CN103146774A - Preparation method of aglycone-type isoflavones - Google Patents
Preparation method of aglycone-type isoflavones Download PDFInfo
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- CN103146774A CN103146774A CN2011104016804A CN201110401680A CN103146774A CN 103146774 A CN103146774 A CN 103146774A CN 2011104016804 A CN2011104016804 A CN 2011104016804A CN 201110401680 A CN201110401680 A CN 201110401680A CN 103146774 A CN103146774 A CN 103146774A
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Abstract
The invention relates to a preparation method of aglycone-type isoflavones. According to the invention, the aglycone-type isoflavones is prepared by hydrolyzing glycosidic bonds of isoflavone glucosides by using beta-glucosidase of Thermofilum pendens in aqueous dimethyl sulfoxide under a high temperature. The invention firstly establishes the method that glucoside-type isoflavones with a total glycoside content of 40 % (w/w) or more are fully converted into the aglycone-type isoflavones through enzymatic hydrolysis in vitro; and the method has advantages of small reaction solution volume, fast reaction speed and high enzymatic hydrolysis efficiency.
Description
Technical field
The present invention relates to a kind of preparation method of aglycon type soybean isoflavones, refer in particular to and utilize that a kind of extreme heat is stable, the beta-glucuroide of organic solvent-resistant, in the moisture organic solvent of high temperature, the glycosidic link of hydrolyzed soy bean isoflavone glucosides, preparation aglycon type soybean isoflavones.
Background technology
Soybean isoflavones has weak estrogen activity to human body, can alleviate the incidence of the tumor diseases such as the uncomfortable disease of Woman climacteric, preventing osteoporosis disease, reduction mammary cancer and prostate cancer.At present, the soybean isoflavone protective foods has been widely used in controversies in hormone replacement in the elderly, to replenish the oestrogenic hormon in the menopausal woman body.
Soybean isoflavones has glucoside type and two kinds of molecular architectures of aglycon type.The bioavailability of aglycon type soybean isoflavones in human body is higher, and he can directly be absorbed by human intestinal and enter blood, and Plasma Concentration is high, time to peak is short.The bioavailability of soybean isoflavone glycoside is lower, and he can not directly be absorbed by human intestinal, only has small part under the effect of intestinal microflora Secretases, is hydrolyzed lower glucosyl residue, is transformed into the aglycon form, just can be absorbed.
Isoflavones in soybean and alcohol extract thereof is mainly glucoside type.In order to improve the oral absorption rate of soybean isoflavones, usually use the beta-glucuroide to be converted into isoflavone genin at the external alcohol extract that will hang down soybean isoflavone glucoside content in advance.Yet water-soluble lower (for example at room temperature, Genistoside water-soluble less than 25 μ M) of soybean isoflavones, the direct alcohol extract of enzymatic conversion method soybean isoflavone glucoside content more than 40% (w/w) still so far.
Summary of the invention
The present invention adopts Pyrococcus furiosus beta-glucuroide, the soybean isoflavone glycoside of catalysis high density hydrolysis in high temperature and moisture organic solvent, thereby preparation aglycon type soybean isoflavones (Fig. 1).
The soybean isoflavone glucoside that this invention is used comprises Genistoside (genistin), daidzin (daidzin), glycitin (glycitin) and composition thereof, and total glucosides content is more than 40% (w/w).
The organic solvent that this invention is used is dimethyl sulfoxide (DMSO); Dimethyl sulfoxide (DMSO) can be dissolved each other with water, and soybean isoflavones has higher solubleness in moisture dimethyl sulfoxide (DMSO).
The extreme heat that this invention is used is stable, the beta-glucuroide (EC 3.2.1.21) of organic solvent-resistant, belong to carbohydrate activity enzyme GH3 family, originate from and contain the intestinal bacteria that external source relies on heated filament bacterium Thermofilum pendens beta-alpha-glucosidase gene (Tpbgl).Namely adopt conventional gene cloning (Sambrook and Russell, Molucular Cloning:A Laboratory Manual, third edition.Cold Spring Harbor Laboratory Press.2001.ISBN 978-087969577-4): rely on heated filament bacterium Thermofilum pendens genomic dna as masterplate (10ng) take 1 μ L, add four kinds of dNTP mixtures of 8 μ L (every kind of 2.5mM), 2 μ L * 10 μ M forward primers (Tpbgl-NdeI:5 '-GGG
CATATGAGGGGAAGGGGCCCTG-3 '), 2 μ L * 10 μ M reverse primers (Tpbgl-HindIII:5 '-CCG
AAGCTTTCAGGGCGCGAATCTCA-3 '), 20 μ L damping fluids (
Buffer Mg
2+Plus), 66 μ L sterile distilled waters and 1 μ LDNA polysaccharase (
HS DNA polymerase, 2.5U/ μ L), under the condition of 98 ℃ of denaturation temperatures, 62 ℃ of annealing temperatures, 72 ℃ of elongating temperatures, carry out the polymerase chain reaction, amplify the Tpbgl gene of 2.1kb; Will be mixed with Tpbgl gene and the p6 * His119 carrier of restriction enzyme NdeI and HindIII difference double digestion, add the T4DNA ligase, construction recombination plasmid p6 * His119-Tpbgl, then transform intestinal bacteria E.coli MC1061[F-, araD139, recA13, Δ (araABC-leu) 7696, galU, galK lacX74, rpsL, thi, hsdR2, and mcrB]; The method of the preparation of the cultivation of gene recombined escherichia coli, beta-glucuroide, purifying and determination of activity is the method for reporting in document " Choi; Ki-Won et al.Modulation of the Regioselectivity of a Thermotoga neapolitana β-Glucosidase by Site-Directed Mutagenesis.Journal of Microbiology and Biotechnology.2008,18 (5): 901-907 ".Adopting the resulting dependence heated filament of aforesaid method bacterium Thermofilum pendens beta-glucuroide, is a kind of novel gene recombination Pyrococcus furiosus beta-glucuroide; The suitableeest catalyzed reaction temperature of this enzymic hydrolysis p-nitrophenyl-β-D-glycopyranoside is that 90 ℃, the suitableeest catalyzed reaction pH are 3.5; And, in the dimethyl sulfoxide (DMSO) of 10-60% (v/v), still have higher hydrolytic activity.The solubleness that helps to strengthen soybean isoflavone glucoside due to high temperature and moisture dimethyl sulfoxide (DMSO), so in the hydrolysis reaction that relies on the enzyme catalysis of heated filament bacterium Thermofilum pendens beta-glucoside, can use the soybean isoflavone glucoside of higher concentration of substrate.
The preparation method of aglycon type soybean isoflavones involved in the present invention comprises the following steps:
(1) with the soybean isoflavones of total glucosides content more than 40% (w/w), join in the dimethyl sulfoxide (DMSO) that concentration is 10-60% (v/v) (contain the pH value and be the damping fluid of 3-7), mixing, the concentration that makes substrate are 0.05-0.55% (w/v);
(2) with above-mentioned substrate solution at 50-100 ℃ of preheating 3min-1hr;
(3) adding enzyme activity is the dependence heated filament bacterium Thermofilum pendens beta-glucuroide of 10-500U/mL reaction solution, mixing;
(4) continue then to be cooled to room temperature at 50-100 ℃ of insulation 10min-2hr;
(5) by carbon 18 posts or the macroporous adsorptive resins of activation, adsorb isoflavones, wash except sugared salt again, resolve isoflavones with 70% ethanol (v/v) at last.
Carbon 18 posts involved in the present invention or the activation of macroporous adsorptive resins be after adopting 70% ethanol (v/v) to spend the night to soak, then washing are except alcohol; Then with reaction solution upper prop, absorption isoflavones; Remove sugared salt, resolve isoflavones with two column volume 70% ethanol (v/v) with two column volume washings again; Flow velocity is one hour two column volume.Macroporous adsorbent resin can be selected Rohm﹠amp; Haas Amberlite
TMXAD16 macroporous adsorbent resin or the close macroporous adsorbent resin of other polarity.
The detection method of soybean isoflavones involved in the present invention is high performance liquid chromatography, and testing conditions is:
Adopt high performance liquid chromatography (Prominence LC 20AT, Shimadzu, Japan); UV-detector, wavelength 254nm; Chromatographic column, carbon 18 (Zorbax Eclipse XDB, 150 * 4.6mm i.d., 3.5 μ m particle size, Agilent, the U.S.); Moving phase, A: methanol/water/formic acid (20: 80: 0.1) and B: methanol/water/formic acid (80: 20: 0.1); Gradient: 0min, 100%A:0～30min, B is increased to 100% by zero line; Flow velocity 0.8mL/min; 25 ℃ of column temperatures; Sampling volume 20 μ L.
Owing to having used this novel beta-glucuroide, so can be in water-containing organic solvent at higher temperature, carry out the enzymatic hydrolysis reaction of high concentration substrate, can make soybean isoflavone glucoside all be converted into corresponding isoflavone genin, dwindled the volume of reaction solution, improve enzymolysis efficiency, had using value preferably.
Description of drawings
Fig. 1 is the Genistoside high-efficient liquid phase chromatogram.In figure, retention time is that the chromatographic peak of 15.131min is Genistoside.
Fig. 2 is that after adding dependence heated filament bacterium Thermofilum pendens beta-glucuroide, Genistoside is hydrolyzed to the high-efficient liquid phase chromatogram of genistein fully.In figure, retention time is that the chromatographic peak of 24.178min is genistein.
Embodiment
1.6mg soybean isoflavones (Genistoside content is 95.8% (w/w)) and 200 μ L dimethyl sulfoxide (DMSO) are joined in the Eppendorf tube of 1.5mL, the Britton-Robinson damping fluid that adds again 200 μ L pH3.5, after mixing, be put into preheating 5min in the water-bath of 90 ℃; Then adding vigor is the dependence heated filament bacterium Thermofilumpendens beta-glucuroide of 150U, with the blank tube that do not add enzyme in contrast, continues to be cooled to room temperature after 90 ℃ of insulation 1hr; With carbon ten eight posts (SEP-Pak C18cartridge, Mi Libo, the U.S.) of above-mentioned mixed solution by activation, washing is except sugar and salt; After again 70% ethanol (v/v) desorbed solution being used respectively the micro-pore-film filtration in 0.45 μ M aperture, carry out efficient liquid phase chromatographic analysis, obtain respectively the color atlas 1 of control sample (in Fig. 1, retention time 15.131min chromatographic peak is Genistoside) and the color atlas 2 (in Fig. 2, the chromatographic peak of retention time 24.178min is genistein) of response sample; Result shows: under above-mentioned reaction conditions, rely on heated filament bacterium Thermofilum pendens beta-glucuroide all Genistosides all are hydrolyzed into genistein, transformation efficiency is 100%.
Claims (4)
1. the preparation method of an aglycon type soybean isoflavones is characterized in that adopting following steps:
(1) with the soybean isoflavones of total glucosides content more than 40% (w/w), join in the dimethyl sulfoxide (DMSO) that concentration is 10-60% (v/v) (contain the pH value and be the damping fluid of 3-7), mixing, the concentration that makes substrate are 0.05-0.55% (w/v);
(2) with above-mentioned substrate solution at 50-100 ℃ of preheating 3min-1hr;
(3) adding enzyme activity is the dependence heated filament bacterium Thermofilum pendens beta-glucuroide of 10-500U/mL reaction solution, mixing;
(4) continue then to be cooled to room temperature at 50-100 ℃ of insulation 10min-2hr;
(5) by carbon 18 posts or the macroporous adsorptive resins of activation, adsorb isoflavones, wash except sugar and salt again, resolve isoflavones with 70% (v/v) ethanol at last.
2. the preparation method of a kind of aglycon type soybean isoflavones according to claim 1, is characterized in that the enzyme that uses is to rely on heated filament bacterium Thernofilum pendens beta-glucuroide.
3. the preparation method of a kind of aglycon type soybean isoflavones according to claim 1, is characterized in that the organic solvent that uses is dimethyl sulfoxide (DMSO).
4. the preparation method of a kind of aglycon type soybean isoflavones according to claim 1, is characterized in that the soybean isoflavone glucoside that uses is Genistoside, daidzin, glycitin or its mixture of total glucosides content more than 40% (w/w).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115669862A (en) * | 2022-11-15 | 2023-02-03 | 苏州金记食品有限公司 | Preparation method of bean curd gel rich in soybean isoflavone |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6444239B2 (en) * | 2000-01-28 | 2002-09-03 | Kikkoman Corporation | Process for producing isoflavone aglycone-containing composition |
| CN101086002A (en) * | 2007-06-22 | 2007-12-12 | 清华大学 | Method for hydrolyzing soybean isoflavone by enzyme |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6444239B2 (en) * | 2000-01-28 | 2002-09-03 | Kikkoman Corporation | Process for producing isoflavone aglycone-containing composition |
| CN101086002A (en) * | 2007-06-22 | 2007-12-12 | 清华大学 | Method for hydrolyzing soybean isoflavone by enzyme |
Non-Patent Citations (1)
| Title |
|---|
| YEMIN XUE, ET AL.: "Hydrolysis of soy isoflavone glycosides by recombinant β-glucosidase from hyperthermophile Thermotoga maritima", 《J IND MICROBIOL BIOTECHNOL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115669862A (en) * | 2022-11-15 | 2023-02-03 | 苏州金记食品有限公司 | Preparation method of bean curd gel rich in soybean isoflavone |
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Application publication date: 20130612 |