CN110540557B

CN110540557B - Method for preparing glycitein by taking soybean navel powder as raw material

Info

Publication number: CN110540557B
Application number: CN201910522546.6A
Authority: CN
Inventors: 刘汝萃; 鲁绪强; 韩亮; 刘代成; 董欣伟; 时玉强; 崔玉涛; 刘杰
Original assignee: Shandong Normal University; Shandong Yuwang Ecological Food Industry Co Ltd
Current assignee: Shandong Normal University; Shandong Yuwang Ecological Food Industry Co Ltd
Priority date: 2019-06-17
Filing date: 2019-06-17
Publication date: 2020-12-08
Anticipated expiration: 2039-06-17
Also published as: CN110540557A

Abstract

The invention discloses a method for preparing glycitein by taking soybean navel powder as a raw material, which comprises the following steps: extracting 80-100 mesh bean navel powder in 55-65% ethanol for 45-60min, adding ethanol solution with volume of 500-600ml into per 100g bean navel powder to obtain extractive solution, and evaporating to dry to obtain dry residue; adding n-hexane into the dry residue, extracting, and removing the n-hexane supernatant to obtain residue; adding ethyl acetate into the residue, extracting, centrifuging to obtain supernatant, and evaporating the supernatant to dryness to obtain glycitein. When the soybean navel powder is extracted by a specific process, isoflavone, glycitein, with a single component can be extracted, and a pure glycitein product is obtained after n-hexane degreasing and ethyl acetate impurity removal. The method is simple, rapid, low in cost and suitable for industrial production.

Description

Method for preparing glycitein by taking soybean navel powder as raw material

Technical Field

The invention particularly relates to a method for preparing glycitein by taking soybean navel powder as a raw material.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The soybean is a traditional food in China, contains a large amount of physiologically active substances including oligosaccharides, phospholipids, vitamins, isoflavone, soyasaponin and the like, and the comprehensive development and utilization of the soybean are more and more concerned by various countries in the world along with the continuous and deep research on the functional components of the soybean. The hilum of soybean, also known as the germ, is an important component of soybean seeds. The soybean germ contains 28% protein, 8.7% fat (containing unsaturated fatty acid up to 80%, such as linoleic acid, linolenic acid and oleic acid), large amount of vitamin E, 1.4-1.76% soybean isoflavone and 0.4% sterol. The isoflavone content in the soybean cotyledon is 0.15-0.32%, and the isoflavone content in the seed coat is 0.01-0.02%, so the isoflavone in the soybean is mainly enriched in the germ. The germ can be used to produce soy isoflavones.

The inventor finds that the soybean isoflavone has common basic structures of all components, and the differences of the structures and the properties of all the components are small, so that the soybean isoflavone seems to coexist symbiotically and is difficult to separate. And the oil in the soybean umbilicus is more in variety and is not easy to separate from the soybean isoflavone with weak polarity. The primary extraction methods reported in the literature mainly include an alkali extraction and acid precipitation method, an organic solvent extraction method and a carbon dioxide supercritical extraction method, and a solvent extraction method is more commonly used. The solvent extraction method adopts ethanol, methanol, acetone and weak alkaline aqueous solution as solvent, and the extract contains isoflavone components, and also contains impurities such as oil, protein, cellulose, monosaccharide and polysaccharide. In the prior art, when n-hexane or petroleum ether or 6# gasoline is used as a solvent to remove the oil in the soybean meal and the soybeans, no soybean isoflavone is detected in the extracted oil.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention aims to provide a method for preparing glycitein by taking the soybean navel powder as a raw material.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the invention aims to provide a method for preparing glycitein by taking soybean navel powder as a raw material, which comprises the following steps:

extracting 80-100 mesh bean navel powder in 55-65% ethanol for 45-60min, adding ethanol solution with volume of 500-600ml into per 100g bean navel powder to obtain extractive solution, and evaporating to dry to obtain dry residue;

adding n-hexane into the dry residue, extracting, and removing the n-hexane supernatant to obtain residue;

adding ethyl acetate into the residue, extracting, centrifuging to obtain supernatant, and evaporating the supernatant to dryness to obtain glycitein.

The inventors have unexpectedly found in experiments that when soy navel flour is extracted with 55-65% (volume fraction) ethanol for 45-60min, a single ingredient isoflavone-glycitein can be extracted, and if the concentration of ethanol deviates from this range (less than or greater than this range) or the volume of ethanol deviates from this range (less than or greater than this range) or the extraction time deviates from this range (less than or greater than this range), the extract is an isoflavone complex rather than a single ingredient isoflavone-glycitein.

Because the extract contains partial grease, salt, cysteine, polypeptide and other components, the grease in the extract can be removed after n-hexane extraction, and the salt, the cysteine, the polypeptide and other components in the glycitein can be removed after ethyl acetate selective extraction, so that the pure glycitein can be obtained.

In some embodiments, 15-20ml of n-hexane is added per 100g of dry residue prepared from the hilum powder for degreasing.

The oil cannot be completely removed due to the addition of too little n-hexane, and the waste of n-hexane is easily caused when the addition of too much n-hexane is performed.

Further, the extraction time is 10-20min after adding n-hexane.

Further, after the system after n-hexane extraction is subjected to centrifugal separation, n-hexane supernatant is discarded, the centrifugal rotation speed is 1000-1500r/min, and the centrifugal time is 5-10 min. The separation of the n-hexane supernatant and the solid is easier to realize by centrifugal separation, and the loss of the solid is avoided when the supernatant is discarded.

In some embodiments, the time for ethyl acetate extraction is 10-20 min.

The second purpose of the invention is to provide a second method for preparing glycitein by taking the soybean navel powder as a raw material, which comprises the following steps:

adding n-hexane into the bean navel powder, stirring and extracting to obtain an extracting solution, and evaporating the extracting solution to dryness to obtain bean navel oil;

adding anhydrous ethanol into the Sophora umbilicalis oil at a volume ratio of the anhydrous ethanol to the Sophora umbilicalis oil of 9-15:1, extracting for 0.5-1h, standing for layering, collecting the upper layer solution, and steaming to obtain paste;

adding anhydrous ethanol into the paste for extraction, taking supernatant, and evaporating the supernatant to dryness to obtain glycitein.

Before extracting the hilum powder by using normal hexane, in order to verify the prior art, the inventor adopts normal hexane or petroleum ether or No. 6 gasoline to extract soybean meal, soybeans and soybean oil residues, and detects that no soybean isoflavone exists in an extracting solution. The inventor tries to extract the hilum of the soybean by using n-hexane, petroleum ether or No. 6 gasoline, and unexpectedly finds that when the extraction solvent is n-hexane, the extract contains a small amount of glycitein but does not contain other isoflavone. Probably the grease in the soybean umbilicus can promote the dissolution of glycitein in normal hexane.

In order to further separate oil and fat and glycitein in the extract and prepare a pure glycitein product, the inventor carries out a series of experiments and finds that when absolute ethyl alcohol is added into the prepared glycitein oil, the volume ratio of the absolute ethyl alcohol to the glycitein oil is 9-15:1, and the extraction is carried out for 0.5-1h, the glycitein in the glycitein oil can be selectively extracted and further purified, so that the pure glycitein product can be obtained.

In some embodiments, the soybean navel powder is extracted by n-hexane for 5 to 8 times, and the amount ratio of n-hexane to soybean navel powder is: 1.5-3: 1, V/M.

Because the extraction amount of glycitein in the soybean umbilicus by n-hexane is low once, the glycitein powder needs to be repeatedly extracted to completely extract the glycitein in the soybean umbilicus powder as much as possible, and when the dosage ratio of the n-hexane to the glycitein powder extracted each time is as follows: 1.5-3: 1, V/M, the glycitein in the soybean umbilicus can be selectively extracted, and tests show that the selective extraction of glycitein is not facilitated when the n-hexane content is too large or too small.

Further, the extraction time of the bean navel powder by n-hexane is 1-1.5h each time, and the extraction temperature is 15-30 ℃.

In some embodiments, the volume ratio of the soy navel oil to the absolute ethanol added thereto is 1: 8-12.

In some embodiments, the mass ratio of the paste to absolute ethyl alcohol is 100 times.

The beneficial technical effects of the invention are as follows:

the inventor unexpectedly finds that when the soybean navel powder is extracted by a specific process (ethanol extraction with specific concentration, normal hexane degreasing, ethyl acetate impurity removal, or normal hexane extraction with specific volume ratio and absolute ethanol impurity removal), isoflavone, glycitein, with a single component can be extracted, and a pure glycitein product is obtained after degreasing and impurity removal. The method is simple, rapid, low in cost and suitable for industrial production.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.

FIG. 1 is a thin layer separation 3D scan of glycitin, genistin and daidzin;

FIG. 2 is a scanning peak diagram of the thin layer development of the product prepared in example 1.

FIG. 3 is a plot of the thin-layer spread scan peaks of a glycitein standard solution.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Example 1

100g of 80-100-mesh bean navel powder is added with 550mL of 60% ethanol, stirred and extracted at 50 ℃ for 1h, and 400mL of filtrate is steamed in a rotary manner until the dry matter is 2 g. Adding 20mL of n-hexane into the dry matter, shaking for 10min, centrifuging for 5min at 1000-1500r/min, and discarding the supernatant to obtain the residue. Adding 20mL of ethyl acetate into the residue, shaking for extraction for 15min, centrifuging at 1000-1500r/min for 10min to obtain supernatant, and removing ethyl acetate by rotary evaporation to obtain 10.7mg of glycitein with purity of 88%.

Example 2

Adding 600mL of 55% ethanol into 100g of 80-100-mesh bean navel powder, stirring and extracting at 60 ℃ for 1h, and rotatably steaming 450mL of filtrate until the dry matter is 1.5 g. Adding 15mL of n-hexane into the dry matter, shaking for extraction for 20min, centrifuging for 10min at 1000-1500r/min, and discarding the supernatant to obtain the residue. Adding 15mL of ethyl acetate into the residue, shaking for extraction for 20min, centrifuging for 5min at 1000-1500r/min to obtain supernatant, and removing ethyl acetate by rotary evaporation to obtain 11mg of glycitein with purity of 90%.

Example 3

And (3) adding 500mL of 65% ethanol into 100g of 80-100-mesh bean navel powder, stirring and extracting at 55 ℃ for 45 minutes, and rotatably steaming 350mL of filtrate until the dry matter is 2 g. Adding 20mL of n-hexane into the dry matter, shaking for extraction for 15min, centrifuging for 8min at 1000-1500r/min, and discarding the supernatant to obtain the residue. Adding 20mL of ethyl acetate into the residue, shaking for extraction for 20min, centrifuging at 1000-1500r/min for 8min to obtain supernatant, and removing ethyl acetate by rotary evaporation to obtain 10.4mg of glycitein with purity of 88%.

Comparative example 1

The differences from example 1 are: the particle size of the bean navel powder is 60-75 meshes, and the rest is the same as that of the example 1. The mass of the prepared glycitein is 7.6 mg.

Comparative example 2

The differences from example 1 are: the particle size of the bean navel powder is 110-120 meshes, and the rest is the same as that of the embodiment 1. The product contains more fatty acids, and has increased content of leucine, isoleucine, glycine, valine and polypeptide impurities, and glycitein purity of 63%.

Comparative example 3

The differences from example 1 are: the concentration of ethanol was 50%, and the rest was the same as in example 1. The extracted product contains glycitin.

Comparative example 4

The differences from example 1 are: the concentration of ethanol was 70%, and the rest was the same as in example 1. The product extracted by the concentration has more impurities such as triglyceride and the like, and the purity of the glycitein is 65%.

Comparative example 5

The differences from example 1 are: the volume of ethanol added was 450ml, otherwise the same as in example 1. The glycitein product contains more amino acid and polypeptide impurities, and the purity of glycitein is 54%.

Comparative example 6

The differences from example 1 are: the volume of ethanol added was 600ml, the rest being the same as in example 1. The impurities of grease, small peptide and fatty acid are more, and the purity of glycitein is 56%.

Example 4

Taking 110g of ground (80-mesh) soybean hilum powder, adding 300mL of n-hexane, stirring and extracting at room temperature for 1h, pouring out 150mL of supernatant after standing, adding 150mL of n-hexane, stirring and extracting at room temperature for 1h, and pouring out 150mL of supernatant after standing. This was repeated 3 more times, and extracted 5 times in total. The supernatant fluid is combined and rotated to evaporate n-hexane, thus obtaining 218mL of the soybean navel oil.

Adding anhydrous ethanol 200mL into soybean navel oil 18mL, extracting under stirring for 1h, standing, pouring out ethanol extractive solution (other oil and fat below), and rotary evaporating to remove ethanol to obtain small amount of paste (about less than 0.1 g).

Adding 10mL of anhydrous ethanol into the paste, shaking for extraction for 12min, pouring out ethanol supernatant (discarding oil stuck to the bottom of the bottle), rotary evaporating to dry, and detecting glycitein to be 19.88mg (serving as a standard substance for detecting soybean isoflavone) and glycitein purity to be 90.4%.

Example 5

Taking 110g of ground (80-mesh) soybean hilum powder, adding 250mL of n-hexane, stirring and extracting at room temperature for 1.5h, pouring out 150mL of supernatant after standing, adding 150mL of n-hexane, stirring and extracting at room temperature for 1.5h, and pouring out 150mL of supernatant after standing. This was repeated 5 more times, for a total of 7 extractions. And combining the supernatants, and performing rotary evaporation to remove n-hexane to obtain 21mL of the soybean navel oil.

Adding 250mL of anhydrous ethanol into 21mL of soybean navel oil, stirring and extracting for 45min, standing, pouring out ethanol extract (other oil and fat are below), and rotary evaporating to remove ethanol to obtain a small amount of paste (about less than 0.1 g).

Adding 10mL of anhydrous ethanol into the paste, shaking for 10min, pouring out ethanol supernatant (discarding oil stuck to the bottom of the bottle), rotary evaporating to dry, and detecting glycitein to be 21mg (serving as a standard substance for detecting soybean isoflavone) and glycitein purity to be 90.8%.

Example 6

Taking 110g of ground (80-mesh) soybean hilum powder, adding 200mL of n-hexane, stirring and extracting at room temperature for 1.3h, pouring out 150mL of supernatant after standing, adding 150mL of n-hexane, stirring and extracting at room temperature for 1.3h, and pouring out 150mL of supernatant after standing. This was repeated 4 more times, for a total of 6 extractions. And (4) combining the supernatants, and performing rotary evaporation to remove n-hexane to obtain 20mL of the soybean navel oil.

Adding 300mL of anhydrous ethanol into 20mL of soybean navel oil, stirring and extracting for 0.5h, standing, pouring out ethanol extract (other oil and fat are below), and rotary evaporating to remove ethanol to obtain a small amount of paste (about less than 0.1 g).

Adding 10mL of anhydrous ethanol into the paste, shaking for 15min, pouring out ethanol supernatant (discarding oil stuck to the bottom of the bottle), rotary evaporating to dry, and detecting glycitein to be 19.5mg (serving as a standard product for detecting soybean isoflavone) and glycitein purity to be 90.4%.

Comparative example 7

The differences from example 1 are: petroleum ether is used for replacing n-hexane, other steps are the same as those in the example 1, and the product does not contain glycitein.

Comparative example 8

The differences from example 1 are: the volume of n-hexane added to the hilum powder was 330ml, and the other steps were the same as in example 1. The solvent consumption is large, the product has more impurities, and the purity of the glycitein is 67 percent.

Comparative example 9

The differences from example 1 are: the volume of absolute ethyl alcohol added into the soybean navel oil is 150ml, other steps are the same as the example 1, the soybean navel oil is not well dissolved, the liquid is sticky, and the extraction is not complete.

The products prepared in examples 1-3 and comparative examples 1-9 were tested as follows:

1. 6 standard substances of daidzein, daidzin, genistein, genistin, glycitein and glycitin are respectively prepared into 6 standard substance solutions of 1 mg/ml.

2. The samples prepared in examples 1 to 3 and comparative examples 1 to 9 were dissolved in 10ml of ethyl acetate, respectively, to prepare sample solutions.

3. Using activated silica gel GF254 high efficiency thin layer plate (HPTLC)10cm by 20cm, taking 1 mul, 2 mul, 3 mul, 4 mul and 5 mul of each of 6 standard solutions to prepare 13 standard curves, and obtaining 6 standard curve equations correspondingly.

4. Detection of soybean isoflavone glycoside (daidzein glycoside, genistin, glycitin): external standard three standard solutions each 5. mu.l. Samples of the products of examples 1 to 6 and comparative examples 1 to 9 were taken at 5. mu.l each in which ethyl acetate was dissolved. Spot on GF254HPTLC plate and air dry. Spreading in a double-groove glass spreading cylinder, adding 20ml of a spreading agent, wherein the formula of the spreading agent is as follows: dichloromethane: methanol: acetic acid 10:2:0.1, putting the HPTLC plate into a developing cylinder, covering, saturating for 10 minutes, starting to develop, developing distance: 13 cm. Taking out after finishing. After being dried, the mixture is observed under an ultraviolet lamp of 365 nm.

5. Scanning conditions were performed on a thin layer scanner TLC scanner III with wincats1.4.1 software (CAMAG, switzerland): scanning speed 80nm/s, resolution 200 μm/step, irradiation lamp D2 lamp, wavelength 260nm, scanning width 6.00mm 0.90 mm. As shown in fig. 1, it was found that the three substances were completely separated from each other, i.e., daidzin Rf was 0.41, genistin Rf was 0.52, and glycitin Rf was 0.50.

6. Detection of soybean isoflavone aglycone (daidzein, genistein, glycitein): external standard three standard solutions each 5. mu.l. Samples of the products of examples 1 to 6 and comparative examples 1 to 9 were taken at 5. mu.l each in which ethyl acetate was dissolved. Spot on GF254HPTLC plate and air dry. Spreading in a double-groove glass spreading cylinder, adding 20ml of a spreading agent, wherein the formula of the spreading agent is as follows: trichloromethane: methanol: acetic acid 9:1:0.15, putting HPTLC plate into developing cylinder, covering, saturating for 10min, developing distance: 10cm, taking out after the exhibition is finished, airing, and observing under a 365nm ultraviolet lamp.

7. Scanning conditions were performed on a thin layer scanner TLC scanner III with wincats1.4.1 software (CAMAG, switzerland): scanning speed 80nm/s, resolution 200 μm/step, irradiation lamp D2 lamp, wavelength 260nm, scanning width 6.00mm 0.90mm, daidzein Rf 0.50, genistein Rf 0.67, and glycitein Rf 0.62.

The sample liquids of examples 1 to 6 and comparative examples 1 to 9 were tested, and the test results were as follows: the sample solutions of examples 1-6 contained only glycitein and no other soy isoflavones. As shown in FIGS. 2 and 3, the peak positions of the product prepared in example 1 and the glycitein standard substance are the same, and the scanning chart in FIG. 2 has no other impurity peaks, which indicates that the glycitein product is relatively pure.

The compositions and purities of the products prepared in examples 1-6 and comparative examples 1-9 are shown in Table 1.

TABLE 1

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing glycitein by taking soybean navel powder as a raw material is characterized by comprising the following steps: the method comprises the following steps:

2. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 1, characterized in that: adding 15-20ml of n-hexane into dry residue per 100g of bean navel powder for removing oil and fat.

3. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 1, characterized in that: adding n-hexane, and extracting for 10-20 min.

4. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 1, characterized in that: and (3) centrifugally separating the system after n-hexane extraction, discarding the n-hexane supernatant, wherein the centrifugal rotation speed is 1000-1500r/min, and the centrifugal time is 5-10 min.

5. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 1, characterized in that: the extraction time of ethyl acetate is 10-20 min.

6. A method for preparing glycitein by taking soybean navel powder as a raw material is characterized by comprising the following steps: the method comprises the following steps:

adding anhydrous ethanol into the paste for extraction, taking supernatant, and evaporating the supernatant to dryness to obtain glycitein;

and (3) repeatedly extracting the bean navel powder by using n-hexane for 5-8 times, wherein the dosage ratio of the n-hexane to the bean navel powder is as follows: 1.5-3: 1, V/M.

7. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 6, characterized in that: the extraction time of the soybean navel powder by n-hexane is 1-1.5h each time, and the extraction temperature is 15-30 ℃.

8. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 6, characterized in that: the volume ratio of the soybean navel oil to the absolute ethyl alcohol added into the soybean navel oil is 1: 8-12.

9. The method for preparing glycitein from soy bean paste powder as a raw material according to claim 6, characterized in that: the mass ratio of the paste to the absolute ethyl alcohol is 100 times.