CN101348811B - Method for preparing soybean isoflavone glycoside from soybean isoflavones aglycone - Google Patents
Method for preparing soybean isoflavone glycoside from soybean isoflavones aglycone Download PDFInfo
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- CN101348811B CN101348811B CN2008100131093A CN200810013109A CN101348811B CN 101348811 B CN101348811 B CN 101348811B CN 2008100131093 A CN2008100131093 A CN 2008100131093A CN 200810013109 A CN200810013109 A CN 200810013109A CN 101348811 B CN101348811 B CN 101348811B
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- chicken gizzard
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- buffer solution
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- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 235000008696 isoflavones Nutrition 0.000 title claims abstract description 52
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 43
- 244000068988 Glycine max Species 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 25
- 150000002515 isoflavone derivatives Chemical class 0.000 title claims description 12
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 title abstract 4
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 title abstract 4
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 title abstract 4
- ZWSNUPOSLDAWJS-QNDFHXLGSA-N 6,7-dihydroxy-3-[4-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]chromen-4-one Chemical compound OC[C@H]1O[C@@H](Oc2ccc(cc2)-c2coc3cc(O)c(O)cc3c2=O)[C@H](O)[C@@H](O)[C@@H]1O ZWSNUPOSLDAWJS-QNDFHXLGSA-N 0.000 title 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 50
- 108090000790 Enzymes Proteins 0.000 claims abstract description 40
- 102000004190 Enzymes Human genes 0.000 claims abstract description 40
- 239000000243 solution Substances 0.000 claims abstract description 27
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- -1 isoflavone glucoside Chemical class 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 239000002699 waste material Substances 0.000 claims abstract description 14
- 239000000843 powder Substances 0.000 claims abstract description 12
- 239000000203 mixture Substances 0.000 claims abstract description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 60
- 239000012528 membrane Substances 0.000 claims description 41
- 244000118681 Iresine herbstii Species 0.000 claims description 40
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 claims description 29
- 239000008351 acetate buffer Substances 0.000 claims description 27
- 229930182470 glycoside Natural products 0.000 claims description 23
- ZQSIJRDFPHDXIC-UHFFFAOYSA-N Daidzein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZQSIJRDFPHDXIC-UHFFFAOYSA-N 0.000 claims description 22
- GMTUGPYJRUMVTC-UHFFFAOYSA-N Daidzin Natural products OC(COc1ccc2C(=O)C(=COc2c1)c3ccc(O)cc3)C(O)C(O)C(O)C=O GMTUGPYJRUMVTC-UHFFFAOYSA-N 0.000 claims description 22
- KYQZWONCHDNPDP-UHFFFAOYSA-N Daidzoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 KYQZWONCHDNPDP-UHFFFAOYSA-N 0.000 claims description 22
- KYQZWONCHDNPDP-QNDFHXLGSA-N daidzein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 KYQZWONCHDNPDP-QNDFHXLGSA-N 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 21
- ZCOLJUOHXJRHDI-FZHKGVQDSA-N Genistein 7-O-glucoside Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)c1cc(O)c2C(=O)C(c3ccc(O)cc3)=COc2c1 ZCOLJUOHXJRHDI-FZHKGVQDSA-N 0.000 claims description 20
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 claims description 20
- 229930183217 Genin Natural products 0.000 claims description 15
- 230000009466 transformation Effects 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 230000009514 concussion Effects 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 238000004062 sedimentation Methods 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 229920000298 Cellophane Polymers 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims 3
- 239000012295 chemical reaction liquid Substances 0.000 claims 1
- 235000019580 granularity Nutrition 0.000 claims 1
- 230000007062 hydrolysis Effects 0.000 abstract description 10
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 10
- 229930182478 glucoside Natural products 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000002253 acid Substances 0.000 abstract description 3
- 239000003513 alkali Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract 6
- 239000007853 buffer solution Substances 0.000 abstract 5
- 230000007547 defect Effects 0.000 abstract 2
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000002893 slag Substances 0.000 abstract 1
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 description 9
- 235000006539 genistein Nutrition 0.000 description 9
- 229940045109 genistein Drugs 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 239000007795 chemical reaction product Substances 0.000 description 7
- 125000003147 glycosyl group Chemical group 0.000 description 7
- 239000011541 reaction mixture Substances 0.000 description 7
- 101710094902 Legumin Proteins 0.000 description 5
- NEKNNCABDXGBEN-UHFFFAOYSA-L disodium;4-(4-chloro-2-methylphenoxy)butanoate;4-(2,4-dichlorophenoxy)butanoate Chemical compound [Na+].[Na+].CC1=CC(Cl)=CC=C1OCCCC([O-])=O.[O-]C(=O)CCCOC1=CC=C(Cl)C=C1Cl NEKNNCABDXGBEN-UHFFFAOYSA-L 0.000 description 5
- NNUVCMKMNCKPKN-UHFFFAOYSA-N glycitein Natural products COc1c(O)ccc2OC=C(C(=O)c12)c3ccc(O)cc3 NNUVCMKMNCKPKN-UHFFFAOYSA-N 0.000 description 5
- 235000008466 glycitein Nutrition 0.000 description 5
- DXYUAIFZCFRPTH-UHFFFAOYSA-N glycitein Chemical compound C1=C(O)C(OC)=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 DXYUAIFZCFRPTH-UHFFFAOYSA-N 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 150000008131 glucosides Chemical class 0.000 description 3
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000001066 destructive effect Effects 0.000 description 2
- 230000013011 mating Effects 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 210000004317 gizzard Anatomy 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 210000002784 stomach Anatomy 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing aglycone from soybean isoflavone glucoside. The method comprises the following steps: galli stomachichum corium powder is mixed with 0.2 milligram per liter phosphoric acid buffer solution or acetic acid buffer solution with a pH value between 3.0 and 7.0 according to the mass ratio of 1 to 3-10; the mixture is leached for 3 to 36 hours at a temperature of between 4 and 40 DEG C and then waste slag is centrifuged; supernatant is added with ammonia sulfate or ethanol, and precipitated galli stomachichum corium zymoprotein is dissolved into the 0.2 milligram per liter phosphoric acid buffer solution or the acetic acid buffer solution with a pH value between 3.0 and 7.0, and then galli stomachichum corium enzyme solution is obtained; enzyme, the soybean isoflavone glucoside and the buffer solution are mixed and reacted for 3 to 30 hours at a temperature of between 10 and 50 DEG C and with a pH value between 3.0 and 7.0; and the soybean isoflavone aglycone is extracted. The method overcomes the defects of large destructiveness, large pollution and poor purposiveness of the acid and alkali hydrolysis method on the aglycone and simultaneously overcomes the defect that the enzyme activity of the microbial enzyme hydrolysis method is generally low. The method has easy, simple and convenient technology, complete zymohydrolysis, high conversion and high edible safety, and is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method for preparing isoflavone genin, extract the Membrane of Chicken Gizzard enzyme especially a kind of Membrane of Chicken Gizzard of the dry gizzard inner membrance from Phasianidae animal man chicken, prepare the method for its aglycon with Membrane of Chicken Gizzard enzyme hydrolysis of soybean isoflavone glycosides glycosyl.
Background technology
Soybean isoflavones is a kind of non-steroidal compound that contains aromatic nucleus, from soybean, isolate 12 kinds of soybean isoflavones isomer up to now altogether, be divided into aglycon two classes of the glucosides and the free type of mating type, the mating type glucosides mainly contains nine kinds of forms such as Genistoside, daidzin and daidzin; Aglycon comprises genistein, big legumin and three kinds of forms of glycitein.These 12 kinds of soybean isoflavones isomer have different titles because of the difference of group on its female ring specific position.The Genistoside that the present invention hereinafter mentions, daidzin and daidzin are one of soybean isoflavones isomeric forms, and the present invention is referred to as the soybean isoflavones glycosides; And the aglycon of pairing corresponding form behind genistein, big legumin and glycitein to be Genistoside, daidzin and daidzin the be hydrolyzed glycosyl, invention is referred to as isoflavone genin.
It is Genistoside that 50%～60% isoflavones is arranged in the soybean approximately, and Genistoside and aglycon genistein thereof are many a kind of soybean isoflavones compositions of research at present.Soybean isoflavones platymiscium oestrogenic hormon, it is a class important physical active substance, many soybean saponins that studies show that have multiple physiologically active and good pharmacological action, as anticancer, regulate immunological competence, prevent cardiovascular disorder, hypotensive, reduce cholesterol in serum content, and effect such as antithrombotic, be mainly used in fields such as medicine, health care and makeup.
Discovering in recent years has close getting in touch between the glycosyl in the soybean isoflavones glycosides and its biological activity.The estrogen receptor activity of isoflavone genin is than at least doubly 30 times of the soybean isoflavone glucoside height of being with glycosyl.But mostly commercially available soybean isoflavones is the glucosides type of band glycosyl at present, and its activity is lower.Glycosyl for how with soybean isoflavone glucoside removes, and makes it be converted into highly active aglycon, has all carried out more research both at home and abroad.
Application number is the preparation method that 200710029119.1 Chinese patent application discloses a kind of soybean isoflavones, this patent application utilizes the method hydrolyzed soy bean isoflavone glycosides glycosyl of hydrochloric acid-alcohol reflux hydrolysis, it is destructive big that yet the acid and alkali hydrolysis method exists aglycon, pollute greatly the shortcoming of purpose difference.
The patent No. is that the Chinese patent of ZL 03133637X discloses the method with the method hydrolyzed soy bean isoflavone glycosides glycosyl of microbial fermentation, and its lytic enzyme derives from microbial fermentation, and transformation efficiency is 50～80%.
Produce soy bean isoflavone glycosidase by microbe fermentation method, cost is lower, but its enzyme activity is generally not high, and complex operation.
Summary of the invention
The present invention is in order to overcome the existing in prior technology technical problem, provides a kind of simple to operate, with low cost and the glycosyl Membrane of Chicken Gizzard enzyme hydrolysis of soybean isoflavone glycosides that transformation efficiency is high prepares the method for its aglycon.
The present invention is achieved in that
(1) producing of Membrane of Chicken Gizzard enzyme:
(a) Membrane of Chicken Gizzard is ground into less than 20 purpose particles with stainless steel pulverizing machine and becomes the Membrane of Chicken Gizzard powder;
(b) with the product of phosphoric acid or acetate buffer solution lixiviate (a), phosphoric acid or acetate buffer solution concentration are 0.2M/L, and pH is in 3.0～7.0 scopes, and the quality that product (a) and phosphoric acid or acetate buffer solution are pressed 1:3～10 is than mixing, at 4 ℃ of lixiviate 3～36h; Centrifugal waste gets supernatant liquor;
(c) add in the product of (b) ammonium sulfate to saturated or supersaturation with precipitation Membrane of Chicken Gizzard zymoprotein, ammonium sulfate concentrations is 35～70%; The centrifugal clear liquid that discards obtains the Membrane of Chicken Gizzard zymoprotein;
(d) with the product of phosphoric acid or hac buffer dissolving (c), phosphoric acid or hac buffer concentration are 0.2M/L, and pH is 3.0～7.0, and product (c) and phosphoric acid or hac buffer compare mixing by the quality of 1:3～5; Remove ammonium sulfate with the cellophane bags dialysis, centrifugal waste promptly gets Membrane of Chicken Gizzard enzyme liquid;
(2) preparation of isoflavone genin:
(e) the soybean isoflavones glycosides is mixed with the working fluid of 1～100g/L with phosphoric acid or acetate buffer solution; Phosphoric acid or hac buffer concentration are 0.2M/L, and pH is 3.0～7.0;
(f) will carry out enzyme reaction behind (d) and the product mixing (e), the enzyme reaction condition is that temperature is 10～40 ℃, and the reaction times is 3～30h, is 1:0.5～5 with (e) proportion of products (d);
(g) after (f) step reaction stops, add ethyl acetate, volume (g) and ethyl acetate ratio are 1:1～3; Adopt machinery concussion or artificial concussion, fully concussion extraction, standing demix, the upper strata is the isoflavone genin ethyl acetate solution that makes; Extract 2 times, merge the upper and lower respectively; The upper strata ethyl acetate comes into operation after reclaiming by evaporation once more; Lower floor's phosphoric acid or hac buffer come into operation by concentrating to reclaim once more;
(h) product of concentrated (g) is isoflavone genin, and soybean isoflavones glycosides transformation efficiency can reach 60～97%.
The present invention can collect Membrane of Chicken Gizzard albumen with ethanol or two kinds of methods of ammonium sulfate.If during with ethanol sedimentation Membrane of Chicken Gizzard zymoprotein, 95% the ethanol that adds 3～6 times of supernatant liquors in above-mentioned (c) step is with precipitation Membrane of Chicken Gizzard zymoprotein; Because precipitation without ammonium sulfate, has been removed ammonium sulfate so just need not dialysis in (d) step.
Facts have proved, below be preferable practice mode:
(1) producing of Membrane of Chicken Gizzard enzyme: the Membrane of Chicken Gizzard powder is pressed the mass ratio of 1:6～8 with the phosphoric acid or the hac buffer of 0.2M/L, pH5.0～7.0,5～25h is got in 4 ℃ of lixiviates, centrifugal waste, toward the middle ethanol sedimentation zymoprotein that adds 3 times 95% of supernatant liquor (containing enzyme mixture), the centrifugal supernatant liquor of abandoning, collect zymoprotein, be dissolved in 0.2M/L, pH5.0～7.0 phosphoric acid or acetate buffer solution of 3 times of vat liquor volumes, be Membrane of Chicken Gizzard enzyme liquid;
(2) preparation of isoflavone genin:
1. enzymolysis: with soybean isoflavones glycosides 0.2M/L, pH5.0～7.0 phosphoric acid or acetate buffer solution are mixed with 10～50g/L, mix with Membrane of Chicken Gizzard enzyme liquid, Membrane of Chicken Gizzard enzyme liquid and phosphoric acid or acetate buffer solution volume ratio are 1:1～3, react 12～24h down for 25～40 ℃ in temperature.
2. extract: add the ethyl acetate of 2～3 times of volumes in the reaction mixture, fully concussion extraction, standing demix, the upper strata is an ethyl acetate layer, lower floor uses ethyl acetate extraction 1～2 time again, merges the upper strata and is the isoflavone genin ethyl acetate solution;
3. concentrate: after volatilizing ethyl acetate layer by evaporation, can obtain the aglycon that transformation efficiency is 60～97% corresponding soybean isoflavones isomer.
Described soybean isoflavones glycosides is the form of mixtures of daidzin, Genistoside and daidzin monomeric form or daidzin, Genistoside and daidzin, is referred to as the soybean isoflavones glycosides usually; Described genistein, big legumin and glycitein are the aglycon that Genistoside, daidzin and daidzin are hydrolyzed pairing corresponding form behind the glycosyl, are referred to as isoflavone genin.
The present invention compares with prior art, has following outstanding feature:
1, has higher edible safety: with animal ferment hydrolyzed soy bean isoflavone glycosides glycosyl, preparation isoflavone genin.It is existing destructive big to aglycon to have overcome prior art acid and alkali hydrolysis method, pollutes greatly shortcomings such as purpose difference;
2, simple and easy to do, the transformation efficiency height: simple than this enzyme extraction of microbial enzyme hydrolysis method, enzyme activity is also higher, and transformation efficiency can reach more than 96%.
3, hydrolysis is more thorough: this enzyme not only can hydrolysis monomer glycosides---daidzin, Genistoside, daidzin, become corresponding aglycon, this enzyme also can be hydrolyzed into big legumin, genistein, glycitein etc. to mixing glycosides such as daidzin, Genistoside, daidzins and mix aglycon.
4, with low cost: take from the animal body in the enzyme source of lytic enzyme, especially Membrane of Chicken Gizzard---abandon when edible dry stomach (ballast inner membrance) inwall of young section animal man chicken in obtain.
Embodiment
The experimental result of following examples detects back calculating by high performance liquid phase and gets:
Embodiment 1:
A. Membrane of Chicken Gizzard powder and 0.2M/L, after the phosphoric acid buffer of pH6.0 is pressed the mass ratio mixing of 1:5,24h is got in 4 ℃ of lixiviates, centrifugal waste, add in the supernatant liquor ammonium sulfate to saturated with the precipitation zymoprotein, collect albumen, be dissolved in 0.2M/L, in the pH6.0 phosphoric acid buffer, remove ammonium sulfate with the cellophane bags dialysis, centrifugal waste is enzyme liquid.
B. the daidzin 100mL and the described enzyme liquid of a. 100mL that with concentration are 10g/L mix, and reaction is 30 hours under 25 ℃ of conditions of temperature.
C. the ethyl acetate that adds 2 times of reaction volumes in the reaction mixture, fully concussion extraction, standing demix.To change into the transformation efficiency of big legumin be 97.1% to daidzin in the reaction product.
Embodiment 2:
A. after the mass ratio that Membrane of Chicken Gizzard powder and 0.2M, the acetate buffer of pH5.0 press 1:6 mixed, 3h was got in 4 ℃ of lixiviates, centrifugal waste, the 95% ethanol sedimentation zymoprotein that adds 3 times of volumes in the supernatant liquor, the centrifugal supernatant liquor of abandoning is collected albumen, be dissolved in acetate buffer solution (0.2M, pH5.0) in, be enzyme liquid.
B. with the Genistoside 100mL acetate buffer solution of 40g/L (0.2M, pH5.0) and the described enzyme liquid of a. 200mL mix, reaction is 30 hours under 40 ℃ of conditions of temperature.
C. the ethyl acetate that adds 3 times of reaction volumes in the reaction mixture, fully concussion extraction, standing demix.To change into the transformation efficiency of genistein be 90.4% to Genistoside in the reaction product.
Embodiment 3:
A. after the mass ratio that Membrane of Chicken Gizzard powder and 0.2M, the acetate buffer of pH6.0 press 1:10 mixed, 36h was got in 4 ℃ of lixiviates, centrifugal waste, the ethanol sedimentation zymoprotein that adds 6 times of volumes 95% in the supernatant liquor, the centrifugal supernatant liquor of abandoning is collected albumen, be dissolved in acetate buffer solution (0.2M, pH7.0) in, be enzyme liquid.
B. (0.2M pH7.0) mixes with the described enzyme liquid of a. 200mL, and reaction is 18 hours under 40 ℃ of conditions of temperature with 70g/L Genistoside 100mL acetate buffer solution.
C. the ethyl acetate that adds 3 times of reaction volumes in the reaction mixture, fully concussion extraction, standing demix.To change into the transformation efficiency of genistein be 93.6% to Genistoside in the reaction product.
Embodiment 4:
A. after the mass ratio that Membrane of Chicken Gizzard powder and 0.2M, the acetate buffer of pH5.0 press 1:8 mixed, 20h was got in 30 ℃ of lixiviates, centrifugal waste, the ethanol sedimentation zymoprotein that adds 4 times of volumes 95% in the supernatant liquor, the centrifugal supernatant liquor of abandoning is collected albumen, be dissolved in acetate buffer solution (0.2M, pH5.0) in, be enzyme liquid.
B. (0.2M pH5.0) mixes with the described enzyme liquid of a. 50mL, and reaction is 20 hours under 40 ℃ of conditions of temperature with 100g/L Genistoside, 100mL acetate buffer solution.
C. the ethyl acetate that adds 3 times of reaction volumes in the reaction mixture, fully concussion extraction, standing demix.To change into the transformation efficiency of genistein be 79.1% to Genistoside in the reaction product.
Embodiment 5:
A. Membrane of Chicken Gizzard powder and 0.2M, after the acetate buffer of pH3.0 is pressed the mass ratio mixing of 1:5,30h is got in 25 ℃ of lixiviates, centrifugal waste is toward the middle ethanol sedimentation zymoprotein that adds 5 times of volumes 95% of supernatant liquor (containing enzyme mixture), the centrifugal supernatant liquor of abandoning, collect albumen, be dissolved in acetate buffer solution (0.2M, pH3.0) in, be enzyme liquid.
B. (0.2M pH3.0) mixes with the described enzyme liquid of a. 20mL, and reaction is 12 hours under 50 ℃ of conditions of temperature with 40g/L Genistoside 100mL acetate buffer solution.
C. the ethyl acetate that adds 3 times of reaction volumes in the reaction mixture, fully concussion extraction, standing demix.To change into the transformation efficiency of genistein be 65.8% to Genistoside in the reaction product.
Embodiment 6:
A. Membrane of Chicken Gizzard powder 0.2M, after the acetate buffer of pH4.0 is pressed the mass ratio mixing of 1:4,18h is got in 4 ℃ of lixiviates, and centrifugal waste adds the saturated ammonium sulphate zymoprotein in supernatant liquor (containing enzyme mixture), the centrifugal supernatant liquor of abandoning, collect albumen, be dissolved in acetate buffer solution (0.2M, pH4.0) in, with the cellophane bags dialysis, be enzyme liquid.
B. handle daidzin with the method for embodiment 1, the result of purification and check enzyme reaction product: daidzin changes into glycitein, transformation efficiency 86.7%.
Embodiment 7:
A. after the mass ratio that Membrane of Chicken Gizzard powder 0.2M, the acetate buffer of pH4.0 press 1:8 mixed, 10h was got in 10 ℃ of lixiviates, centrifugal waste adds the ethanol sedimentation zymoprotein of 4 times of volumes 95%, the centrifugal supernatant liquor of abandoning in the supernatant liquor, collect albumen, albumen is redissolved in the acetate buffer of pH4.0, be enzyme liquid.
B. 60g/L soybean isoflavones glycosides is added gained enzyme liquid among a., reaction is 15 hours under 45 ℃ of conditions of temperature.
C. add the ethyl acetate of 2.5 times of reaction volumes in the reaction mixture, mixing tank fully shakes extraction, standing demix.To change into the transformation efficiency of isoflavone genin be 85.6% to the soybean isoflavones glycosides in the reaction product.
Claims (5)
1. a soybean isoflavones glycosides prepares the method for its aglycon, it is characterized in that comprising following processing step:
(1) producing of Membrane of Chicken Gizzard enzyme:
(a) Membrane of Chicken Gizzard is ground into less than 20 purpose granularities;
(b) with phosphoric acid or acetate buffer solution at 4～30 ℃ of lixiviate Membrane of Chicken Gizzard powder 3～36h; The mass ratio of Membrane of Chicken Gizzard powder and phosphoric acid or acetate buffer solution is 1: 6～10; Centrifugal waste gets supernatant liquor;
(c) in supernatant liquor, add ammonium sulfate to saturated or supersaturation with precipitation Membrane of Chicken Gizzard zymoprotein; The centrifugal clear liquid that discards obtains the Membrane of Chicken Gizzard zymoprotein;
(d) with phosphoric acid or hac buffer dissolving Membrane of Chicken Gizzard zymoprotein, the mass ratio of Membrane of Chicken Gizzard zymoprotein and phosphoric acid or hac buffer is 1: 3～5; Ammonium sulfate is removed in dialysis, and centrifugal waste promptly gets Membrane of Chicken Gizzard enzyme liquid;
(2) preparation of isoflavone genin:
(e) the soybean isoflavones glycosides is mixed with the working fluid of 1～100g/L with phosphoric acid or acetate buffer solution;
(f) with Membrane of Chicken Gizzard enzyme liquid and (e) working fluid by behind 1: 0.5～5 the volume ratio mixing, carry out 12～30h enzyme digestion reaction in 25～50 ℃;
(g) after enzyme digestion reaction stops, add ethyl acetate, the volume ratio of enzyme digestion reaction liquid and ethyl acetate is 1: 1～3, fully concussion extraction, and standing demix extracts 2 times, merges the upper and lower respectively, and the upper strata is the isoflavone genin ethyl acetate solution;
(h) with after isoflavone genin solution is removed ethyl acetate by concentration and evaporation in (g), can obtain the aglycon of transformation efficiency 60～97% corresponding soybean isoflavones;
Described phosphoric acid or acetate buffer solution concentration are 0.2M/L, and pH is 3.0～7.0.
2. the method for preparing its aglycon according to claim 1 soybean isoflavones glycosides is characterized in that wherein substituting with following steps in the step of producing (c) of (1) Membrane of Chicken Gizzard enzyme with (d): (c) with 95% ethanol sedimentation Membrane of Chicken Gizzard zymoprotein; The ethanol consumption is 3～6 times of the supernatant liquors of (b) step; (d) step is that the mass ratio of Membrane of Chicken Gizzard zymoprotein and phosphoric acid or hac buffer is 1: 3～5 with phosphoric acid or hac buffer dissolving Membrane of Chicken Gizzard zymoprotein; Promptly get the Membrane of Chicken Gizzard enzymolysis solution.
3. the method for preparing its aglycon according to claim 1 soybean isoflavones glycosides is characterized in that wherein that in the step of (d) dialysis is carried out with cellophane bags.
4. the method for preparing its aglycon according to claim 1 soybean isoflavones glycosides, it is characterized in that wherein in the step of (g), lower floor after the extraction extracts 1～2 time with ethyl acetate again, merges the upper strata and is the isoflavone genin ethyl acetate solution, carries out (h) and concentrates.
5. prepare the method for its aglycon according to claim 1 soybean isoflavones glycosides, it is characterized in that: described soybean isoflavones glycosides is the form of mixtures of daidzin, Genistoside and daidzin monomeric form or daidzin, Genistoside and daidzin.
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| CN106905392B (en) * | 2017-02-28 | 2020-11-13 | 安徽安科恒益药业有限公司 | Galactoside soybean isoflavone and its synthesis |
| CN108220356A (en) * | 2017-11-20 | 2018-06-29 | 荆门市德爱生物工程股份有限公司 | A kind of production method of isoflavone genin |
| CN113616678A (en) * | 2020-05-09 | 2021-11-09 | 成都恒福禧生物技术有限公司 | Chicken's gizzard membrane zymolyte and preparation method and application thereof |
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| EP0837139A2 (en) * | 1996-10-15 | 1998-04-22 | Protein Technologies International, Inc. | Two step conversion of vegetable protein isoflavone conjugates to isoflavone aglucones |
| CN1483826A (en) * | 2003-08-01 | 2004-03-24 | 金凤燮 | Method for preparing aglycon of soybean isoflavone glycoside base by enzymatic method hydrolyzing soybean |
| CN1928105A (en) * | 2006-09-08 | 2007-03-14 | 东北农业大学 | Method of preparing soybean isoflavone aglycone by hydrolyzing enzyme of natural edible raw material |
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| EP0837139A2 (en) * | 1996-10-15 | 1998-04-22 | Protein Technologies International, Inc. | Two step conversion of vegetable protein isoflavone conjugates to isoflavone aglucones |
| CN1483826A (en) * | 2003-08-01 | 2004-03-24 | 金凤燮 | Method for preparing aglycon of soybean isoflavone glycoside base by enzymatic method hydrolyzing soybean |
| CN1928105A (en) * | 2006-09-08 | 2007-03-14 | 东北农业大学 | Method of preparing soybean isoflavone aglycone by hydrolyzing enzyme of natural edible raw material |
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