/mL, 2mg/mL, 4mg/L, 8mg/mL,.By 7.4
MmoL/LABTS and 2.6 mmoL/L K2S2O8Isometric mixing, 14 h of avoid light place at room temperature, with PBS buffer solution (pH=
7.4) it dilutes 30 times and is used as working solution.Take two sugar juice of fine jade that 0.5 mL various concentration is added in 1.5 mL working solutions, 37 DEG C of water
After 1 h of bath reaction, light absorption value is measured at 734 nm, and using ascorbic acid (Vc) as positive control.It is measured in parallel 3 times；
ABTS+Free radical scavenging ability is calculated by following formula:
Clearance rate (%)=[A1-Ai/A1]×100%
In formula: A1For sample is not added, the absorbance of ABTS is added；Ai is the absorbance that sample and ABTS is added；
ABTS stock solution (7.4 mmol/L) is prepared: being taken 96 mg of ABTS, is added 25 mL of deionized water.K2S2O8Stock solution (2.6
Mmol/L it) prepares: taking K2S2O8 378.4 mg add from 10 mL of water；
PBS buffer solution is prepared: weighing 8g NaCl, 0.2g KCl, 1.44g Na2HPO4、0.24gKH2PO4Be dissolved in 800mL go from
In sub- water, with the pH value of HCl adjusting solution to 7.4, finally plus deionized water is settled to 1L.Steam sterilization is (extremely under high pressure
It is 20 minutes few), it is stored in room temperature or 4 DEG C of refrigerators；
2 fine jade disaccharides of table is to ABTS+Free radical scavenging activity
By table 2 as it can be seen that as fine jade disaccharides concentration gradually increases, ABTS is removed+The ability of free radical gradually increases, when fine jade two
When sugar is 8mg/mL, ABTS is removed+The ability of free radical is maximum, reaches 22 times of positive control Vc.
3, the measurement of OH free radical ability is removed
Prepare two sugar juice of fine jade of various concentration, concentration series 1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL.0.4 is taken respectively
Two sugar juice of fine jade of mL various concentration, sequentially adds 0.5 mL FeSO4(9 mmol/L), salicylic acid (with 50% ethanol as solvent,
9 mmol/L), two sugar juice of fine jade and H2O2(8.8 mmol/L) reacts 60 min in 37 DEG C of water-baths, at 510 nm respectively
Measure the absorbance value A of each group mixed solutionS, using deionized water replace sample as blank group survey absorbance value Ab, with go from
Sub- water substitutes H2O2As damage group, absorbance A n is surveyed, replaces sample as positive control using ascorbic acid, antioxidant
OH free radical scavenging activity calculates as follows:
OH free radical scavenging activity (%)=[(As-An)/(Ab-An)] × 100
9 mmol/L FeSO4Solution is prepared: weighing anhydrous FeSO41.37g deionized water dissolving, is settled to 1L；
Salicylic acid solution is prepared: being weighed 1.342g salicylic acid and is dissolved with 50% ethyl alcohol, is settled to 1L；
8.8 mmol/LH2O2Solution is prepared: the H of accurate measuring 30%2O20.9mL is settled to 1L；
3 fine jade disaccharides of table is to OH base free radical scavenging activity
Seen from table 3, as fine jade disaccharides concentration gradually increases, the ability for removing OH free radical is gradually increased, when fine jade disaccharides
When concentration is 8mg/mL, the ability for removing OH free radical is maximum, reaches 25 times of positive control Vc.
The active fine jade disaccharides of 4 high anti-oxidation of embodiment measures Penaeus Vannmei meat fresh-keeping effect
Penaeus Vannmei is pre-processed, takes 1,3,5,7,10 g fine jade disaccharides to be added in 1 L water respectively and various concentration is made
Oligosaccharide solution carries out high-temperature sterilization.The Penaeus Vannmei meat sample product handled well are immersed in above-mentioned oligosaccharide solution, soaking time
For 30 min, while shrimp is impregnated as a control group with sterile water, take out after shrimp 10 d of preservation in 4 DEG C of refrigerators；
1, pH value measures
Shrimp is smashed to pieces, 100 mL deionized waters are added, stir evenly, stands, pH meter is inserted perpendicularly into meat and is measured.Each sample
Product are primary every 2 d measurement, as a result as shown in Figure 3；
From the figure 3, it may be seen that the pH value of blank control and test group is all in the trend risen after falling before, the reason is that testing South America used
After the processing of white shrimp meat, decomposition glucide, the acid that when decomposition generates reduce the pH value of lower shrimp first.Amino is decomposed again
The alkaline matter of acid, generation can make the pH value of shrimp become larger.The fresh and alive shrimp pH value of in-situ processing is subacidity, close to neutrality.
In addition, the pH value change procedure of control group is that the fine jade disaccharides processing group of 1 g/L is similar to concentration, and by comparing, fine jade disaccharides concentration
Fresh-keeping effect is preferable between (5~7) g/L.This explanation is not that fine jade disaccharides concentration is bigger, and fresh-keeping effect is better.But a certain concentration
Fine jade disaccharides have inhibit microbial reproduction, delay the nutriments such as protein degrade ability, thus effectively keep shrimp product
Matter plays preferable fresh-keeping effect；
2, TVB-N value measures
TVB-N value is in the world for evaluating unique physical and chemical index of meat freshness.The fresher TVB-N value of meat is smaller.Meat by
The effect of ectoenzyme is decomposed in its own enzyme or other putrefactive microorganisms, drops the nitrogenous compound of protein and nonprotein
Solution, the volatile basics nitrogen substance such as ammonia and amine of generation of degrading can make TVB-N value become larger.TVB-N values determination method is such as
Under:
1. the preparation of sample liquid: weighing the Penaeus Vannmei meat 1g sample handled well with 1,3,5,7,10g/L fine jade disaccharides respectively in cone
In shape bottle, add 10mL deionized water, shake frequently, is filtered after impregnating 30min, filtrate is placed in spare in refrigerator；
2. measurement: 10mL absorbing liquid will be filled in advance and the conical flask for mixing indicator solution added with 5-6 drop is placed in condenser pipe lower end, and
It is inserted into its lower end under the systemic liquid level of conical flask, precision draws the above-mentioned sample filtrate of 5ml in distiller reaction chamber, adds
5mL1% magnesia suspension, lid is filled in rapidly, and adds water with anti-gas-leak, is passed through steam, is closed when steam is full of in distiller
Steam outlet pipe, timing there is the first drop condensed water by cold collateral vessel, distillation 5min stop, absorbing liquid 0.01mol/
The titration of L hydrochloric acid standard solution, terminal are in bluish violet.Reagent blank test is done simultaneously；
Absorbing liquid: 2% boric acid solution.
Mix indicator solution: 0.2% methyl red ethanol solution of a liquid, 0.1% aqueous solution of methylene blue of b liquid face the used time for a liquid b liquid
Mixed in equal amounts is mixing indicator solution.
3. calculating
In formula: X1--- the content of Volatile Base Nitrogen, mg/100g in sample
V1--- measurement consumes hydrochloric acid or sulfuric acid standard solution volume, mL with sample liquid
V2--- reagent blank consumes hydrochloric acid or sulfuric acid standard solution volume, mL
N1--- the molar concentration of hydrochloric acid or sulfuric acid standard solution, mol/L
m1--- sample quality, g
14 --- the milligram number of the suitable nitrogen of 1mol/L hydrochloric acid standard solution 1mL
Test result is as shown in Figure 4；Figure 4, it is seen that the TVB-N value of blank control and test group is all with fresh keeping time
Increase it is in rising trend, but the rate of climb through the processed shrimp TVB-N value of fine jade disaccharides is smaller than blank control group.Through fine jade
The processed shrimp of glue oligosaccharides can effectively reduce the decomposition of protein, to extend the shelf life of Penaeus Vannmei；
3, TBA value measures
It is one of catabolite of lipid peroxidation that the principle of TBA method measurement, which is malonaldehyde (MDA), can be with thio barbital
Acid colour developing.Colorimetric estimation is carried out at 520 nm, can detect the relative amount of malonaldehyde in sample, according to colour developing situation with judgement
The degree of biomembrane lipid peroxidation.TBA value is judge fat oxidation degree important for measuring fat oxidation degree of spoilage
Index；
The Penaeus Vannmei meat 1g handled well with 1,3,5,7,10g/L fine jade disaccharides is weighed respectively, wherein 5 mL concentration are added being
7.5% trichloroacetic acid (containing 0.1%EDTA), shakes 30min, is centrifuged 5 under the conditions of 4 DEG C, 8000 r/min after mixing
min.0.5 mL centrifuged supernatant is taken, 0.5 mL TBA solution is added, 40 min are reacted in boiling water bath, are cooled down after reaction
5 min are centrifuged under the conditions of 1 h, 8000 r/min, 0.5 mL chloroform is added in supernatant, and stratification takes supernatant in 532 Hes
600nm measures light absorption value.Calculate TBA value；
TBA value (mg/100g)=(A532-A600)/155 × 1/10 × 72.6 × 100
TBA solution: it is soluble in water to accurately weigh TBA0.288g, and is diluted to 100ml, is equivalent to 0.02M.Trichloroacetic acid mixing
Liquid: trichloroacetic acid (analysis is pure) 7.5g and 0.1gEDTA is accurately weighed, is dissolved with water, is diluted to 100ml；
Test result is as shown in Figure 5.Fig. 5 is it is found that the TBA value of blank control and test group all becomes in rising as time increases
Gesture, but the TBA value of the processed shrimp of fine jade disaccharides is smaller than blank control group.Show fine jade disaccharides to the fat in shrimp preservation process
Oxidation has obvious inhibiting effect, it may be possible to since fine jade disaccharides can form hydrogen bond with biomolecule, consolidate instead of space structure
Some hydrones.It is all wrapped in water membrane around protein, lipid and other biological macromolecular in Penaeus Vannmei meat, when
Shrimp in preservation process can lose moisture film, and two glycan molecule of fine jade energy and large biological molecule are connected with hydrogen bond, to form one
Layer protective film replaces the moisture film lost to play preferable fresh-keeping effect to maintain its original structure and activity.
The above examples are only used to illustrate the technical scheme of the present invention, rather than is limited；Although referring to aforementioned reality
It applies example to be described in detail to by invention, but for those of ordinary skill in the art, it still can be to aforementioned reality
Technical solution documented by example is applied to modify or equivalent replacement of some of the technical features；And to these modifications
Or replacement, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of preparation method with high anti-oxidation activity fine jade disaccharides, it is characterised in that specifically includes the following steps:
(1) preparation of fine jade disaccharides oligomer
1) dissolution of agaropectin oligose: the solution that mass concentration is 1% is configured by agaropectin oligose with deionized water, in 30 DEG C of water-baths
Heating stirring is allowed to be completely dissolved；
2) enzymatic hydrolysis of agaropectin oligose: crude enzyme liquid is mixed with the agaropectin oligose of dissolution according to the volume ratio of 1:5~10, in 30 DEG C of water
Bathe 5~10min of heating stirring；
3) enzymolysis liquid inactivates: by the liquid after enzymatic hydrolysis in 100 DEG C of 10~20min of inactivation；
4) enzymolysis liquid is centrifuged: by the product of enzymatic hydrolysis inactivation in 6000~8000r/min, 10~15min of high speed centrifugation；
5) enzymolysis liquid is lyophilized: enzymolysis liquid is centrifuged object vacuum freeze drying, obtains fine jade disaccharides oligomer powder；
(2) preparation of fine jade disaccharides
1) dissolution of fine jade disaccharides oligomer: configuring the solution that mass concentration is 1% for fine jade disaccharides oligomer with deionized water,
30 DEG C of heating water bath stirrings are allowed to be completely dissolved；
2) enzymatic hydrolysis of fine jade disaccharides oligomer: crude enzyme liquid is mixed with the fine jade disaccharides oligomer of dissolution according to the volume ratio of 1:5~10,
5~10min is stirred in 30 DEG C of heating water baths；
3) enzymolysis liquid inactivates: by the liquid after enzymatic hydrolysis in 100 DEG C of 10~20min of inactivation；
4) enzymolysis liquid is centrifuged: by the product of enzymatic hydrolysis inactivation in 6000~8000r/min, 10~15min of high speed centrifugation；
5) enzymolysis liquid is lyophilized: enzymolysis liquid is centrifuged object vacuum freeze drying, obtains fine jade disaccharides powder.
2. a kind of preparation method with high anti-oxidation activity fine jade disaccharides according to claim 1, it is characterised in that described
Two sugar degrading enzyme crude enzyme liquid of fine jade is made by the steps:
1) willAgarivorans albusStrain is inoculated in activation medium, in 25~35 DEG C, 150-250rpm/min shaking table
40~50h of middle culture, carries out culture activation, obtains seed liquor；
2) seed liquor is inoculated into fermentation medium by 5~10% inoculum concentration, is shaken in 25~35 DEG C, 150~250rpm/min
40~50h is cultivated in bed, obtains fermentation liquid；
3) by fermentation liquid under the conditions of 4 DEG C, in 4000~5000r/min, 15~30min of refrigerated centrifuge, supernatant is taken after centrifugation,
Obtain two sugar degrading enzyme crude enzyme liquid of fine jade.
3. a kind of preparation method with high anti-oxidation activity fine jade disaccharides according to claim 2, it is characterised in that described
Activation medium at being grouped as are as follows: agar-agar 5%, NaCl 3.0%, urea 0.1%, yeast powder 0.1%, MgSO4 0.05%、K2HPO4
0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
4. a kind of preparation method with high anti-oxidation activity fine jade disaccharides according to claim 2, it is characterised in that described
Fermentation medium at being grouped as are as follows: glucose 1%, agar-agar 5%, NaCl 3.0%, urea 0.1%, yeast powder 0.1%, MgSO4
0.05%、K2HPO4 0.1%、FeSO4 0.002%、Fe2(SO4)3 0.001%、CaCl2 0.02%。
5. by the fine jade disaccharides of claim 1 the method preparation.
6. application of the fine jade disaccharides in preservation of fishery described in claim 5.
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Application publication date: 20191025 |