CN101086002A - Method for hydrolyzing soybean isoflavone by enzyme - Google Patents
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Abstract
The invention discloses a method for hydrolyzing soybean isoflavone with biphase biological method. It is to hydrolyze glycoside soybean isoflavone by using immobilized soybean isoflavone glycoside hydrolase in biphase system that is composed of organic solvent and buffering solution, and get aglycone soybean isoflavone. The solubility of said organic solvent in said soybean isoflavone is better than that in water. The invention is characterized by effective hydrolysis of soybean isoflavone, no speical smell of product, and simple device and process.
Description
Technical field
The present invention relates to a kind of method of enzyme hydrolysis of soybean isoflavone.
Background technology
The immobilization of enzyme is that the biological catalyst enzyme is fixed on certain carrier.Along with development of biology, enzyme has specificity good as catalyzer, and catalytic efficiency height, and advantage such as reaction conditions gentleness also substitute traditional catalyzer increasing on industrial application.But enzyme is exactly the price comparison costliness as the main drawback of the industrial application of catalyzer, and can not reuse.Nelson in 1916 and Griffin are adsorbed onto sucrase on activated carbon and the alumina first, realize first enzyme immobilization (R.F.Taylor, Protein immobilization, Marcel Dekker, Inc:New York, 1991, v).The immobilization technology of enzyme has just obtained very fast development from then on, and up to the present very sophisticated process for fixation has been arranged: absorption, covalent cross-linking, embedding etc. also draw the stability that immobilized enzyme can also improve enzyme to a certain extent simultaneously.The immobilization of the eighties of last century enzyme seventies begun be applied to industrialization (R.F.Taylor, Protein immobilization, MarcelDekker, Inc:New York, 1991, v).
Soybean isoflavones is a kind of material with physiologically active in the soybean, its major function has two aspects, phytoestrogen function and anti-oxidant function (Song T T, Gendirch S, Murphy P A.Estrogenic activityof glycitein, a soy isoflavone, J.Agric.Food Chem., 1999,47:1607-1610; Michael Naim, Benjamin Gesterner, Shmuel Zilkah.Soy isoflavones, characterization, determination, and antifungal activity, J.Agric.FoodChem., 1974,22 (5): 806-810).Mainly show as: (1) has the estrogenic function of human body, thereby prevents and treats female dimacteric syndrome effectively.(2) anti-curing cancers.The activity of arrestin Tyrosylprotein kinase (TPK) improves the activity that human body is separated toxenzyme specifically, prevents and treats mammary cancer, uterus carcinoma, intestinal cancer, leukemia, prostate cancer etc. effectively.(3) but two-ways regulation human body inner estrogen level has the effect of beauty and health care.(4) protect against osteoporosis.Can suppress bone and absorb, be the good medicine of control skeleton bulking.(5) has significant antiserum(antisera) lipoprotein lipid peroxidation, effect even be better than vitamin-E.The disease that prevention and the peroxidation of assisting therapy body lipid cause is as hyperlipidemia, arteriosclerosis, coronary heart disease etc.(6) because of it has stronger antioxygenation, suppress or removing activity in vivo free radical, can delay the aging of body.
In the soybean isoflavones, effective constituent mainly contains glucosides and aglycon two classes.Glucosides mainly comprises daidzin (Daidzin) (structural formula is suc as formula I) and Genistoside (Genistin) (structural formula is suc as formula II), its hydrolysate is respectively daidzein (Daidzein) (structural formula such as formula III) and genistein (Genistein) (structural formula is suc as formula IV), just free aglycon.Directly extracting the crude soya bean isoflavones main component that obtains from soybean is glucosides, aglycon only accounts for a very little part, but that mainly play physiologically active is aglycon (MizukiOnozawa, Kazunori Fukuda, Mikinobu Ohtani.Effects of Soybean Isoflavoneson Cell Growth and Apoptosis of the Human Prostatic Cancer Cell Line LNCaP, Jpn.J.Clin.Oncol., 1998,28:360-363), so the research of hydrolyzed soy bean isoflavone is just become a focus.Hydrolyzed soy bean isoflavone mainly contains acid system hydrolysis, alkali process hydrolysis and enzymatic hydrolysis at present.Because acid system and alkali process hydrolysis obtain the stability of product and have obtained query (Zhang Chen, Yang Xiaoquan, Kong Huiqing. soybean isoflavones component Study on Stability present situation under the different processing conditionss. the soybean science, 2006,25 (1): 73-76), and because a large amount of soda acid in the system is not easy to realize industrialization.The enzymatic hydrolysis soybean isoflavones has the advantage of enzymic catalytic reaction such as mild condition, but exists enzyme not recycle, cost price height, and problem such as later stage product separation difficulty.
Summary of the invention
The method that the purpose of this invention is to provide a kind of enzyme hydrolysis of soybean isoflavone.
The method of enzyme hydrolysis of soybean isoflavone provided by the present invention is the soybean isoflavones that the soybean isoflavone glycoside hydrolysis is obtained the aglycon type with the immobilization soybean isoflavone glucoside hydrolase in the diphasic system of being made up of organic solvent and damping fluid; Described organic solvent to the solubleness of the aglycon of described soybean isoflavones greater than water.
Described organic solvent can be alcohols, ester class or organic solvent of ketone, is preferably the ester class, especially is preferably ethyl acetate; Described damping fluid can be phosphate buffered saline buffer, Tirs-hydrochloride buffer, citrate buffer or carbonate buffer solution.
Described soybean isoflavone glucoside hydrolase specifically can be beta-glucosidase; Described damping fluid specifically can be citrate buffer.
The pH value of described damping fluid can be 3-9, is preferably 3-7, especially is preferably 5-6.5.
The solution of described citrate buffer for being made into by citric acid and trisodium citrate; The volumetric molar concentration of the solution of described citric acid and trisodium citrate can be 0.05-2M, is preferably 0.1-0.5M, especially is preferably 0.1M.
The organic solvent volume content is preferably 10%-80% in the described diphasic system, especially is preferably 40%-60%.
The temperature of reaction of described hydrolysis reaction can be 10-90 ℃, is preferably 30 ℃-60 ℃, especially is preferably 40-45 ℃.
In the described method, the proportioning of described immobilization beta-glucosidase and soybean isoflavones can be every gram soybean isoflavones 1U-1000U immobilization beta-glucosidase, is preferably every gram soybean isoflavones 10-50U immobilization beta-glucosidase.
In the described method, reaction can be removed described organic solvent with organic phase and aqueous phase separation after finishing, and obtains the soybean isoflavones hydrolysate.
In order to solve the cost price height that exists in the existing enzymatic hydrolysis soybean isoflavones and the problem of later stage separation difficulty, the present invention adopts immobilized enzyme hydrolysis soybean isoflavones and diphasic system hydrolyzed soy bean isoflavone.In the immobilized enzyme, the enzyme of hydrolyzed soy bean isoflavone is selected beta-glucosidase for use; And carrier is selected the abundant and good chitosan of biocompatibility in source for use; It is made bead, then beta-glucosidase is fixed on the bead with the ordinary method covalent cross-linking.Immobilized enzyme can reuse, and is easy to separate from product.Soybean isoflavones is dissolved in the ester class easily, alcohols, and organic solvent of ketone is insoluble in water, and sherwood oil, in the non-polar solvents such as normal hexane; And enzyme easy inactivation in organic solvent, its hydrolysis needs suitable buffered soln.Therefore diphasic system can be realized the efficient utilization of enzyme and the analysis of product.This biphasic reaction system mechanism as shown in Figure 1, in the reaction, consumption along with substrate in the aqueous phase solution (daidzin and Genistoside), substrate in the precipitation enters aqueous phase solution and replenishes, for ethyl acetate, the solubleness of glucosides in the soybean isoflavones in water is higher than ethyl acetate, replenishes so the substrate of most of solid phase directly enters in the water.The product aglycon that generates after the hydrolysis in diphasic system (daidzein and genistein) constantly is extracted into (daidzein and genistein are very little in aqueous phase solubleness) in the organic phase, again because glucosides solubleness in organic phase is very little, so reaction after product aglycon all is enriched in the organic phase, and contains glucosides hardly.Reaction finishes to take out organic phase, evaporates organic solvent and can obtain desired aglycon.
The present invention also provides simultaneously respectively in single-phase (damping fluid) and diphasic system, with the experimental result of immobilization and free beta-glucoside enzyme hydrolysis of soybean isoflavone.Find that the immobilized enzyme hydrolysis soybean isoflavones is faster than speed in single-phase in the diphasic system, the yield height, and the product that obtains does not have peculiar smell; Resolvase is easy to inactivation in two-phase, hydrolyzed soy bean isoflavone is not thorough.
The method of enzyme hydrolysis of soybean isoflavone of the present invention has not only realized the effective hydrolyzed soy bean isoflavone of enzyme, and the product that this method obtains do not have peculiar smell, need not purifying, and apparatus and process is simple, has good industrial amplification prospect.
Description of drawings
Fig. 1 is a diphasic system immobilized enzyme hydrolysis soybean isoflavones schematic diagram of the present invention
Fig. 2 is the HPLC collection of illustrative plates of daidzin, Genistoside, daidzein and genistein standard substance
Fig. 3 is the dripping and leaking device synoptic diagram
Fig. 4 is the relative reactivity of free and immobilization beta-glucosidase under the different pH values
Fig. 5 is the stability of 40 ℃ of immobilizations and free beta-glucosidase
Fig. 6 is the stability of 50 ℃ of immobilizations and free beta-glucosidase
Fig. 7 is the stability of 60 ℃ of immobilizations and free beta-glucosidase
Fig. 8 lives for the relative enzyme of 16 pNPG of immobilization beta-glucosidase continuous hydrolysis
Fig. 9 is the hydrolysising experiment result of immobilized enzyme in the single_phase system
Figure 10 is the hydrolysising experiment result of immobilized enzyme in the diphasic system
Figure 11 is easy hydrolysis isoflavones reactor synoptic diagram
Embodiment
The method of enzyme hydrolysis of soybean isoflavone of the present invention may further comprise the steps:
(1) enzyme is fixed on the carrier according to a conventional method, the enzyme here can be various soybean isoflavone glucoside hydrolases, is preferably beta-glucosidase, and carrier can also be the carrier of multiple routine, is preferably chitosan.
(2) in diphasic system, utilize immobilized enzyme, carry out the hydrolysis of soybean isoflavone glycoside.The diphasic system here is made up of a kind of organic solvent and a kind of damping fluid, and wherein organic phase can be for dissolving good ester class to the aglycon type, alcohols, and organic solvent of ketone, as n-hexyl alcohol, ethyl acetate, methyl acetates etc. are preferably ethyl acetate here.Water can be for the suitable damping fluid of multiple enzyme commonly used, as phosphoric acid buffer, and the Tirs hydrochloride buffer, citrate buffer, carbonate buffer solutions etc. are preferably citrate buffer here.Further, selected citrate buffer specifically can be the solution of citric acid and trisodium citrate, and its pH can be 3.0-9.0, is preferably pH=5.0, and volumetric molar concentration is 0.1M.
Component proportioning arbitrarily in the diphasic system is preferably 1: 1 described in (3) second steps.
(4) in the described method, the proportioning of described immobilization beta-glucosidase and soybean isoflavones can be every gram soybean isoflavones 1U-1000U enzyme, is preferably every gram soybean isoflavones 10U.
(5) temperature of reaction of the described hydrolysis reaction of present method can be 10-90 ℃, is preferably 40 ℃.
(6) in the described method, after reaction finishes, organic phase and water are decomposed, remove the organic solvent in the organic phase,, can obtain the soybean isoflavones hydrolysate of aglycon type as preferred ethyl acetate.
Specifically illustrate the method for enzyme hydrolysis of soybean isoflavone of the present invention below in conjunction with embodiment.
Used material among the following embodiment:
Chitosan: available from Jinan Haidebei Marine Organism Engineering Co., Ltd., deacetylation 90.2%.
Beta-glucosidase: available from Fluka, article No. 49290.
Beta-glucoside enzyme substrates pNPG (p-Nitrophenyl-β-D-galactopyranoside, BBI packing).
Daidzin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 111738-200501.
Genistoside: Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 111709-200501.
Daidzein: Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 1502-200101.
Genistein: Nat'l Pharmaceutical ﹠ Biological Products Control Institute, 111704-200501.
Soybean isoflavones:, be conventional 30% content product available from Zhejiang Xinxin Bio-tech Co., Ltd..Its component concentration is to measure with HPLC (day the island proper Tianjin), and moving phase is to contain methanol solution and the pure water that volumn concentration is 0.5% acetate, and the volume ratio of methanol solution and pure water is 37: 63, and flow velocity is 1ml.min
-1, 40 ℃ of column temperatures.Chromatographic column is the C18 reversed-phase column of the Diamonsil of Dikma company, and model is 5U, 250 * 4.6mm.Be HPLC such as Fig. 2 with this understanding with standard substance.And do typical curve with external standard method with this understanding, get linear regression equation:
Daidzin: Y=21872.46+3.552E7*X, R=0.99992, X:0002-0.064/mg.ml
-1, Y is a peak area.
Genistoside: Y=18902.34+4.9223E7*X, R=0.99978, X:0.002-0.048/mg.ml
-1, Y is a peak area.
Daidzein: Y=-28376.86+6.4507E7*X, R=0.99903, X:0.002-0.048/mg.ml
-1, Y is a peak area.
Genistein: Y=33896.50+6.7382E7*X, R=0.99975, X:0.002-0.08/mg.ml
-1, Y is a peak area.
HPLC condition in this patent is all the same, and data computation is all by this equation.
The quality percentage composition of this soybean isoflavones component: daidzin (Daidzin): 10.6%, Genistoside (Genistin): 17.9%, daidzein (Daidzein): 1.7%, genistein (Genistein): 1.5%.Mention the equal specification for this reason of soybean isoflavones before the hydrolysis if no special instructions.
Below in each example immobilized enzyme or resolvase hydrolyzed soy bean isoflavone obtain the yield of aglycon, be defined as follows:
The preparation of embodiment 1, immobilization beta-glucosidase
One, the preparation of immobilization beta-glucosidase
Prepare about 2.5mm of diameter and very uniform chitosan bead (Peng Zhiying according to the method for describing in the document, Yue Zhenfeng, Zhang Xuebing. the preparation of microspheroidal immobilization alpha-glucosidase. South China Science ﹠ Engineering University's journal (natural science edition), 2001,29 (6): 56-59 and Nitin W.Fadnavis, Gurrala Sheelu, BezavadaMani Kumar.Gelatin Blends with Alginate:Gels for Lipase Immobilizationand Purification, Biotechnol.Prog.2003,19:557-564).Concrete grammar is as follows: with volumn concentration is the chitosan solution that 1% acetate is made into 25g/100ml, static 30 minutes, cooperate little funnel to make as shown in Figure 3 dripping and leaking device with suction nozzle, suction nozzle is gloomy 1ml of gill and 200 μ l suction nozzles, and funnel is the little funnel of 10ml.
Apart from being to be the condensation water that 3: 2 2M NaOH and formaldehyde are made under the suction nozzle 20cm, chitosan solution is splashed into the condensation water that is stirring promptly get the chitosan bead by volume ratio.
With chitosan bead flushing with clean water 3-5 time that makes, in the 0.1M of pH=6.5 citric acid-sodium citrate damping fluid, at room temperature proportioning adding chitosan bead and the beta-glucosidase according to every gram chitosan 10U beta-glucosidase carries out physical adsorption 10h, added volumn concentration then and be 0.4% glutaraldehyde cross-linking 20 minutes, the beta-glucosidase of being fixed.
Two, the characteristic of immobilization beta-glucosidase
1, the relation of immobilization beta-glucosidase enzymic activity and pH
With immobilized enzyme and the resolvase that obtains, respectively at 40 ℃, the pNPG of hydrolysis 10mM in the 0.1M citric acid-sodium citrate damping fluid of different pH values, hydrolysis 7 minutes, the concentration of hydrolysate (right-nitrophenols) in the working sample obtains the relative reactivity at different pH enzymes then.The relative reactivity here refers under certain pH that will measure high reactivity and is defined as 1, and the relative reactivity other pH under be its activity and the most highly active ratio.Because the slope of the variation of sample absorbancy follows the activity of enzyme to be directly proportional, derivation is as follows again:
A＝KCL
A is an absorbancy, C solution mole solubility, and L light path, K are instrument parameter, are definite value.
Because: dA/dt=K (dC/dt) L
That is: Slope=K[(dm/V)/dt] (V/S)
So: dm/dt=slope.S/K
Here m is the quality of product, and S is the hole area (definite value) of enzyme plate.
So the slope of the variation of mensuration absorbancy can be calculated the relation between each activity under the identical situation of sampling.
The result shows with this understanding that as shown in Figure 4 the optimal reaction pH of immobilized enzyme is 5.
2, the relation of immobilization beta-glucosidase enzymic activity and temperature
Respectively at 40 ℃, 50 ℃, place the free and the immobilized beta-glucosidase of some equivalent in 60 ℃ the water-bath, the residing microenvironment of enzyme is the citric acid-sodium citrate damping fluid of the 0.1M of pH=5.Took out the enzyme of equivalent every two hours, in the citric acid-sodium citrate damping fluid of the 0.1M of pH=5, under 40 ℃ the condition, the pNPG of hydrolysis 10mM, hydrolysis 7 minutes, the concentration of hydrolysate (right-nitrophenols) in the working sample then.Obtain the thermostability under each temperature in 8 hours.The result shows that immobilized enzyme is more stable when being lower than 40 ℃ shown in Fig. 5-7, enzyme work had reduced by 83.8% after enzyme work had reduced by 25.2%, 60 ℃ of heating 8h behind 50 ℃ of heating 8h.
3, the repeat performance of immobilization beta-glucosidase enzyme
At 40 ℃, the pNPG of continuous hydrolysis 10mM in the 0.1M citric acid-sodium citrate damping fluid of pH=5, each hydrolysis was carried out 7 minutes, the concentration of hydrolysate in the working sample then, the relative enzyme of being fixed is lived.With the enzyme work of mensuration for the first time is 1, measures enzyme enzyme alive and that measure for the first time ratio alive later on as relative reactivity.From reaction solution, take out immobilized enzyme, give a baby a bath on the third day after its birth time, repeat above-mentioned survey enzyme and live, and then take out, wash again, repeat this process with damping fluid.Being fixed enzyme is in the activity change of using through 16 times.The immobilization activity of beta-glucosidase is not loss (as Fig. 8) almost.
The soybean isoflavones and the 1.48U immobilization beta-glucosidase that in the citric acid-sodium citrate damping fluid of 5ml 0.1M pH=5 and 5ml ethyl acetate, add 30% specification of 14.8mg.Reaction is all carried out in 40 ℃ constant temperature shaking table, reacts three hours, measures daidzin, Genistoside, daidzein and genistein content by above-mentioned HPLC method.
The amount 0.96mg of the amount 0.97mg, reaction that the result shows the daidzein that immobilized enzyme hydrolysis obtains at last deserved daidzein of amount 0.25mg, the daidzin complete hydrolysis of daidzein when initial; The amount 1.64mg of the amount 1.50mg of the genistein that obtains at last, the reaction deserved genistein of amount 0.22mg, the Genistoside complete hydrolysis of genistein when initial.As calculated, the immobilization beta-glucosidase is hydrolyzed soy bean isoflavone in this diphasic system, and the yield of daidzein is 75.4%, and the yield of genistein is 77.9%.
The application contrast in single_phase system and diphasic system of embodiment 3, immobilization beta-glucosidase
Two processing are established in experiment, and one of them is treated to single_phase system, at 10ml 0.1M, add the soybean isoflavones and the 1.48U immobilization beta-glucosidase of 30% specification of 14.8mg in the pH=5 citric acid-sodium citrate damping fluid; Another is treated to diphasic system, adds the soybean isoflavones and the 1.48U immobilization beta-glucosidase of 30% specification of 14.8mg in the citric acid-sodium citrate damping fluid of 5ml 0.1M pH=5 and 5ml ethyl acetate.Reaction is all carried out in 40 ℃ constant temperature shaking table, and per hour sampling is measured daidzin, Genistoside, daidzein and genistein content by above-mentioned HPLC method, reacts three hours.Result such as Fig. 9 and shown in Figure 10 not only can not get product in the single_phase system, and hydrolysis rate is also a little less than the speed in diphasic system.
Hydrolyzed soy bean isoflavone in embodiment 4, the immobilized enzyme diphasic system
The soybean isoflavones and the 1.48U immobilization beta-glucosidase that in the citric acid-sodium citrate damping fluid of 1ml 0.1M pH=5 and 9ml ethyl acetate, add 30% specification of 14.8mg.Reaction is all carried out in 40 ℃ constant temperature shaking table, reacts three hours, measures daidzin, Genistoside, daidzein and genistein content by above-mentioned HPLC method.
The amount 0.96mg of the amount 0.96mg, reaction that the result shows the daidzein that immobilized enzyme hydrolysis obtains at last deserved daidzein of amount 0.25mg, the daidzin complete hydrolysis of daidzein when initial; The amount 1.64mg of the amount 1.20mg of the genistein that obtains at last, the reaction deserved genistein of amount 0.22mg, the Genistoside complete hydrolysis of genistein when initial.As calculated, the immobilization beta-glucosidase is hydrolyzed soy bean isoflavone in this diphasic system, and the yield of daidzein is 73.9%, and the yield of genistein is 59.8%.
The application contrast in diphasic system of embodiment 5, immobilization and free beta-glucosidase
With the flask of 100ml, thermostatic mixer, magneton makes up an easy intermittently continuous stir reactor reactor (Figure 11).Two processing are established in experiment, one of them is treated to free beta-glucosidase diphasic system, adds the soybean isoflavones and the 5U free beta-glucosidase of 30% specification of 500mg in the citric acid-sodium citrate damping fluid of 15ml 0.1MpH=5 and 15ml ethyl acetate; Another is treated to immobilization beta-glucosidase diphasic system, adds the soybean isoflavones and the 5U immobilization beta-glucosidase of 30% specification of 500mg in the citric acid-sodium citrate damping fluid of 15ml 0.1M pH=5 and 15ml ethyl acetate.At 40 ℃, magneton reacted respectively under the condition of 800rpm/min 20 hours.After reaction finished, sampling was measured daidzin, Genistoside, daidzein and genistein content by above-mentioned HPLC method.
The amount 32.3mg of the amount 30.9mg, reaction that the result shows the daidzein that immobilized enzyme hydrolysis obtains at last deserved daidzein of amount 8.5mg, the daidzin complete hydrolysis of daidzein when initial; The amount 55.5mg of the amount 52.3mg of the genistein that obtains at last, the reaction deserved genistein of amount 7.5mg, the Genistoside complete hydrolysis of genistein when initial; As calculated, the immobilization beta-glucosidase is hydrolyzed soy bean isoflavone in this diphasic system, and the yield of daidzein is 69.2%, and the yield of genistein is 80.8%.The amount 26.8mg of the daidzein that the resolvase hydrolysis obtains at last, the amount of genistein is 25.4mg; As calculated, resolvase hydrolyzed soy bean isoflavone, the yield of its daidzein are 56.7%, genistein is 32.3%, and along with the reaction times prolongs, its yield does not further improve, and illustrates that free beta-glucosidase is at the easy inactivation of hydrolytic process in this system.
Claims (9)
1, a kind of method of enzyme hydrolysis of soybean isoflavone is the soybean isoflavones that the soybean isoflavone glycoside hydrolysis is obtained the aglycon type with the immobilization soybean isoflavone glucoside hydrolase in the diphasic system of being made up of organic solvent and damping fluid; Described organic solvent to the solubleness of described isoflavone genin greater than water.
2, method according to claim 1 is characterized in that: described organic solvent is alcohols, ester class or organic solvent of ketone, is preferably the ester class, especially is preferably ethyl acetate; Described damping fluid is phosphate buffered saline buffer, Tirs-hydrochloride buffer, citrate buffer or carbonate buffer solution.
3, method according to claim 1 is characterized in that: described soybean isoflavone glucoside hydrolase is a beta-glucosidase; Described damping fluid is a citrate buffer.
4, method according to claim 3 is characterized in that: the pH value of described damping fluid is 3-9, is preferably 3-7, especially is preferably 5-6.5.
5, method according to claim 4 is characterized in that: the solution of described citrate buffer for being made into by citric acid and trisodium citrate; The volumetric molar concentration of the solution of described citric acid and trisodium citrate is 0.05-2M, is preferably 0.1-0.5M, especially is preferably 0.1M.
6, method according to claim 1 is characterized in that: the organic solvent volume content is 10%-80% described in the described diphasic system, is preferably 40%-60%.
7, method according to claim 1 is characterized in that: the temperature of reaction of described hydrolysis reaction is 10-90 ℃, is preferably 30 ℃-60 ℃, especially is preferably 40-45 ℃.
8, method according to claim 1, it is characterized in that: in the described method, the proportioning of described immobilization beta-glucosidase and soybean isoflavones is every gram soybean isoflavones 1U-1000U immobilization beta-glucosidase, is preferably every gram soybean isoflavones 10-50U immobilization beta-glucosidase.
9, method according to claim 1 is characterized in that: after reaction finishes in the described method, with organic phase and aqueous phase separation, remove described organic solvent, obtain the soybean isoflavones hydrolysate.
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| CN101974577A (en) * | 2010-09-27 | 2011-02-16 | 徐州技源天然保健品有限公司 | Novel production process for extracting soy isoflavones aglycones |
| CN102094052A (en) * | 2010-12-12 | 2011-06-15 | 大连理工大学 | Method for preparing natural medicines by coupling glycosidase catalysis and salting-out extraction |
| CN102094052B (en) * | 2010-12-12 | 2013-04-17 | 大连理工大学 | Method for preparing natural medicines by coupling glycosidase catalysis and salting-out extraction |
| CN103146774A (en) * | 2011-12-07 | 2013-06-12 | 长春大学 | Preparation method of aglycone-type isoflavones |
| CN103262941A (en) * | 2013-05-14 | 2013-08-28 | 华南理工大学 | Isoflavone aglycone-enriched soybean protein and preparation method thereof |
| CN103262941B (en) * | 2013-05-14 | 2014-07-02 | 华南理工大学 | Isoflavone aglycone-enriched soybean protein and preparation method thereof |
| CN103667403A (en) * | 2013-12-17 | 2014-03-26 | 江苏科技大学 | Segmented flow microreaction technology for enzymatic synthesis of isoquercitrin in biphasic system |
| CN106755214A (en) * | 2016-12-08 | 2017-05-31 | 江苏大学 | A kind of method that two-phase enzyme hydrolysis obtains precious leaves of pulse plants glycosides I |
| CN106755214B (en) * | 2016-12-08 | 2020-06-26 | 江苏大学 | Method for obtaining baohuoside I by means of two-phase enzymatic hydrolysis |
| 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 |
| WO2020107629A1 (en) * | 2018-11-28 | 2020-06-04 | 山东省分析测试中心 | Use of high-speed countercurrent chromatograph in bioconversion of glycoside component |
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