CN1261586C - Method for preparing aglycon of soybean isoflavone glycoside base by enzymatic method hydrolyzing soybean - Google Patents
Method for preparing aglycon of soybean isoflavone glycoside base by enzymatic method hydrolyzing soybean Download PDFInfo
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- CN1261586C CN1261586C CN 03133637 CN03133637A CN1261586C CN 1261586 C CN1261586 C CN 1261586C CN 03133637 CN03133637 CN 03133637 CN 03133637 A CN03133637 A CN 03133637A CN 1261586 C CN1261586 C CN 1261586C
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Abstract
The present invention discloses a method for preparing the aglycone of a soybean isoflavone glycoside base by hydrolyzing the soybean isoflavone glycoside base with an enzyme method, which prepares soybean isoflavone aglycone by hydrolyzing a soybean isoflavone glycoside base with enzyme. The present invention comprises the following steps: enzyme which can hydrolyze a soybean isoflavone glycoside base is taken; the enzyme, the soybean isoflavone glycoside and a buffer solution are mixed for reaction at 4 to 75 DEG C at the pH value of 3 to 9 for 1 to 40 hours to extract soybean isoflavone aglycone. The present invention overcomes the defects of large destructibility to aglycone, large pollution, poor purposefulness, etc. existing in the prior art (an acid and alkali hydrolysis method); the present invention has the advantages of no pollution, strong purposefulness and high conversion rate, specifically, an inducer of enzyme preparation is added to microorganism fermentation so as to greatly improve the yield of enzyme thereof, help to sufficiently hydrolyze the soybean isoflavone glycoside base and all the more raise the conversion rate of the soybean isoflavone aglycone. The present invention can adopt enzyme for hydrolyzing monomer glycoside such as soybean glycoside, genistin and daidzin to prepare the corresponding aglycone, and can also adopt enzyme for hydrolyzing the mixed glycoside of soybean glycoside, genistin, daidzin, etc. to prepare the mixed aglycone of daidzein, soybean isoflavone, glycitein, etc. The product can be used as the raw material of medicine and health products.
Description
Technical field:
The present invention relates to a kind of method for preparing isoflavone aglycones, especially a kind ofly prepare the method for its glucoside unit with enzyme hydrolysis of soybean isoflavone glucoside glycosyl.
Background technology:
Soybean isoflavones content in soybean and soybean cotyledon is higher, mainly contains three kinds of glucoside units (big legumin glucoside unit, soybean isoflavone aglycones and the plain glucoside of soya bean unit), is referred to as soybean isoflavone aglycones; Also contain three kinds of glucosides (Daidzin, genistin and soya bean glucoside), be referred to as isoflavone glycoside.Its chemical structure is as follows respectively:
Big legumin (Daidzein) soybean isoflavones (Genistein) soya bean element (Glycitein)
Daidzin (Daidzin) genistin (Genistin)
Soya bean glucoside (Glycitin)
Soybean isoflavones is a phytoestrogen, can promote the secretion of sexual hormoue, somatomedin, has the menopausal women of improvement syndromes, preventing osteoporosis and cancer isoreactivity.The physiologically active of soybean isoflavone aglycones is far above isoflavone glycoside, but in the soybean isoflavones content higher but be genistin (Cenistin) and Daidzin, the content of glucoside unit is very low, has only the 2-3% of general glycoside.In order to obtain active high soybean isoflavone aglycones, once reported the peracid alkali hydrolysis method (need the life chief editor, the natural goods chemistry, Science Press, 1996, p603-604), but big, pollute also more serious to the broken ring of soybean isoflavone aglycones.
Although United States Patent (USP) (US5554519A), Japanese Patent (JP11-89589, JP8-214787) all disclose the method for producing soybean isoflavone aglycones with the glycosyl of enzyme hydrolysis of soybean isoflavone glucoside.But, because the ability of used enzyme hydrolysis of soybean isoflavone glucoside glycosyl causes the transformation efficiency of soybean isoflavone aglycones low.
Summary of the invention:
The present invention provides a kind of glycosyl of enzymatic hydrolysis isoflavone glycoside to prepare the method for its glucoside unit in order to solve the existing in prior technology technical problem, pollute little, purpose is strong, transformation efficiency is high.
Technical solution of the present invention is: a kind of enzymatic hydrolysis isoflavone glycoside glycosyl prepares the method for its glucoside unit, it is characterized in that comprising the steps:
A. be high-temperature aerobic bacterium, black-koji mould, candiyeast, Agaricus bitorqui bacterium enzymatic production inductor with the soybean, obtain containing the enzyme mixed solution behind liquid state fermentation or the solid state fermentation, in containing the enzyme mixed solution, add ammonium sulfate or extraction using alcohol enzyme;
B. with the enzyme, isoflavone glycoside and the acetic acid that are extracted or phosphoric acid hybrid reaction 1-40 hour, reaction conditions was temperature 4-75 ℃, pH value 3-9;
C. extract soybean isoflavone aglycones.
The isoflavone glycoside concentration of described b step is the 0.1-10% of total reactant volume.
Described isoflavone glycoside is the mixture of Daidzin, genistin and soya bean glucoside monomer or Daidzin, genistin and soya bean glucoside.
Described c step is for extracting or use n-butanol extraction with ethanol sedimentation.
The present invention compares with prior art, and maximum characteristics are to be the enzyme hydrolysis of soybean isoflavone glucoside glycosyl that high-temperature aerobic bacterium, black-koji mould, candiyeast, Agaricus bitorqui bacterium enzymatic production inductor are produced with the soybean, the preparation soybean isoflavone aglycones.Not only available enzyme hydrolysis monomer glucoside---Daidzin, genistin, soya bean glucoside become corresponding glucoside unit; Also can become big legumin, soybean isoflavone aglycones, soya bean element etc. and mix glucoside unit with mixing glucosides such as enzyme hydrolysis of soybean glucoside, genistin, soya bean glucosides, it is destructive big to glucoside unit to have overcome existing in prior technology, pollute big, shortcomings such as purpose difference have advantage pollution-free, that purpose is strong, transformation efficiency is high.
Embodiment:
Embodiment 1:
A. containing 3% Semen Maydis powder, 1% product enzyme induction thing with high-temperature aerobic bacterium Clostridium thermocopriea (document 1)---in soybean extract (dry) substratum, under 60 ℃ of conditions of temperature shaking culture 30-60 hour, bactofugation, toward the middle ammonium sulfate precipitation zymoprotein that adds 65% saturation ratio of supernatant liquor (containing the enzyme mixed solution), with 0.02M, the dialysis of pH5 acetate buffer solution, centrifugal slagging-off, freeze-drying obtains the isoflavone glycoside enzyme.
B. with 2 gram isoflavone glycoside enzymes, 30 gram genistins (Genistin), 1500 milliliters of acetate buffer solution (0.2M, pH5.0) and 150 milliliters of ethanol mix, make genistin account for the 0.1-10% of total reaction volume, stirring reaction is 1 hour under 75 ℃ of conditions of temperature.
C. react Hou and add 6000 milliliters of ethanol, remove by filter albumen precipitation, the filtrate decompression evaporate to dryness obtains 24 gram glucoside unit crude products, changes into soybean isoflavones (Genistein) glucoside unit with the glucoside of thin layer chromatography (document 2) detected result more than 80%.
Handle Daidzin and soya bean glucoside (Glycitin) with above-mentioned b step method, can obtain same result.Above-mentioned enzyme also can be handled the isoflavone glycoside mixture, and 80-90% becomes soybean isoflavones (Genistein) glucoside unit.
Above-mentioned enzyme liquid DEAE-Cellulose ion exchange resin column method is separated purifying soybean isoflavones zymoprotein with BioRed protein Preparation chromatographic instrument (document 3), and with SDS electrophoresis method determining molecular weight (document 4), the molecular weight of enzyme is 40,000.
Embodiment 2:
A. containing 4% Fructus Hordei Germinatus extract, 1% product enzyme induction thing with black-koji mould (Aspegillus niger)---in the substratum of soybean extract, stirring under temperature 28-30 ℃ condition ventilates cultivated 50-100 hour, bactofugation, in supernatant liquor (containing the enzyme mixed solution), precipitate zymoprotein, collect albumen with the ammonium sulfate of 60-75% saturation ratio, be dissolved in the phosphoric acid buffer (0.02M of 1/10 fermentating liquid volume, pH5.0) in, ammonium sulfate is removed in dialysis, and centrifugal slagging-off is enzyme liquid.
B. with 4 gram soybean genistins (Genistin), 100 milliliters of phosphoric acid buffers (0.02M, pH5.0) and 50 milliliters of above-mentioned enzyme liquid mix, make genistin account for the 0.1-10% of total reaction volume, reaction is 40 hours under 4 ℃ of conditions of temperature.
C. the n-butanol extraction three times that adds 1/3 total reaction volume, evaporated under reduced pressure obtains glucoside unit crude product.Obtain the pure soybean isoflavone aglycones of 1.4 grams with silicagel column separation method (document 3).
Above-mentioned enzyme is handled Daidzin and soya bean glucoside (Glycitin), obtains same result.
Above-mentioned enzyme is handled the isoflavone glycoside mixture, and 80-90% becomes soybean isoflavones (Genistein) glucoside unit.
Above-mentioned enzyme liquid DEAE-Cellulose ion exchange resin column method is separated purifying soybean daidzin zymoprotein with BioRed protein Preparation chromatographic instrument (document 3), and with SDS electrophoresis method (document 4) determining molecular weight, the molecular weight of enzyme is 3.5 ten thousand.
Embodiment 3:
A. containing 5% wheat bran extract, 0.2% product enzyme induction thing---soybean isoflavones or rutin with aspergillus oryzae (Aspegillus oryzae), perhaps in the substratum of the extract of 1% its flavonoid source plant, stirring under temperature 28-30 ℃ condition ventilates cultivated 50-100 hour, bactofugation must contain the enzyme mixed solution, add ethanol to 70% precipitation zymoprotein, collect albumen, freeze-drying is enzyme.
B. handle three kinds of isoflavone glycosides or mix glucoside with embodiment 1, embodiment 2 methods, change into glucoside unit more than 80%.
Above-mentioned enzyme separates purifying soybean isoflavones zymoprotein through DEAE-Cellulose ion exchange resin column method with BioRed protein Preparation chromatographic instrument (document 3), and with SDS electrophoresis method (document 4) determining molecular weight, the molecular weight of enzyme is 5.3 ten thousand.
Embodiment 4:
A. candiyeast is containing 10% product enzyme induction thing---and the wheat bran substratum (dry 1000 grams) of defatted soyflour is gone up inoculation, be divided in the eggplant bottle 30 ℃ of solid culture 3～6 days, collect culture, the physiological saline that in the substratum dry, adds 6000 milliliters, soaked one hour, centrifuging and taking supernatant (containing the enzyme mixed solution), the ammonium sulfate that adds saturation ratio 60～75 ‰, make the zymoprotein precipitation, collecting precipitation, with 800 milliliters pH5, the dissolving of 0.02M sodium-acetate buffer, ammonium sulfate is removed in dialysis, centrifugal slagging-off promptly gets the enzyme liquid about 1000 milliliters.
B. above-mentioned enzyme liquid is handled the result of soybean isoflavones, purification and measurement of enzymatic reaction products by the method for embodiment 1: soybean isoflavones changes into glucoside unit, and the molecular weight of enzyme is 50,000.
Embodiment 5:
A. cultivate on defatted soyflour and each wheat bran substratum of 10% of rice skin with Agaricus bitorqui bacterium (Agaricus bitorguis), under 25 ℃ of conditions of temperature, cultivated 6-8 days, press the method for embodiment 4 and extract enzyme liquid.
The method of b. pressing embodiment 2,3 is handled soybean isoflavones, is separated and purify and measurement of enzymatic reaction products, and 50% soybean isoflavones changes into glucoside unit.
Document described in the embodiment is:
1.Fengxie?Jin?et?al.：J.Int.Syst.Bact，1988，38，279-281。
2. Cui Hongbin edits, the exploitation of soybean biological active substance and application, China Light Industry Press, 2001, p39-40.
3.Hongshan Yu, et al (the red sudden strain of a muscle of fish etc.): Chem.Pharm.Bull., 50 (2), 175-178.
4. open Long Xiang etc.: distillation experimental technique and technology, Higher Education Publishing House, 1997, p100-111.
Claims (4)
1. an enzymatic hydrolysis isoflavone glycoside glycosyl prepares the method for its glucoside unit, it is characterized in that comprising the steps:
A. be the enzymatic production inductor of high-temperature aerobic bacterium, black-koji mould, candiyeast, Agaricus bitorqui bacterium with the soybean, obtain containing the enzyme mixed solution behind liquid state fermentation or the solid state fermentation, in containing the enzyme mixed solution, add ammonium sulfate or extraction using alcohol enzyme;
B. with the enzyme, isoflavone glycoside and the acetic acid that are extracted or phosphoric acid hybrid reaction 1-40 hour, reaction conditions was temperature 4-75 ℃, pH value 3-9;
C. extract soybean isoflavone aglycones.
2. enzymatic hydrolysis isoflavone glycoside glycosyl according to claim 1 prepares the method for its glucoside unit, it is characterized in that: the isoflavone glycoside concentration of described b step is the 0.1-10% of total reactant volume.
3. enzymatic hydrolysis isoflavone glycoside glycosyl according to claim 1 prepares the method for its glucoside unit, it is characterized in that: described isoflavone glycoside is the mixture of Daidzin, genistin and soya bean glucoside monomer or Daidzin, genistin and soya bean glucoside.
4. enzymatic hydrolysis isoflavone glycoside glycosyl according to claim 1 prepares the method for its glucoside unit, it is characterized in that: described c step is for extracting or use n-butanol extraction with ethanol sedimentation.
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| CN 03133637 CN1261586C (en) | 2003-08-01 | 2003-08-01 | Method for preparing aglycon of soybean isoflavone glycoside base by enzymatic method hydrolyzing soybean |
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| CN1261586C true CN1261586C (en) | 2006-06-28 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100351388C (en) * | 2004-08-13 | 2007-11-28 | 黑龙江双河松嫩大豆生物工程有限责任公司 | Method for producing soybean isoflavones aglycone using glucosidase and membrane technology |
| CN100454003C (en) * | 2005-05-26 | 2009-01-21 | 中国科学院成都生物研究所 | Method for deciding hydrolyzation or not or hydrolyzation degree of soybean isoflavone glucoside |
| CN101200744B (en) * | 2007-12-07 | 2011-12-21 | 南京师范大学 | High-effective clean method for preparing soybean isoflavone aglycone |
| CN101348811B (en) * | 2008-09-04 | 2011-05-11 | 大连工业大学 | Method for preparing soybean isoflavone glycoside from soybean isoflavones aglycone |
| 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 |
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Assignee: SHANDONG LONCT ENZYMES Co.,Ltd. Assignor: Jin Fengxie Contract record no.: 2011370000227 Denomination of invention: Method for preparing aglycon of soybean isoflavone glycoside base by enzymatic method hydrolyzing soybean Granted publication date: 20060628 License type: Exclusive License Open date: 20040324 Record date: 20110530 |
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Effective date of registration: 20180517 Address after: No. 29, Ji Xiang street, Sujiatun District, Shenyang, Liaoning Patentee after: SHENYANG TIANLEBAO CHEMICAL PRODUCTS CO.,LTD. Address before: 116034 College of biology and food engineering, Dalian Light Industry Institute, No. 1 Light Industrial Park, Ganjingzi District, Dalian, Liaoning Patentee before: Jin Fengxie |
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