Method for extracting isoflavone aglycone from soybean germs

Abstract

The invention discloses a method for extracting isoflavone aglycone from soybean germs.The method comprises the following steps that S1, soybean germ powder obtained after superfine grinding is used as a raw material, the soybean germ powder and a weak acid buffer solution are mixed to be uniform, and a mixed solution with the soybean germ powder mass concentration being 5-15% is prepared; s2, performing ultrasonic and / or microwave-assisted solvent extraction operation on the mixed solution obtained in the step S1 to obtain an isoflavone-rich extracting solution; and S3, carrying out an enzymolysis reaction on the isoflavone-rich extracting solution obtained in the step S2 in a multi-stage biological enzyme reactor to obtain a high-purity isoflavone aglycone product. According to the extraction method of the isoflavone aglycone, the biological enzyme is high in enzyme activity and can be repeatedly used, and the obtained isoflavone aglycone is high in extraction rate and purity.

Description

Technical Field

[0001] The invention relates to the technical field of extracting food functional active substances, in particular to a method for extracting isoflavone aglycones from soybean germs. Background Art

[0002] Soy isoflavones are active ingredients in soybeans, which have similar effects to estrogen. They can play an antioxidant, anti-tumor, prevent cardiovascular and cerebrovascular diseases, and anti-aging role, and have great market value. There are 12 types of soy isoflavones discovered so far, which are divided into free aglycones and bound glycosides. Among them, free aglycones include daidzein, genistein and soybean yellow, accounting for 2-3% of the total; while bound glycosides mainly exist in the form of glucoside, acetylglucoside and malonyl glucoside, accounting for 97-98% of the total. The absorption rate and quantity of soy isoflavone aglycones by the human body are much greater than those of soy isoflavone glycosides.

[0003] There are two main ways to obtain free aglycones: one is direct extraction from soybeans, and the other is conversion from bound soybean isoflavone glycosides. In the existing technology, the extraction rate of soybean isoflavone aglycones is low with a single extraction method; the technologies related to conversion mainly include acid-base hydrolysis and enzyme hydrolysis, but there is still a problem of low extraction rate and conversion rate of soybean isoflavone aglycones, which cannot maximize its market value. Summary of the invention

[0004] In view of the above shortcomings of the prior art, the present invention provides a method for extracting isoflavone aglycones from soybean germ to solve the problems of the existing isoflavone aglycone preparation method, in which the biological enzyme used not only has low enzyme activity and is difficult to reuse, but also has low extraction rate and purity of the obtained isoflavone aglycones.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A method for extracting isoflavone aglycones from soybean germ, the method comprising the following steps:

[0007] S1: using ultrafinely crushed soybean germ powder as a raw material, mixing the soybean germ powder with a short-chain alcohol aqueous solution with a mass concentration of 80-90% to prepare a mixed solution with a mass concentration of 5-15% of soybean germ powder;

[0008] S2: subjecting the mixed solution obtained in step S1 to ultrasonic and / or microwave-assisted solvent extraction to obtain an isoflavone-rich extract;

[0009] S3: subjecting the isoflavone-rich extract obtained in step S2 to an enzymatic hydrolysis reaction in a multi-stage bio-enzyme reactor to obtain a high-purity isoflavone aglycone product.

[0010] The method for extracting isoflavone aglycones from soybean germ in the present application should be understood by those skilled in the art that the term "extraction" in the present application is not a physical extraction operation in a narrow sense, but is interpreted in a broad sense, that is, it includes the enzymatic hydrolysis reaction step of step S3 of the present application, so as to convert the bound soybean isoflavone glycoside components into isoflavone aglycone products as much as possible after enzymatic hydrolysis and transformation of the raw soybean germ powder, thereby improving its economic value.

[0011] Preferably, the short-chain alcohol is at least one of methanol, ethanol and propanol.

[0012] Preferably, the solvent used in the solvent extraction operation is a short-chain alcohol, the extraction temperature is 50-60° C., and the extraction time is 1-3 hours.

[0013] Preferably, the ultrasonic power of the ultrasonic-assisted solvent extraction operation is 100 to 500 W, and the working time is 30 to 180 min.

[0014] Preferably, the microwave power of the microwave-assisted solvent extraction operation is 100 to 1000 W, and the working time is 10 to 60 minutes.

[0015] Preferably, the multi-stage bio-enzyme reactor comprises a first-stage bio-enzyme reactor and a second-stage bio-enzyme reactor; wherein, after the isoflavone-rich extract is subjected to enzymatic hydrolysis in the first-stage bio-enzyme reactor, a first-stage filtrate and a first-stage concentrated solution are obtained; the first-stage concentrated solution is flushed out of the enzymatic hydrolysis in the first-stage bio-enzyme reactor by a backwash operation, and then fed into the inlet of the second-stage bio-enzyme reactor, and after enzymatic hydrolysis in the second-stage bio-enzyme reactor, a second-stage filtrate and a second-stage retentate are obtained; the first-stage filtrate and the second-stage filtrate are collected, vacuum-heated and dried, and then repeatedly rinsed with deionized water to obtain a high-purity isoflavone aglycone product.

[0016] Preferably, the bio-enzyme reactor is a reactor obtained by immobilizing β-glucosidase on the surface of a nanofiltration membrane.

[0017] Preferably, the preparation method of the bio-enzyme reactor is: evenly dispersing β-glucosidase in a weakly acidic buffer solution, adding sodium alginate and a small amount of silane coupling agent, stirring evenly, and preparing a bio-enzyme solution; then immersing the rolled nanofiltration membrane in the bio-enzyme solution, and after immersion for 1 to 5 hours, obtaining a nanofiltration membrane with bio-enzyme immobilized on the membrane surface; and obtaining a bio-enzyme reactor after winding and assembling with a central collecting tube and a porous support material.

[0018] Preferably, the weakly acidic buffer solution is a citric acid-sodium citrate buffer solution with a pH of 5.5 to 6.5.

[0019] Preferably, the mass ratio of the biological enzyme to the weakly acidic buffer solution is 1:15-20, and the immobilized amount of the biological enzyme on the biological enzyme reactor is 10-100 mg / g.

[0020] Preferably, the membrane material of the nanofiltration membrane is polyamide, and the molecular weight cutoff of the nanofiltration membrane is 300 to 500 Daltons.

[0021] Beneficial effects of the present invention:

[0022] The method for extracting isoflavone aglycones from soybean germs of the present invention comprises the following steps: firstly, an isoflavone-rich extract is obtained by ultrasonic and / or microwave-assisted solvent extraction, and then an enzymatic hydrolysis reaction is carried out in a multi-stage bio-enzyme reactor. The final extraction rate of the isoflavone aglycones obtained is greater than 92%, and the purity is greater than 97%. Moreover, the bio-enzyme used has high enzyme activity and can be reused. DETAILED DESCRIPTION

[0023] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0024] Example 1

[0025] The method for extracting isoflavone aglycones from soybean germ in this embodiment comprises the following steps:

[0026] S1: Using ultrafinely crushed soybean germ powder as a raw material, the soybean germ powder is evenly mixed with an ethanol aqueous solution with a mass concentration of 80% to prepare 500 mL of a mixed solution with a mass concentration of 5% soybean germ powder;

[0027] S2: subjecting the mixed solution obtained in step S1 to ultrasonic-assisted solvent extraction to obtain an isoflavone-rich extract; the solvent used in the solvent extraction operation is short-chain alcohol ethanol, the extraction temperature is 50° C., and the extraction time is 3 hours. The ultrasonic power of the ultrasonic-assisted solvent extraction operation is 200W, the operation is intermittent, and the working time is 60 minutes.

[0028] S3: subjecting the isoflavone-rich extract obtained in step S2 to an enzymatic hydrolysis reaction in a multi-stage bio-enzyme reactor to obtain a high-purity isoflavone aglycone product.

[0029] The multistage bio-enzyme reactor includes a first-stage bio-enzyme reactor and a second-stage bio-enzyme reactor; wherein, after the isoflavone-rich extract is hydrolyzed by the first-stage bio-enzyme reactor, a first-stage filtrate and a first-stage concentrated solution are obtained; the first-stage concentrated solution is then sent to the inlet of the second-stage bio-enzyme reactor, and after the enzymatic hydrolysis by the second-stage bio-enzyme reactor, a second-stage filtrate and a small amount of second-stage concentrated solution are obtained (the second-stage concentrated solution is circulated back to the inlet of the first-stage bio-enzyme reactor); the first-stage filtrate and the second-stage filtrate are collected, vacuum heated and dried, and then repeatedly rinsed with deionized water to obtain a high-purity isoflavone aglycone product. The present application utilizes a multistage bio-enzyme reactor, which is conducive to the thorough conversion of combined soybean isoflavone glycosides into isoflavone aglycone products. The feed flow rate of the first-stage bio-enzyme reactor is 60mL / min, and the operating pressure is 0.15MPa; the feed flow rate of the second-stage bio-enzyme reactor is 30mL / min, and the operating pressure is 0.25MPa.

[0030] The bio-enzyme reactor is a reactor obtained by immobilizing β-glucosidase on the surface of a nanofiltration membrane.

[0031] The preparation method of the bio-enzyme reactor is as follows: β-glucosidase is uniformly dispersed in a weakly acidic buffer solution, sodium alginate (sodium alginate accounts for 1.5% of the mass percentage of the bio-enzyme solution, the same below) and a small amount of silane coupling agent (the silane coupling agent is KH550, and the mass percentage of the silane coupling agent in the bio-enzyme solution is 0.1%, the same below), and after stirring evenly, a bio-enzyme solution with a mass percentage of 1.5% of β-glucosidase is prepared; then a rolled nanofiltration membrane is immersed in the bio-enzyme solution, and after immersion for 1 hour, a nanofiltration membrane with bio-enzyme immobilized on the membrane surface is obtained; and a bio-enzyme reactor is obtained after winding and assembling with a central collecting tube and a porous support material. The mass ratio of the bio-enzyme to the weakly acidic buffer solution is 1:20, and the immobilized amount of the bio-enzyme on the bio-enzyme reactor is 30 mg / g.

[0032] The weakly acidic buffer solution is a citric acid-sodium citrate buffer solution with a pH of 6.2. After the weakly acidic buffer solution prepared in the present application is treated with bioenzyme immobilization, interference from components such as phospholipids and proteins is avoided, the purity of soybean isoflavone aglycones is further improved, and the deterioration of soybean isoflavone aglycones is also avoided.

[0033] The membrane material of the nanofiltration membrane is polyamide. The molecular weight cutoff of the nanofiltration membrane is 300-500 Daltons. The isoflavone-rich extract of the present application flows under pressure outside the nanofiltration membrane, and the isoflavone aglycone products are collected and flow out through the central collection tube, while the soybean isoflavone glycosides that are not completely enzymatically hydrolyzed are retained outside the membrane.

[0034] In this embodiment, the nanofiltration membrane after immobilization of β-glucosidase with weak acidic buffer solution, sodium alginate and silane coupling agent is used as a reactor bioenzyme reactor for enzymatic hydrolysis reaction, and the bioenzyme used has high enzyme activity, and the final extraction rate of isoflavone aglycones obtained is 93.5% and the purity is 98.1%. The extraction rate of this application refers to the percentage of isoflavones in soybean germ raw materials that are finally converted into isoflavone aglycones.

[0035] With the same raw materials and operating conditions, the operation of this example was repeated 5 times, each time using the same bio-enzyme reactor without changing the bio-enzyme reactor, and the final extraction rate of isoflavone aglycones was measured to be 92.5% and the purity was 97.2%.

[0036] Example 2

[0037] The method for extracting isoflavone aglycones from soybean germ in this embodiment comprises the following steps:

[0038] S1: Using ultrafinely crushed soybean germ powder as a raw material, the soybean germ powder is evenly mixed with an ethanol aqueous solution with a mass concentration of 80% to prepare 500 mL of a mixed solution with a mass concentration of 10% soybean germ powder;

[0039] S2: subjecting the mixed solution obtained in step S1 to microwave-assisted solvent extraction to obtain an isoflavone-rich extract; the solvent used in the solvent extraction operation is short-chain alcohol ethanol, the extraction temperature is 60° C., and the extraction time is 2 hours. The microwave power of the microwave-assisted solvent extraction operation is 500 W, the operation is intermittent, and the working time is 60 minutes.

[0040] S3: subjecting the isoflavone-rich extract obtained in step S2 to an enzymatic hydrolysis reaction in a multi-stage bio-enzyme reactor to obtain a high-purity isoflavone aglycone product.

[0041] The multi-stage bio-enzyme reactor comprises a first-stage bio-enzyme reactor and a second-stage bio-enzyme reactor; wherein, after the isoflavone-rich extract is subjected to enzymatic hydrolysis in the first-stage bio-enzyme reactor, a first-stage filtrate and a first-stage concentrated solution are obtained; the first-stage concentrated solution is then fed to the inlet of the second-stage bio-enzyme reactor, and after enzymatic hydrolysis in the second-stage bio-enzyme reactor, a second-stage filtrate and a small amount of second-stage concentrated solution are obtained (the second-stage concentrated solution is circulated back to the inlet of the first-stage bio-enzyme reactor); the first-stage filtrate and the second-stage filtrate are collected, vacuum heated and dried, and then repeatedly rinsed with deionized water to obtain a high-purity isoflavone aglycone product. The feed flow rate of the first-stage bio-enzyme reactor is 50 mL / min, and the operating pressure is 0.2 MPa; the feed flow rate of the second-stage bio-enzyme reactor is 20 mL / min, and the operating pressure is 0.3 MPa.

[0042] The bio-enzyme reactor is a reactor obtained by immobilizing β-glucosidase on the surface of a nanofiltration membrane.

[0043] The preparation method of the bio-enzyme reactor is as follows: β-glucosidase is uniformly dispersed in a weakly acidic buffer solution, sodium alginate and a small amount of silane coupling agent are added, and after being stirred evenly, a bio-enzyme solution with a mass proportion of β-glucosidase of 1.2% is prepared; then a rolled nanofiltration membrane is immersed in the bio-enzyme solution, and after immersion for 3 hours, a nanofiltration membrane with bio-enzyme immobilized on the membrane surface is obtained; after winding and assembling with a central collecting tube and a porous support material, a bio-enzyme reactor is obtained. The mass ratio of the bio-enzyme to the weakly acidic buffer solution is 1:18, and the immobilized amount of the bio-enzyme on the bio-enzyme reactor is 50 mg / g.

[0044] The weakly acidic buffer solution is a citric acid-sodium citrate buffer solution with a pH of 5.5.

[0045] The membrane material of the nanofiltration membrane is polyamide and the molecular weight cutoff of the nanofiltration membrane is 300 to 500 Daltons.

[0046] In this embodiment, the nanofiltration membrane immobilized with β-glucosidase, weak acidic buffer solution, sodium alginate and silane coupling agent is used as a reactor bioenzyme reactor for enzymatic hydrolysis reaction. The bioenzyme used has high enzyme activity, and the final extraction rate of isoflavone aglycones obtained is 93.9% and the purity is 98.4%.

[0047] With the same raw materials and operating conditions, the operation of this example was repeated 5 times, each time using the same bio-enzyme reactor without changing the bio-enzyme reactor, and the final extraction rate of isoflavone aglycones was measured to be 93.1% and the purity was 97.7%.

[0048] Example 3

[0049] The method for extracting isoflavone aglycones from soybean germ in this embodiment comprises the following steps:

[0050] S1: Using ultrafinely crushed soybean germ powder as a raw material, the soybean germ powder is evenly mixed with an ethanol aqueous solution with a mass concentration of 80% to prepare 500 mL of a mixed solution with a mass concentration of 15% soybean germ powder;

[0051] S2: subjecting the mixed solution obtained in step S1 to ultrasonic-assisted solvent extraction to obtain an isoflavone-rich extract; the solvent used in the solvent extraction is short-chain alcohol ethanol, the extraction temperature is 60° C., and the extraction time is 3 hours. The ultrasonic power of the ultrasonic-assisted solvent extraction is 500 W, and the working time is 180 minutes.

[0052] S3: subjecting the isoflavone-rich extract obtained in step S2 to an enzymatic hydrolysis reaction in a multi-stage bio-enzyme reactor to obtain a high-purity isoflavone aglycone product.

[0053] The multi-stage bio-enzyme reactor comprises a first-stage bio-enzyme reactor and a second-stage bio-enzyme reactor; wherein, after the isoflavone-rich extract is subjected to enzymatic hydrolysis in the first-stage bio-enzyme reactor, a first-stage filtrate and a first-stage concentrated solution are obtained; the first-stage concentrated solution is then fed to the inlet of the second-stage bio-enzyme reactor, and after enzymatic hydrolysis in the second-stage bio-enzyme reactor, a second-stage filtrate and a small amount of second-stage concentrated solution are obtained (the second-stage concentrated solution is circulated back to the inlet of the first-stage bio-enzyme reactor); the first-stage filtrate and the second-stage filtrate are collected, vacuum heated and dried, and then repeatedly rinsed with deionized water to obtain a high-purity isoflavone aglycone product. The feed flow rate of the first-stage bio-enzyme reactor is 60 mL / min, and the operating pressure is 0.15 MPa; the feed flow rate of the second-stage bio-enzyme reactor is 30 mL / min, and the operating pressure is 0.25 MPa.

[0054] The bio-enzyme reactor is a reactor obtained by immobilizing β-glucosidase on the surface of a nanofiltration membrane.

[0055] The preparation method of the bio-enzyme reactor is as follows: β-glucosidase is uniformly dispersed in a weakly acidic buffer solution, sodium alginate and a small amount of silane coupling agent are added, and after being stirred evenly, a bio-enzyme solution with a mass proportion of β-glucosidase of 1.0% is prepared; then a rolled nanofiltration membrane is immersed in the bio-enzyme solution, and after immersion for 5 hours, a nanofiltration membrane with bio-enzyme immobilized on the membrane surface is obtained; after winding and assembling with a central collecting tube and a porous support material, a bio-enzyme reactor is obtained. The mass ratio of the bio-enzyme to the weakly acidic buffer solution is 1:20, and the immobilized amount of the bio-enzyme on the bio-enzyme reactor is 60 mg / g.

[0056] The weakly acidic buffer solution is a citric acid-sodium citrate buffer solution with a pH of 6.0.

[0057] The membrane material of the nanofiltration membrane is polyamide, and the molecular weight cutoff of the nanofiltration membrane is 300 to 500 Daltons.

[0058] In this embodiment, the nanofiltration membrane immobilized with β-glucosidase, weak acidic buffer solution, sodium alginate and silane coupling agent is used as a reactor bioenzyme reactor for enzymatic hydrolysis reaction. The bioenzyme used has high enzyme activity, and the final extraction rate of isoflavone aglycones obtained is 94.2% and the purity is 97.8%.

[0059] With the same raw materials and operating conditions, the operation of this example was repeated 5 times, each time using the same bio-enzyme reactor without changing the bio-enzyme reactor, and the final extraction rate of isoflavone aglycones was measured to be 92.5% and the purity was 97.1%.

[0060] Comparative Example 1

[0061] The method for extracting isoflavone aglycones from soybean germ in this comparative example has the same raw material composition and operation steps as those in Example 1, except that in the method of this comparative example, the biological enzyme in step S2 is directly added to the isoflavone-rich extract obtained in step S2 for enzymolysis reaction, rather than being subjected to enzymolysis reaction in a biological enzyme reactor. The final extraction rate of isoflavone aglycones was measured to be 93.8% and the purity was 92.4%.

[0062] Comparative Example 2

[0063] The method for extracting isoflavone aglycones from soybean germ in this comparative example has the same raw material composition and operation steps as those in Example 1, except that in the method of this comparative example, the biological enzyme in step S2 is directly added to the isoflavone-rich extract obtained in step S2 for enzymolysis reaction, rather than being subjected to an enzyme reactor for enzymolysis reaction. Under the same raw materials and operation conditions, the operation of this comparative example is repeated 5 times, and each time the solution containing the biological enzyme after the previous purification, separation and concentration is added to the isoflavone-rich extract obtained in step S2 for enzymolysis reaction (i.e., the biological enzyme is reused as much as possible, rather than adding new biological enzymes), and the final extraction rate of isoflavone aglycones is measured to be 66.7%, and the purity is 76.5%.

[0064] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A method for extracting isoflavone aglycones from soybean germ, characterized in that: The method comprises the following steps: S1: using ultrafinely crushed soybean germ powder as a raw material, mixing the soybean germ powder with a short-chain alcohol aqueous solution with a mass concentration of 80-90% to prepare a mixed solution with a mass concentration of 5-15% of soybean germ powder; S2: subjecting the mixed solution obtained in step S1 to ultrasonic and / or microwave-assisted solvent extraction to obtain an isoflavone-rich extract; S3: subjecting the isoflavone-rich extract obtained in step S2 to an enzymatic hydrolysis reaction in a multi-stage bio-enzyme reactor to obtain a high-purity isoflavone aglycone product.

2. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The short-chain alcohol is at least one of methanol, ethanol and propanol.

3. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The solvent used in the solvent extraction operation is a short-chain alcohol, the extraction temperature is 50-60° C., and the extraction time is 1-3 hours.

4. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The ultrasonic power of the ultrasonic-assisted solvent extraction operation is 100 to 500 W, and the working time is 30 to 180 minutes.

5. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The microwave power of the microwave-assisted solvent extraction operation is 100-1000W, and the working time is 10-60min.

6. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The multi-stage bio-enzyme reactor comprises a first-stage bio-enzyme reactor and a second-stage bio-enzyme reactor; wherein, after the isoflavone-rich extract is subjected to enzymatic hydrolysis in the first-stage bio-enzyme reactor, a first-stage filtrate and a first-stage concentrated solution are obtained; the first-stage concentrated solution is flushed out of the enzymatic hydrolysis in the first-stage bio-enzyme reactor by a reverse flushing operation, and then fed into the inlet of the second-stage bio-enzyme reactor, and after enzymatic hydrolysis in the second-stage bio-enzyme reactor, a second-stage filtrate and a second-stage retentate are obtained; the first-stage filtrate and the second-stage filtrate are collected, vacuum-heated and dried, and then repeatedly rinsed with deionized water to obtain a high-purity isoflavone aglycone product.

7. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The bio-enzyme reactor is a reactor obtained by immobilizing β-glucosidase on the surface of a nanofiltration membrane.

8. The method for extracting isoflavone aglycones from soybean germ according to claim 1, characterized in that: The preparation method of the bio-enzyme reactor is as follows: beta-glucosidase is uniformly dispersed in a weakly acidic buffer solution, sodium alginate and a small amount of silane coupling agent are added, and the mixture is stirred evenly to prepare a bio-enzyme solution; then a rolled nanofiltration membrane is immersed in the bio-enzyme solution for 1 to 5 hours to obtain a nanofiltration membrane with bio-enzyme immobilized on the membrane surface; The bio-enzyme reactor is obtained after being wound and assembled with a central collecting tube and a porous supporting material.

9. The method for extracting isoflavone aglycones from soybean germ according to claim 8, characterized in that: The weakly acidic buffer solution is a citric acid-sodium citrate buffer solution with a pH value of 5.5 to 6.

5.

10. The method for extracting isoflavone aglycones from soybean germ according to claim 8, characterized in that: The mass ratio of the biological enzyme to the weakly acidic buffer solution is 1:15-20, and the immobilized amount of the biological enzyme on the biological enzyme reactor is 10-100 mg / g.