CN101532039A - Magnetic nano immobilized enzyme catalysis production method of soya isoflavone - Google Patents
Magnetic nano immobilized enzyme catalysis production method of soya isoflavone Download PDFInfo
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Abstract
The invention relates to a magnetic nano immobilized enzyme catalysis production method of soya isoflavone, comprising the following steps: (1) concentrating glycoside type soya isoflavone raw material using supercritical fluid extraction; (2) extracting soya isoflavone glycoside hydrolase; (3) preparing the magnetic nano immobilized enzyme; (4) catalyzing the glycoside type soya isoflavone raw material using magnetic nano immobilized enzyme; (5) extracting soya isoflavone aglycone. The preparation method has features of simple and easily-operable supercritical extraction technology, innocuity and no-residue of carbon dioxide in operation process, high glycoside extraction ratio; simple preparation method of magnetic nano immobilized enzyme, simple recovery and repeat use of the enzyme using the external magnetic field; no destruction of the soya isoflavone aglycone and biological activity, high content of the soya isoflavone aglycone. The sum of the soya isoflavone aglycone concentration is 82.7% in the soya isoflavone aglycone product and the content of genistein is 90.3%.
Description
Technical field
The present invention relates to the method that a kind of soybean isoflavone glucoside that utilizes the enrichment of magnetic nano immobilized enzyme biocatalysis supercritical fluid extraction is converted into isoflavone genin.
Background technology
Soybean isoflavones is a class secondary metabolite that forms in the soybeans they grow process, totally 12 kinds, by Sophoricol, Daidezin, 3 kinds of isoflavone genins of glycitein, see Fig. 1, and they and glucose, acetyl glucosamine, malonyl-glucose forms with 9 kinds of glucosides type isoflavones (soybean isoflavone glucoside) that β-glycosidic link is connected to form, and sees Fig. 2.Studies have shown that isoflavone genin is the activity form of soybean isoflavones performance pharmacological function, they are active aglycons, and are the strongest with the Sophoricol activity.But active aglycon content is extremely low, only accounts for the 2-3% of soybean isoflavones total amount.Soybean isoflavones mainly is to exist with the glucosides form, and these glucosides are absorbed hardly, and they only are converted into isoflavone genin and just can be absorbed to enter in the body and play a role.Although soybean isoflavones is shown great attention to the effect of HUMAN HEALTH, but because active aglycon proportion in soybean isoflavones is few, the separation and purification difficulty is big, productive rate is low, the cost height, so current soybean isoflavones processing is still to extract the crude extract of producing 40-80% content from de-fatted soybean dregs and is used as foodstuff additive, level of processing is elementary, added value of product is not high, because mainly be the glucosides form, often not obvious because of crowd's individual difference health-care effect, bioavailability is low, has seriously limited the Application and Development of soybean isoflavones in fields such as medicine and makeup.
Unexpectedly carry out the research that the iso-flavone glucoside that content in the soybean is many is converted into active aglycon both at home and abroad mutually.At present, the soybean transformation iso-flavone glucoside is that the method for active aglycon mainly contains two kinds of acid-hydrolysis method and enzyme hydrolysis methods.Acid-hydrolysis method requires the temperature height, hydrolysis reaction lacks specificity, except that the hydrolysis glycosidic link, also can destroy other group on the isoflavones, cause the isoflavone structural instability to cause active the disappearance, and the acid hydrolysis meeting produces environment pollutes, though so acid-hydrolysis method research is many, effect is unsatisfactory.Enzyme hydrolysis method is that the β-glycosidic link with beta-glucoside enzyme hydrolysis of soybean isoflavone glucosides generates active aglycon, and hydrolysis reaction has β-glycosidic link specificity, mild condition, efficient height, the active aglycon Stability Analysis of Structures of isoflavones.Enzyme hydrolysis method research at present also rests on the research resolvase hydrolysis iso-flavone glucoside stage, and enzyme stability is poor, can not reclaim repeateds use, residual enzyme increases the product separating difficulty, enzyme itself costliness in addition, and the production cost height does not still have practical value.Therefore, the key issue of current urgent need solution restriction soybean isoflavones level of processing and Application Areas is how efficient, stable, the active aglycon of low cost production isoflavones.
Immobilized enzyme will provide new technology for the active aglycon of suitability for industrialized production soybean isoflavones.Immobilized enzyme is meant and is fixed on the enzyme that is fit on the carrier and can carries out catalyzed reaction continuously in certain spatial dimension.Immobilized enzyme has not only kept the catalysis characteristics of enzyme, and has overcome the deficiency of resolvase, has the stability that increases enzyme, enzyme can be repeatedly or continuously use and enzyme and reaction product be easy to isolating advantage, thereby efficient height, cost are low.Since Japan's sixties in 20th century continuous production of immobilized aminoacylase industrialization first L-amino acid, enzyme immobilization technology is that the industrial applications of enzyme has been opened up wide development space.2002 " American Chemical Society's proceedings " report, the researchist of U.S.'s Pacific Northwest National Laboratory successfully utilizes nano material with enzyme immobilization, and can keep its activity, stability simultaneously, this becomes the application of nano material zymetology and has opened possibility, has also brought technological breakthrough for enzyme immobilization.So far, magnetic nano immobilized enzyme is that relevant patent and report are not seen in application aspect the isoflavone genin as yet in catalysis soybean isoflavone glycoside feedstock conversion.
Summary of the invention
At the blank in the above research, the invention provides that a kind of technology is simple, easy to operate, environmental protection, can provide high purity
The magnetic nano immobilized enzyme catalysis of isoflavone genin is produced the method for isoflavone genin.
The present invention is to realize by such technical scheme for reaching above purpose:
The method that magnetic nano immobilized enzyme catalysis is produced isoflavone genin comprises following operation steps:
(1), the supercritical fluid extraction enrichment of soybean isoflavone glycoside raw material:
Take fully wetting soybean isoflavone glycoside raw material of entrainment agent, supercritical co (CO under the condition of entrainment agent, extracting pressure 20-40Mpa, extraction temperature 30-50 ℃
2) behind the fluid extraction 2-4h, from separating still, emit extract; Remove the entrainment agent in the extract, obtain the soybean isoflavone glycoside raw material;
(2), the extraction of soybean isoflavone glucoside hydrolase:
By wheat bran 2.0g/L, ammonium sulfate ((NH
4)
2SO
4) 1g/L, potassium primary phosphate (KH
2PO
4) 2g/L, sal epsom (MgSO
47H
2O) 0.2g/L, ferrous sulfate (FeSO
47H
2O) inoculated aspergillus niger on the substratum of 0.01g/L composition, inoculum size 15%, liquid amount 70mL/100mL triangular flask; Shake bottle rotating speed a 200 ± 5r/min, temperature is 30 ± 0.5 ℃ of condition bottom fermentations 3 days; With fermented liquid in 10000r/min, centrifugal 10min under 4 ℃ of conditions, getting supernatant liquor is the beta-glucosidase crude enzyme liquid; Ammonium sulfate precipitation beta-glucosidase crude enzyme liquid with 30%～80% saturation ratio; The acetate buffer that throw out is dissolved in 0.1mol/L, pH5.0 is in 4 ℃ of centrifugal desalinations down of temperature, concentrated; Ion exchange resin DEAE-52 resin isolation post on the concentrated solution, phosphoric acid buffer wash-out resin isolation post with 0.2mol/L, pH6.8, collect activity of beta-glucosidase and partly obtain collecting liquid A, collect liquid A through molecular weight cut-off after to be that 500-1 000 daltonian ultrafiltration membrane system is centrifugal concentrate, carry out dextrane gel (Sephadex G-100) column chromatography, collect activity of beta-glucosidase and partly obtain collecting liquid B; Collecting liquid B is that 500-1 000 daltonian ultrafiltration membrane system is centrifugal concentrated through molecular weight cut-off, obtains the beta-glucoside enzyme solution; Regulate the beta-glucoside enzyme solution with the acetate buffer of 0.1mol/L, pH5.0, the soybean isoflavone glucoside hydrolase solution that obtains enzyme activity and be 1500-2000U/mL is aspergillus niger beta-glucoside enzyme solution;
(3) preparation of magnetic nano immobilized enzyme:
Get in the pH4.0-6.0 acetate buffer solution that the 0.5-1.0g magnetic nano-particle is added to 4.5ml, adding volume fraction then is 2% (volume fraction, v/v) glutaraldehyde solution 0.5ml, medium and low frequency ultra-sonic dispersion; Ratio according to the 0.5-10mL/0.5-1.0g magnetic nano-particle adds aspergillus niger beta-glucoside enzyme solution again, place in the constant temperature water bath oscillator, immobilization 2-4h under temperature 20-30 ℃, rotating speed 100-150r/min condition utilizes permanent magnet to collect the immobilized enzyme particle; The immobilized enzyme particle cleans with the pH4.0-6.0 acetate buffer solution, does not have albumen until effluent liquid and detects, and obtains magnetic nano immobilized enzyme;
(4), the magnetic nano immobilized enzyme catalysis of soybean isoflavone glycoside raw material transforms:
The soybean isoflavone glycoside raw material is mixed with the solution of 0.5-1.0mg/mL with the pH4.5 acetate buffer solution, and regulates the pH to 4.9-5.1 of described solution, obtain soybean isoflavone glucoside solution with the hydrochloric acid (HCl) of 1.0mol//L; In the ratio adding pH5.0 acetate buffer solution of magnetic nano immobilized enzyme with gained in the above-mentioned steps (3), fully stir and make its suspension evenly, formation magnetic nano immobilized enzyme suspension according to 0.8-1.2g/0.8-1.2mL; Add magnetic nano immobilized enzyme suspension and form mixed solution in described soybean isoflavone glucoside solution, the magnetic nano immobilized enzyme suspension vol of adding is 0.5～1 times of described soybean isoflavone glucoside liquor capacity; Described mixed solution at temperature 20-50 ℃ of conditioned response 20min-60min, is obtained the conversion fluid of high isoflavone genin content;
(5), the extraction of isoflavone genin:
Centrifugal or externally-applied magnetic field separates the magnetic nano immobilized enzyme in the conversion fluid of high isoflavone genin content, obtains isoflavone genin solution; The isoflavone genin solution decompression concentrates and obtains high-purity soybean isoflavone aglycon finished product.
Described soybean isoflavone glycoside raw material is defatted soyflour or soybean germ powder or defatted soybean meal.
The used entrainment agent of the supercritical fluid extraction process of described step (1) is mixed solvent or the ethanol and the water mixed solvent of methyl alcohol or ethanol or ethyl acetate or methyl alcohol and water, and add-on is for dropping into the 100-300% of material quantity.
Described magnetic nano-particle is aluminium nitride or Z 250 or titanium oxide, its median size 15-100nm.
The centrifugal condition of described step (5) is 5000-10000r/min, 2-8 ℃ of centrifugal 15-20min.
The isolating concrete operation method of described externally-applied magnetic field is, the permanent magnet bar put into the conversion fluid that above-mentioned steps (4) obtains stirred 1-2 minute, and magnetic nano immobilized enzyme is attracted on the permanent magnet bar.
The extraction of described soybean isoflavone glucoside hydrolase:
Get the garbanzo of germination, the long 1-1.5cm of bud, add liquid nitrogen and grind to form the homogenate shape, and at 0.1Mol/L acetic acid homogeneous 24h in the liquid (pH5.0, down with), centrifugal 10min under 4 ℃ of temperature, 12000r/min condition gets supernatant liquor; Slowly add 30%-80% saturation ratio ammonium sulfate ((NH to supernatant liquor
4)
2SO
4) carry out classification; Get precipitation and be dissolved in the 0.1Mol/L acetate buffer solution, 4 ℃ of refrigerator dialysis promptly got crude enzyme liquid in 18-24 hour; In 4 ℃ of conditions of temperature, the centrifugal desalination of crude enzyme liquid, concentrated; Ion exchange resin on the concentrated solution (DEAE-52) resin isolation post, phosphoric acid buffer wash-out resin isolation post with 0.2mol/L, pH6.8, collect activity of beta-glucosidase and partly obtain collecting liquid C, collect liquid C through molecular weight cut-off after to be that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrate, carry out Sephadex G-100 column chromatography, collect activity of beta-glucosidase and partly obtain collecting liquid D; Collecting liquid D is that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrated through molecular weight cut-off, and concentrated solution is garbanzo beta-glucoside enzyme solution.
The present invention combines supercritical liquid extraction technique and enzyme immobilization technology, at first extract enrichment soybean isoflavone glycoside raw material with supercritical liquid extraction technique, with magnetic nano-particle enzyme being carried out immobilization and obtain magnetic nano immobilized enzyme, is isoflavone genin with magnetic nano immobilized enzyme catalysis soybean isoflavone glycoside feedstock conversion again.
The isoflavone genin total content is higher in the isoflavone genin product that employing the inventive method is prepared from, and wherein Sophoricol content has comparative advantage.Concrete method and data are as follows: accurately prepare Sophoricol, Daidezin, glycitein concentration respectively for the isoflavone genin product solution of present method of the standard specimen solution of 0.1mg/mL and 0.2mg/mL preparation with chromatographically pure methyl alcohol, carry out HPLC.
The HPLC analysis condition: chromatographic column is a U.S. Merck company
STARC18 post (4.6mmi.d. * 250mm, 5 μ m), moving phase are methanol-water-acetate (volume ratio, 10: 10: 1) solution, 25 ℃ of column temperatures, and sampling volume 20 μ L, flow velocity 1.0mL/min detects wavelength 260nm, and sensitivity is 3.0AUFS,
Calculation formula is:
Record that isoflavone genin concentration sum is 82.7% in the isoflavone genin product that the inventive method produces, Sophoricol content is 90.3%.
Use preparation method of the present invention, supercritical extraction process is simple to operation, nontoxic, the noresidue of operating process carbonic acid gas, glucosides percentage extraction height; The magnetic nano immobilized enzyme preparation method is simple, and is easily separated behind the externally-applied magnetic field; The product isoflavone genin structure and the physiologically active of preparation are not damaged the content height.
Description of drawings
Fig. 1 is the molecular structure of isoflavone genin.
Fig. 2 is the molecular structure of soybean isoflavone glucoside.
Embodiment
Below by embodiment the present invention is further described.
Embodiment 1:
The method that magnetic nano immobilized enzyme transforms soybean isoflavone glycoside raw material production isoflavone genin comprises following operation steps:
Step 1: the supercritical fluid extraction enrichment of soybean isoflavone glycoside raw material
Extracting degreasing dregs of beans 100g, put into the extraction kettle of supercritical fluid extraction equipment after fully wetting with 95% ethanol entrainment agent 100mL, at extracting pressure 30MPa, 50 ℃ of extraction temperature, carbon dioxide flow 6L/h, the quiet extraction 120min of elder generation under the 95% ethanol entrainment agent 200mL condition, the moving extraction in back 60min.Emit extract from separating still, reduction vaporization separates the ethanol entrainment agent with extract, obtains entrainment agent and extraction liquid, and the entrainment agent of gained is reusable, and extraction liquid for the glucosides type is main soybean isoflavones raw material, is seen Fig. 2 after lyophilize.
Step 2: the producing of soybean isoflavone glucoside hydrolase
(Aspergillus niger) is seeded in by wheat bran 2.0g/L with the fungi aspergillus niger, ammonium sulfate ((NH
4)
2SO
4) 1g/L, potassium primary phosphate (KH
2PO
4) 2g/L, sal epsom (MgSO
47H
2O) 0.2g/L, ferrous sulfate (FeSO
47H
2O) on the substratum that 0.01g/L forms, inoculum size 15%, liquid amount 70mL/100mL triangular flask, fermentation condition for shake bottle rotating speed a 200 ± 5r/min, temperature is 30 ± 0.5 ℃, fermented 3 days.With fermented liquid in rotating speed 10 000r/min, centrifugal 10min under 4 ℃ of conditions of temperature, getting supernatant liquor is the beta-glucosidase crude enzyme liquid.Ammonium sulfate precipitation beta-glucosidase crude enzyme liquid with 30%～80% saturation ratio; Throw out is dissolved in acetate buffer (0.1mol/L, pH5.0; Down together) back is used
The type ultra-fine filter is in 4 ℃ of centrifugal desalinations down of temperature, concentrated; Ion exchange resin on the concentrated solution (DEAE-52) resin isolation post, phosphoric acid buffer wash-out resin isolation post with 0.2mol/L, pH6.8, collect activity of beta-glucosidase and partly obtain collecting liquid A, collect liquid A through molecular weight cut-off after to be that 500-1 000 daltonian ultrafiltration membrane system is centrifugal concentrate, carry out the SephadexG-100 column chromatography, collect activity of beta-glucosidase and partly obtain collecting liquid B; Collecting liquid B is that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrated through molecular weight cut-off, obtains the beta-glucoside enzyme solution.Regulating the beta-glucoside enzyme solution with the acetate buffer of 0.1mol/L, pH5.0, to obtain enzyme activity be that the soybean isoflavone glucoside hydrolase solution of 1500-2000U/mL is aspergillus niger beta-glucoside enzyme solution.
Aspergillus niger beta-glucosidase liquid state fermentation condition and enzyme activity determination are seen document (Luo Jianping, Wang Guijuan, Pan Lihua. fermentation of Aspergillus niger wheat bran is produced the process optimization and the dynamics research of beta-glucosidase. Transactions of the Chinese Society of Agricultural Engineering, 2007,23 (12): 252-257).
Step 3: the preparation of magnetic nano immobilized enzyme
Getting 1.0g aluminium nitride magnetic nano-particle is added in the 4.5ml acetate buffer solution (pH6.0), the aluminum nitride particle median size is 15-100nm, adding volume fraction (v/v) is 2% glutaraldehyde solution 0.5ml (making the final concentration of glutaraldehyde in the preparation system of magnetic nano immobilized enzyme is 0.1-0.2%), ultrasonic dispersing 3-5 minute, add 50mg/mL aspergillus niger beta-glucoside enzyme solution 5mL, 20 ℃ of temperature, immobilized 3.5h under pH5.0 and the rotating speed 100r/min condition utilizes permanent magnet to collect the immobilized enzyme particle.The immobilized enzyme particle does not have albumen with the cleaning of pH4.0-6.0 acetate buffer solution until effluent liquid and detects, and obtains magnetic nano immobilized enzyme.
The preparation method of magnetic nano immobilized enzyme see document (Pan Lihua, Luo Jianping, Wang Guijuan, as if Xu Xueling is cloud tints. the character of magnetic nanoparticle aluminum nitride particle immobilization beta-glucosidase. catalysis journal, 2008,29 (10): 1021-1026).
Step 4: the magnetic nano immobilized enzyme catalysis of soybean isoflavone glycoside raw material transforms
The soybean isoflavone glycoside raw material that above-mentioned steps 1 is obtained is mixed with the solution of 0.5mg/mL with the pH4.5 acetate buffer solution, and regulates the pH to 4.9-5.1 of described solution, formation soybean isoflavone glucoside solution ie in solution D with the hydrochloric acid of 1.0mol//L; The magnetic nano immobilized enzyme 5.0g that gets gained in the above-mentioned steps 3 adds in the pH5.0 acetate buffer solution (ie in solution E), fully stirs it is suspended evenly, forms magnetic nano immobilized enzyme suspension ie in solution F; Get solution D 5mL, add described solution F2.5mL and solution E 2.5mL, form reaction mixture; Described reaction mixture is reacted 30min under 45 ℃ of conditions of temperature, obtain the conversion fluid of high isoflavone genin content.
Step 5: the extraction of isoflavone genin
The permanent magnet bar is put into the conversion fluid that above-mentioned steps 4 obtains to be stirred 1-2 minute, magnetic nano immobilized enzyme is attracted on the permanent magnet bar, take out the permanent magnet bar and obtain isoflavone genin solution and the magnetic nano immobilized enzyme that is adsorbed on the permanent magnet bar, magnetic nano immobilized enzyme can be reused.Gained isoflavone genin solution obtains high-purity soybean isoflavone aglycon finished product 1.5mg with the centrifugal desalination of reverse osmosis membrane system, concentrated back concentrating under reduced pressure.
Get the raw material that soybean germ powder is a supercritical fluid extraction enrichment soybean isoflavone glycoside, adopt ferroferric oxide magnetic nano-particles, other are with embodiment 1.
Promptly obtain high-purity soybean isoflavone aglycon finished product 1.1mg.
Embodiment 3:
The extracting degreasing soyflour is the raw material of supercritical fluid extraction enrichment soybean isoflavone glycoside, adopts the titanium oxide magnetic nano-particle, and other are with embodiment 1.
Promptly obtain high-purity soybean isoflavone aglycon finished product 0.9mg.
Embodiment 4:
The method that a kind of magnetic nano immobilized enzyme transforms soybean isoflavone glycoside raw material production isoflavone genin comprises following operation steps:
Producing of step 2, soybean isoflavone glucoside hydrolase
Get complete garbanzo seed in the culture dish that is lined with four layers of gauze (diameter 12cm), be placed in 25 ℃ of culturing room and sprout.Every day early, evening is with the tap water flushing and keep certain humidity.After treating that bud grows to 1～1.5cm, use the tap water rinsing, add liquid nitrogen and grind to form the homogenate shape, and at 0.1M acetic acid homogeneous 24h in the liquid (pH5.0, down with), in 4 ℃, the centrifugal 10min of 12000r/min gets supernatant liquor behind the homogeneous; Slowly add 30%～80% saturation ratio (NH to supernatant liquor
4)
2SO
4Carry out classification; Get precipitation and be dissolved in the 0.1M acetate buffer solution, 4 ℃ of refrigerator dialysis promptly got crude enzyme liquid in 18-24 hour.Crude enzyme liquid is used
The type ultra-fine filter is in 4 ℃ of centrifugal desalinations down, concentrated; Ion exchange resin on the concentrated solution (DEAE-52) resin isolation post, phosphoric acid buffer wash-out resin isolation post with 0.2mol/L, pH6.8, collect activity of beta-glucosidase and partly obtain collecting liquid C, collect liquid C through molecular weight cut-off after to be that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrate, carry out Sephadex G-100 column chromatography, collect activity of beta-glucosidase and partly obtain collecting liquid D; Collecting liquid D molecular weight cut-off is that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrated, and concentrated solution is the beta-glucoside enzyme solution.Regulate the beta-glucoside enzyme solution and obtain the soybean isoflavone glucoside hydrolase solution that enzyme activity is 1500-2000U/mL.
Garbanzo beta-glucoside enzyme extraction see document (Pan Lihua, Luo Jianping. separate raw materials screening, purifying and the property research of high activity soy bean isoflavone glycosidase. Food science, 2009,30 (1): 164-168).
The preparation of step 3, magnetic nano immobilized enzyme
Get in the pH5.0 acetate buffer solution that 0.5 magnetic nano-particle is added to 4.5ml, adding volume fraction (v/v) then is 2% glutaraldehyde solution 0.5ml, medium and low frequency ultra-sonic dispersion 3-5 minute; Ratio according to the 0.5-10mL/0.5-1.0g magnetic nano-particle adds garbanzo beta-glucoside enzyme solution 5mL again, place in the constant temperature water bath oscillator, immobilization 2-4h under temperature 20-30 ℃, rotating speed 100-150r/min condition utilizes permanent magnet to collect the immobilized enzyme particle; The immobilized enzyme particle cleans with the pH4.0-6.0 acetate buffer solution, does not have albumen until effluent liquid and detects, and obtains magnetic nano immobilized enzyme;
Other step is with embodiment 1.
Promptly obtain high-purity soybean isoflavone aglycon finished product 1.4mg.
Embodiment 5:
Step 4: the magnetic nano immobilized enzyme catalysis of soybean isoflavone glycoside raw material transforms
The soybean isoflavone glycoside raw material that above-mentioned steps 1 is obtained is mixed with the solution of 0.5mg/mL with the pH4.5 acetate buffer solution, and regulates the pH to 4.9-5.1 of described solution, formation soybean isoflavone glucoside solution ie in solution D with the hydrochloric acid of 1.0mol//L; The magnetic nano immobilized enzyme 5.0g that gets gained in the above-mentioned steps 3 adds in the pH5.0 acetate buffer solution, fully stirs it is suspended evenly, forms magnetic nano immobilized enzyme suspension ie in solution F; Get solution D 5mL, add described solution F5mL, form reaction mixture; Described reaction mixture is reacted 30min under 45 ℃ of conditions of temperature, obtain the conversion fluid of high isoflavone genin content.
Step 5: the extraction of isoflavone genin:
Under rotating speed 8000r/min, 4 ℃ of centrifugal 15min conditions of temperature, separate the magnetic nano immobilized enzyme in the conversion fluid of high isoflavone genin content, obtain isoflavone genin solution and magnetic nano immobilized enzyme; The centrifugal desalination of isoflavone genin solution reverse osmosis membrane system, concentrated back concentrating under reduced pressure obtain high-purity soybean isoflavone aglycon finished product (Fig. 1 product), and magnetic nano immobilized enzyme can be reused.
Other step is with embodiment 1.
Promptly obtain high-purity soybean isoflavone aglycon finished product 1.5mg.
Claims (7)
1, magnetic nano immobilized enzyme catalysis is produced the method for isoflavone genin, it is characterized in that comprising following operation steps:
(1), the supercritical fluid extraction enrichment of soybean isoflavone glycoside raw material:
Take fully wetting soybean isoflavone glycoside raw material of entrainment agent, behind supercritical carbon dioxide extraction 2-4h under the condition of entrainment agent, extracting pressure 20-40Mpa, extraction temperature 30-50 ℃, from separating still, emit extract; Remove the entrainment agent in the extract, obtain the soybean isoflavone glycoside raw material;
(2), the extraction of soybean isoflavone glucoside hydrolase:
By wheat bran 2.0g/L, ammonium sulfate 1g/L, potassium primary phosphate 2g/L, sal epsom 0.2g/L, inoculated aspergillus niger on the substratum that ferrous sulfate 0.01g/L forms, inoculum size 15%, liquid amount 70mL/100mL triangular flask; Shake bottle rotating speed a 200 ± 5r/min, temperature is 30 ± 0.5 ℃ of condition bottom fermentations 3 days; With fermented liquid in 10000r/min, centrifugal 10min under 4 ℃ of conditions, getting supernatant liquor is the beta-glucosidase crude enzyme liquid; Ammonium sulfate precipitation beta-glucosidase crude enzyme liquid with 30%～80% saturation ratio; The acetate buffer that throw out is dissolved in 0.1mol/L, pH5.0 is in 4 ℃ of centrifugal desalinations down of temperature, concentrated; Ion exchange resin DEAE-52 resin isolation post on the concentrated solution, phosphoric acid buffer wash-out resin isolation post with 0.2mol/L, pH6.8, collect activity of beta-glucosidase and partly obtain collecting liquid A, collect liquid A through molecular weight cut-off after to be that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrate, carry out the dextrane gel column chromatography, collect activity of beta-glucosidase and partly obtain collecting liquid B; Collecting liquid B is that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrated through molecular weight cut-off, obtains the beta-glucoside enzyme solution; Regulate the beta-glucoside enzyme solution with the acetate buffer of 0.1mol/L, pH5.0, the soybean isoflavone glucoside hydrolase solution that obtains enzyme activity and be 1500-2000U/mL is aspergillus niger beta-glucoside enzyme solution;
(3), the preparation of magnetic nano immobilized enzyme:
Get in the pH4.0-6.0 acetate buffer solution that the 0.5-1.0g magnetic nano-particle is added to 4.5ml, adding volume fraction then is 2%v/v glutaraldehyde solution 0.5ml, the medium and low frequency ultra-sonic dispersion; Ratio according to the 0.5-10mL/0.5-1.0g magnetic nano-particle adds aspergillus niger beta-glucoside enzyme solution again, place in the constant temperature water bath oscillator, immobilization 2-4h under temperature 20-30 ℃, rotating speed 100-150r/min condition utilizes permanent magnet to collect the immobilized enzyme particle; The immobilized enzyme particle cleans with the pH4.0-6.0 acetate buffer solution, does not have albumen until effluent liquid and detects, and obtains magnetic nano immobilized enzyme;
(4), the magnetic nano immobilized enzyme catalysis of soybean isoflavone glycoside raw material transforms:
The soybean isoflavone glycoside raw material is mixed with the solution of 0.5-1.0mg/mL with the pH4.5 acetate buffer solution, and regulates the pH to 4.9-5.1 of described solution, obtain soybean isoflavone glucoside solution with the hydrochloric acid of 1.0mol//L; With above-mentioned steps 3) in the magnetic nano immobilized enzyme of gained add in the pH5.0 acetate buffer solution according to the ratio of 0.8-1.2g/0.8-1.2mL, fully stir it suspended evenly, form magnetic nano immobilized enzyme suspension; Add magnetic nano immobilized enzyme suspension and form mixed solution in described soybean isoflavone glucoside solution, the magnetic nano immobilized enzyme suspension vol of adding is 0.5～1 times of described soybean isoflavone glucoside liquor capacity; Described mixed solution at temperature 20-50 ℃ of conditioned response 20min-60min, is obtained the conversion fluid of high isoflavone genin content;
(5), the extraction of isoflavone genin:
Centrifugal or externally-applied magnetic field separates the magnetic nano immobilized enzyme in the conversion fluid of high isoflavone genin content, obtains isoflavone genin solution; The isoflavone genin solution decompression concentrates and obtains high-purity soybean isoflavone aglycon finished product.
2, magnetic nano immobilized enzyme catalysis according to claim 1 is produced the method for isoflavone genin, and it is characterized in that: described soybean isoflavone glycoside raw material is defatted soyflour or soybean germ powder or defatted soybean meal.
3, magnetic nano immobilized enzyme catalysis according to claim 1 is produced the method for isoflavone genin, it is characterized in that: the used entrainment agent of the supercritical fluid extraction process of described step (1) is mixed solvent or the ethanol and the water mixed solvent of methyl alcohol or ethanol or ethyl acetate or methyl alcohol and water, and add-on is for dropping into the 100-300% of material quantity.
4, magnetic nano immobilized enzyme catalysis according to claim 1 is produced the method for isoflavone genin, and it is characterized in that: described magnetic nano-particle is aluminium nitride or Z 250 or titanium oxide, its median size 15-100nm.
5, magnetic nano immobilized enzyme catalysis according to claim 1 is produced the method for isoflavone genin, and it is characterized in that: the centrifugal condition of described step (5) is 5000-10000r/min, 2-8 ℃ of centrifugal 15-20min.
6, magnetic nano immobilized enzyme catalysis according to claim 1 is produced the method for isoflavone genin, it is characterized in that: the isolating concrete operation method of described externally-applied magnetic field is, the permanent magnet bar is put into the conversion fluid that above-mentioned steps (4) obtains stirred 1-2 minute, magnetic nano immobilized enzyme is attracted on the permanent magnet bar.
7, magnetic nano immobilized enzyme catalysis according to claim 1 is produced the method for isoflavone genin, it is characterized in that: the extraction of described soybean isoflavone glucoside hydrolase:
Get the garbanzo of germination, the long 1-1.5cm of bud, add liquid nitrogen and grind to form the homogenate shape, and at 0.1Mol/L, pH5.0 acetic acid homogeneous 24h in the liquid, centrifugal 10min under 4 ℃ of temperature, 12000r/min condition gets supernatant liquor; Slowly add 30%-80% saturation ratio ammonium sulfate to supernatant liquor and carry out classification; Get precipitation and be dissolved in the 0.1Mol/L acetate buffer solution, 4 ℃ of refrigerator dialysis promptly got crude enzyme liquid in 18-24 hour; In 4 ℃ of conditions of temperature, the centrifugal desalination of crude enzyme liquid, concentrated; Ion exchange resin resin isolation post on the concentrated solution, phosphoric acid buffer wash-out resin isolation post with 0.2mol/L, pH6.8, collect activity of beta-glucosidase and partly obtain collecting liquid C, collect liquid C through molecular weight cut-off after to be that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrate, carry out Sephadex G-100 column chromatography, collect activity of beta-glucosidase and partly obtain collecting liquid D; Collecting liquid D is that the daltonian ultrafiltration membrane system of 500-1000 is centrifugal concentrated through molecular weight cut-off, and concentrated solution is garbanzo beta-glucoside enzyme solution.
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| CN102676494A (en) * | 2012-05-03 | 2012-09-19 | 天津大学 | Core-shell structure immobilized enzyme particle and preparation method thereof |
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| CN114214374A (en) * | 2021-12-24 | 2022-03-22 | 四川宇奥生物科技有限公司 | Method for preparing soybean isoflavone aglycone by fermentation method |
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| CN102676494B (en) * | 2012-05-03 | 2013-10-30 | 天津大学 | Core-shell structure immobilized enzyme particle and preparation method thereof |
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| CN109295131A (en) * | 2017-12-04 | 2019-02-01 | 合肥工业大学 | A kind of receptor positioning solid phase enzymolysis preparation of dendrobium nobile activated oligosaccharide |
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| CN114214374A (en) * | 2021-12-24 | 2022-03-22 | 四川宇奥生物科技有限公司 | Method for preparing soybean isoflavone aglycone by fermentation method |
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