阅读说明：本技术 一种大豆提取物及其制备方法、应用 (Soybean extract and preparation method and application thereof ) 是由 孙云起 郭朝万 王丽华 聂艳峰 胡露 王娟 于 2021-08-26 设计创作，主要内容包括：本申请涉及领域,具体而言,涉及一种大豆提取物及其制备方法、应用。大豆提取物的制备方法,包括：将大豆破壁,在55～65℃下保温0.5～3h；然后在1～4℃保温6～15h,过滤后取滤液；向所述滤液中加入C1-C4醇、添加剂,调节电导率至40000～80000μs/cm后静置至体系分层,取上层液；在提取制备过程中就实现物质纯化,高效除去蛋白质和卵磷脂。利用不同物质的静电作用、疏水作用和生物亲和作用形成双水相,从而影响不同成分的溶解性有效提高大豆异黄酮的提取率和纯度。(The application relates to the field, in particular to a soybean extract and a preparation method and application thereof. A method of preparing a soy extract comprising: breaking the wall of the soybean, and keeping the temperature at 55-65 ℃ for 0.5-3 h; then preserving heat for 6-15 h at 1-4 ℃, and filtering to obtain filtrate; adding C1-C4 alcohol and an additive into the filtrate, adjusting the conductivity to 40000-80000 mu s/cm, standing until a system is layered, and taking supernatant; the material purification is realized in the extraction preparation process, and the protein and the lecithin are efficiently removed. The double aqueous phases are formed by utilizing the electrostatic action, the hydrophobic action and the biological affinity action of different substances, thereby influencing the solubility of different components and effectively improving the extraction rate and the purity of the soybean isoflavone.)

1. A method for preparing a soybean extract, comprising:

breaking the wall of the soybean, and keeping the temperature at 55-65 ℃ for 0.5-3 h; then preserving heat for 6-15 h at 1-4 ℃, and filtering to obtain filtrate;

adding C1-C4 alcohol and an additive into the filtrate, adjusting the conductivity to 40000-80000 mu s/cm, standing until a system is layered, and taking supernatant;

wherein the additive comprises cetyl PEG/PPG-10/1 polydimethylsiloxane and/or PEG-30 dipolyhydroxystearate; the weight ratio of the additive to the soybean is 10-20%.

2. The method for preparing soybean extract according to claim 1, wherein the step of breaking the wall of soybean comprises: grinding soybeans, a grinding aid and water together;

optionally, the grinding aid comprises at least one of calcium sulfate, calcium carbonate, and barium sulfate.

3. The method for producing a soybean extract according to claim 1, wherein the soybean is soybean germ.

4. The method for producing a soybean extract according to claim 1, wherein the conductivity is adjusted by using a salt insoluble in C1 to C4 alcohol;

optionally, potassium chloride and/or sodium chloride is used to adjust the conductivity.

5. The method for producing a soybean extract according to claim 1,

the C1-C4 alcohol is ethanol;

optionally, the volume ratio of the C1-C4 alcohol to the filtrate is 1-2.

6. The method for producing a soybean extract according to any one of claims 1 to 5, wherein the step of taking the filtrate after filtration is a step of taking the filtrate after filtration by centrifugation.

7. The method for preparing soybean extract according to any one of claims 1 to 5, wherein the step of maintaining the temperature at 55 to 65 ℃ for 0.5 to 3 hours comprises: stirring at 55-65 ℃ and 500-800 rpm for 0.5-3 h.

8. The method for preparing soybean extract according to any one of claims 1 to 5, wherein the step of adding C1-C4 alcohol to the filtrate and the step of adding additives are carried out under the conditions of 2000-5000 rpm.

9. A soybean extract, which is produced by the method for producing a soybean extract according to any one of claims 1 to 8.

10. Use of the soybean extract of claim 9 for the preparation of an antiallergic agent or an antiinflammatory agent.

Technical Field

The application relates to the field, in particular to a soybean extract and a preparation method and application thereof.

Background

The soybean isoflavone has health promotion effects of reducing cholesterol, reducing cardiovascular diseases, preventing osteoporosis and relieving climacteric syndrome; soybean is the most major dietary source of soy isoflavones; however, the extraction rate of the prior art method for extracting isoflavone from soybean needs to be improved.

Disclosure of Invention

The embodiment of the application aims to provide a soybean extract, a preparation method and application thereof, which aim to improve the extraction rate of isoflavone in soybeans.

The application provides a preparation method of a soybean extract, which comprises the following steps:

breaking the wall of the soybean, and keeping the temperature at 55-65 ℃ for 0.5-3 h; then preserving heat for 6-15 h at 1-4 ℃, and filtering to obtain filtrate;

adding C1-C4 alcohol and an additive into the filtrate, adjusting the conductivity to 40000-80000 mu s/cm, standing until a system is layered, and taking supernatant;

wherein the additive comprises cetyl PEG/PPG-10/1 polydimethylsiloxane and/or PEG-30 dipolyhydroxystearate; the weight ratio of the additive to the soybean is 10-20%.

Breaking cell wall of semen glycines, maintaining at high temperature, maintaining at low temperature, and destroying internal tissue of semen glycines to separate soybean isoflavone from semen glycines tissue; adjusting the filtrate by C1-C4 alcohol and an additive to ensure that the soybean protein, the lecithin and the additive enter the same layer, and the C1-C4 alcohol and the soybean isoflavone enter the same layer; the material purification is realized in the extraction preparation process, and the protein and the lecithin are efficiently removed. The double aqueous phases are formed by utilizing the electrostatic action, the hydrophobic action and the biological affinity action of different substances, thereby influencing the solubility of different components and effectively improving the extraction rate and the purity of the soybean isoflavone.

In some embodiments of the present application, the step of breaking the walls of the soybean comprises: grinding soybeans, a grinding aid and water together; the grinding aid comprises at least one of calcium sulfate, calcium carbonate and barium sulfate.

In some embodiments of the present application, the soy is soy germ.

In some embodiments herein, salts insoluble in C1-C4 alcohols are used to adjust conductivity;

optionally, potassium chloride and/or sodium chloride is used to adjust the conductivity.

In some embodiments herein, the C1-C4 alcohol is ethanol;

the volume ratio of the C1-C4 alcohol to the filtrate is 1-2.

In some embodiments of the present application, the step of filtering and then taking the filtrate is performed by centrifuging and then taking the filtrate.

In some embodiments of the present application, the step of maintaining the temperature at 55-65 ℃ for 0.5-3h comprises: stirring at 55-65 ℃ and 500-800 rpm for 0.5-3 h.

In some embodiments of the present application, the step of adding the C1-C4 alcohol to the filtrate, the additive step is performed at 2000-5000 rpm.

The application provides a soybean extract, and the soybean extract is prepared by the preparation method of the soybean extract.

The application also provides an application of the soybean extract in preparing an antiallergic drug or an anti-inflammatory drug.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 shows a physical diagram of the system of example 1 after standing for 1.0 h.

FIG. 2 shows a physical diagram of the system of example 2 after standing for 1.0 h.

FIG. 3 shows a physical representation of the system of example 3 after standing for 1.0 h.

Fig. 4 shows a physical representation of the system of comparative example 1 after standing for 1.0 h.

Fig. 5 shows a physical representation of the system of comparative example 2 after standing for 1.0 h.

Fig. 6 shows a physical representation of the system of comparative example 3 after standing for 1.0 h.

FIG. 7 shows a physical representation of the system of comparative example 4 after 1.0h of standing.

FIG. 8 shows a physical representation of the system of comparative example 5 after 1.0h of standing.

FIG. 9 shows the results of hyaluronidase inhibition by the soybean extracts of examples 1-2 and comparative examples 1-5.

FIG. 10 shows the results of the inhibition ratios of lipoxygenase by the soybean extracts of examples 1-2 and comparative examples 1-5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following provides a detailed description of the soybean extract and the preparation method and application thereof in the examples of the present application.

A method for preparing a soybean extract, comprising:

breaking the wall of the soybean, and keeping the temperature at 55-65 ℃ for 0.5-3 h; then preserving heat for 6-15 h at 1-4 ℃, and filtering to obtain filtrate;

adding C1-C4 alcohol and an additive into the filtrate, adjusting the conductivity to 40000-80000 mu s/cm, standing until the system is layered, and taking supernatant;

wherein the additive is cetyl PEG/PPG-10/1 polydimethylsiloxane and/or PEG-30 dipolyhydroxystearate; the weight ratio of the additive to the soybean is 10-20%.

Illustratively, the soybean isoflavone is introduced into the filtrate by breaking cell walls, then incubating at a high temperature, and then incubating at a low temperature to destroy the internal tissues of the soybeans.

In some embodiments of the present application, the step of breaking the walls of the soybean comprises: grinding the soybeans, grinding aid and water together.

For example, the grinding aid comprises at least one of calcium sulfate, calcium carbonate, and barium sulfate. It will be appreciated that in some other embodiments of the present application, the grinding aid may be selected from other materials that do not chemically react with the soybeans and that do not physically adsorb and are insoluble in water.

It is understood that in other embodiments of the present application, other means of disrupting the cell walls of the soybeans may be used, not limited to grinding.

In some embodiments of the present application, the soy is soy germ, and the soy germ has a higher isoflavone content to obtain more soy isoflavones; it is understood that in other embodiments of the present application, soy may be used directly.

Illustratively, the mass ratio of the grinding aid to the soybean germs is 1-4%; for example, it may be 1%, 2%, 3%, 4%.

The mass ratio of water to soybean may be (0.8 to 2):1, and for example, may be 0.8:1, 0.9:1, 1: 1. 1.2: 1. 1.5: 1. 2: 1, etc.

Grinding the soybeans, the grinding aid and water together, wherein the grinding aid can fully separate soybean isoflavone from other tissues of the soybeans; after grinding, preserving heat for 0.5-3h at 55-65 ℃; and then preserving the heat for 6-15 h at the temperature of 2-4 ℃, and filtering to obtain filtrate.

Preserving the heat at 55-65 ℃ to destroy the protein, and then denaturing the soybean protein at 1-4 ℃.

For example, the temperature at high temperature may be 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 63 ℃, 65 ℃ and the like, and the time for high temperature incubation may be 0.5h, 0.8h, 1h, 1.5h, 2h, 2.5h, 3h and the like.

For example, the heat preservation at 55 to 65 ℃ may be stirring at the temperature, for example, stirring at 500 to 800rpm (for example, 500rpm, 600rpm, 700rpm, 800rpm) at 55 to 65 ℃ for 0.5 to 3 hours. Or directly preserving the ground system at 55-65 ℃ for 0.5-3h without operations such as stirring.

And (3) carrying out low-temperature heat preservation after high-temperature heat preservation, wherein the low-temperature heat preservation temperature is 1-4 ℃, for example, 1 ℃, 2 ℃, 3 ℃,4 ℃ and the like, and the low-temperature heat preservation time is 6-15 h, for example, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 15h and the like. The operation such as stirring and the like can be omitted in the low-temperature heat preservation process.

And filtering after the low-temperature heat preservation is finished.

For example, solid-liquid separation is performed by centrifugation, for example, centrifugation at 10000 g.

Adding C1-C4 alcohol and an additive into the filtrate and mixing.

The additive comprises cetyl PEG/PPG-10/1 polydimethylsiloxane and/or PEG-30 dipolyhydroxystearate. For example, additives include cetyl PEG/PPG-10/1 dimethicone; alternatively, the additive comprises PEG-30 dipolyhydroxystearate; alternatively, the additive comprises cetyl PEG/PPG-10/1 dimethicone and/or PEG-30 dipolyhydroxystearate.

The weight ratio of the additive to the soybean is 10-20%; for example, 10%, 11%, 12%, 13%, 15%, 16%, 18%, 19%, 20%, etc. may be used.

The C1-C4 alcohol may include, for example, at least one of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, in some embodiments of the present application, the C1-C4 alcohol is ethanol.

Illustratively, the volume of the ethanol is 1-2 times of the volume of the filtrate; for example, it may be 1-fold, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, or the like.

It will be appreciated that the amount of ethanol used may be much greater than the filtrate, regardless of factors such as the utilization of ethanol.

For example, in order to fully mix the filtrate, the C1-C4 alcohol and the additive, the mixture can be stirred at 2000-5000rpm for 2-10 min to uniformly mix the system.

Adjusting the conductivity to 40000-80000 mu s/cm, standing until a system is layered, and taking supernatant;

illustratively, the conductivity is adjusted with a salt that is insoluble in C1-C4 alcohols, such as potassium chloride, sodium chloride, calcium chloride, and the like.

The conductivity was adjusted to 40000. mu.s/cm, 50000. mu.s/cm, 60000. mu.s/cm, 70000. mu.s/cm, 80000. mu.s/cm.

For example, the amount of the salt insoluble in C1-C4 alcohol may be 20-32% by weight of the filtrate; it will be appreciated that in other embodiments of the present application, the amount of salt may be selected in an appropriate amount as guided by the conductivity.

And (4) standing until the system is layered after the conductivity is adjusted, and taking supernatant fluid.

The soybean isoflavone is mainly present in the supernatant (alcohol layer). The soybean protein and lecithin are dissolved in the lower layer; the additives may promote the dissolution of the protein and lecithin in the lower layer.

In some embodiments, drying the supernatant to remove the alcohol is also included.

The preparation method of the soybean extract provided by the application at least has the following advantages:

the method has the advantages of low cost, continuous operation and the like, and mild reaction conditions, and realizes substance purification and high-efficiency removal of protein and lecithin in the extraction preparation process by utilizing the principle of similar intermiscibility. The soybean is rich in protein, grease, lecithin and the like, solubilization is carried out through additives, and double aqueous phases are formed by utilizing the electrostatic action, hydrophobic action and biological affinity action of different substances, so that the dissolubility of different components is influenced, and the extraction rate and purity of the soybean isoflavone are effectively improved.

The application also provides a soybean extract, and the soybean extract is prepared by the preparation method of the soybean extract.

The soybean extract prepared by the method has high content of soybean isoflavone and low content of protein and lecithin.

The application also provides an application of the soybean extract in preparing an antiallergic drug or an anti-inflammatory drug.

The soybean extract has good antiallergic and antiinflammatory effects, and can be used for preparing antiallergic drugs or antiinflammatory drugs.

The features and properties of the present application are described in further detail below with reference to examples.

Example 1

This example provides a soy extract, made essentially by the steps of:

(1) 1kg of soybean germ was taken, 0.02kg of calcium sulfate was added, and the mixture was put into a three-roll machine, and 1.02kg of water was added to pulverize it, and the process was repeated three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 20 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 4 ℃, preserving heat for 10h, centrifuging at 10000g, and taking supernatant.

(3) Adding anhydrous ethanol 1 time the weight of the supernatant, adding cetyl PEG/PPG-10/1 polydimethylsiloxane 15% of the weight of soybean sprout, stirring at 4000rpm for 5min, adding potassium chloride 4.5 times the weight of soybean sprout, stirring to dissolve completely, and standing for 1.0 h. Fig. 1 shows the physical image after 1.0h of standing, and obvious delamination can be seen.

(4) Collecting the upper layer liquid (ethanol layer), and vacuum drying to obtain semen glycines extract.

Example 2

This example provides a soy extract, made essentially by the steps of:

(1) 1kg of soybean germ was taken, 0.02kg of calcium sulfate was added, and the mixture was put into a three-roll machine, and 1.02kg of water was added to pulverize it, and the process was repeated three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 10 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 4 ℃, preserving heat for 10h, centrifuging at 10000g, and taking supernatant.

(3) Adding anhydrous ethanol 2.0 times of the weight of the supernatant, adding 15% PEG-30 dipolyhydroxystearate of soybean sprout, stirring at 5000rpm for 5min, adding potassium chloride 5 times of the weight of soybean sprout, stirring to dissolve completely, and standing for 1.0 hr. Fig. 2 shows the physical image after 1.0h of standing, and obvious delamination can be seen.

(4) Collecting the upper layer liquid (ethanol layer), and vacuum drying to obtain semen glycines extract.

Example 3

This example provides a soy extract, made essentially by the steps of:

(1) 1kg of soybean germ was taken, 0.02kg of calcium sulfate was added, and the mixture was put into a three-roll machine, and 1.02kg of water was added to pulverize it, and the process was repeated three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 20 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 4 ℃, preserving heat for 10h, centrifuging at 10000g, and taking supernatant.

(3) Adding methanol 1 time the weight of the supernatant, adding cetyl PEG/PPG-10/1 polydimethylsiloxane 15% of the weight of soybean sprout, stirring at 4000rpm for 5min, adding potassium chloride 4.5 times the weight of soybean sprout, stirring to dissolve completely, and standing for 1.0 h. Fig. 3 shows the physical image after 1.0h of standing, and obvious delamination can be seen.

(4) Collecting the upper layer liquid (methanol layer), and vacuum drying to obtain soybean extract.

Comparative example 1

The present comparative example provides a soy extract, made essentially by the steps of:

(1) taking 1kg of soybean germ, putting into a three-roller machine, adding 1kg of water, crushing, and repeating for three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 15 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 4 ℃, preserving heat for 10h, centrifuging at 10000g, and removing supernatant.

(3) Adding anhydrous ethanol 2.0 times of the weight of the supernatant, adding cetyl PEG/PPG-10/1 polydimethylsiloxane or PEG-30 dipolyhydroxystearate 15% of the weight of the soybean germs, stirring at 5000rpm for 5min, adding potassium chloride 4 times of the weight of the soybean germs, stirring until the soybean germs are completely dissolved, and standing for 1.0 h. Fig. 4 shows the physical image after 1.0h of standing, and obvious delamination can be seen.

(4) Collecting the upper layer liquid (ethanol layer), and vacuum drying to obtain semen glycines extract.

Comparative example 2

The present comparative example provides a soy extract, made essentially by the steps of:

(1) 1kg of soybean germ was taken, 0.02kg of calcium sulfate was added, and the mixture was put into a three-roll machine, and 1.02kg of water was added to pulverize it, and the process was repeated three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 25 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 3 ℃, preserving heat for 10h, centrifuging at 10000g, and taking supernatant.

(3) Adding anhydrous ethanol with the weight twice that of the supernatant, stirring at 2000-5000rpm for 5min, and standing for 1.0 h. Fig. 5 shows the physical image after standing for 1.0h, and it can be seen that no delamination occurred.

(4) Filtering, and vacuum drying the filtrate to obtain semen glycines extract.

Comparative example 3

The present comparative example provides a soy extract, made essentially by the steps of:

(1) 1kg of soybean germ was taken, 0.02kg of calcium sulfate was added, and the mixture was put into a three-roll machine, and 1.02kg of water was added to pulverize it, and the process was repeated three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 15 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, centrifuging at 10000g, and taking the supernatant.

(3) Adding anhydrous ethanol twice the weight of the supernatant, adding 20% cetyl PEG/PPG-10/1 polydimethylsiloxane of soybean sprout weight, stirring at 5000rpm for 5min, adding potassium chloride 4 times the weight of soybean sprout, stirring to dissolve completely, and standing for 1.0 hr. Fig. 6 shows the physical image after 1.0h of standing, and obvious delamination can be seen.

(4) Collecting the upper layer liquid (ethanol layer), and vacuum drying to obtain semen glycines extract.

Comparative example 4

The present comparative example provides a soy extract, made essentially by the steps of:

(1) taking soybean germs, adding 0.02kg of calcium carbonate, putting into a three-roller machine, adding 1:1 water, crushing, and repeating for three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 25 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 3 ℃, preserving heat for 10h, centrifuging at 10000g, and taking supernatant.

(3) Adding anhydrous ethanol 1.0 times of the supernatant, adding 35% cetyl PEG/PPG-10/1 polydimethylsiloxane of soybean sprout weight, stirring at 5000rpm for 5min, adding potassium chloride 2.5 times of soybean sprout weight, and standing for 1.0 h. FIG. 7 shows the physical image after standing for 1.0h, and it can be seen that no delamination occurred.

(4) Filtering, and vacuum drying the filtrate to obtain semen glycines extract.

Comparative example 5

The present comparative example provides a soy extract, made essentially by the steps of:

(1) taking soybean germs, adding 0.02kg of calcium carbonate, putting into a three-roller machine, adding 1:1 water, crushing, and repeating for three times to form homogenate.

(2) Taking the homogenate obtained in the step (1), adding 20 times of water, heating to 60 ℃, stirring at 500rpm for 0.5h, cooling to 3 ℃, preserving heat for 10h, centrifuging at 10000g, and taking supernatant.

(3) Adding cetyl PEG/PPG-10/1 polydimethylsiloxane 20% of the weight of soybean sprout, stirring at 5000rpm for 5min, adding potassium chloride 4.5 times of the weight of soybean sprout, and standing for 1.0 h. FIG. 8 shows the physical image after standing for 1.0h, and it can be seen that no delamination occurred.

(4) Filtering, and vacuum drying the filtrate to obtain semen glycines extract.

Experimental example 1

The soybean extracts of examples 1 to 3 and comparative examples 1 to 5 were tested for protein content using the BCA protein concentration assay method. The principle of the testing method is as follows: the protein reduces divalent copper ions to monovalent copper ions under alkaline conditions, and then reacts with the BCA reagent to produce purple compounds, which are detected at 562 nm. The test results are shown in table 1.

Determination of protein content (%) (sample protein mass (g)/Soybean extract lyophilized powder mass (g)

TABLE 1

Group of	Protein content (%)
		Example 1	0.50
Example 2	0.49
		Example 3	0.42
Comparative example 1	1.25
		Comparative example 2	15.02
Comparative example 3	8.09
		Comparative example 4	3.50
Comparative example 5	12.0

Experimental example 2

The soybean extracts of examples 1 to 3 and comparative examples 1 to 5 were tested for the content of soybean isoflavones, according to the following test principle:

the soybean isoflavone has hydroxyl and aromatic ring functional groups in the structureThe method has the advantages that a conjugated system with strong ultraviolet absorption characteristics is formed, the maximum absorption wavelength difference of each component of the soybean isoflavone is small, the maximum absorption wavelength of the genistein in an ultraviolet region is 260nm, the maximum absorption wavelength of the daidzein is 256nm, and the content of the soybean isoflavone can be detected by adopting an ultraviolet spectrophotometry. Taking genistein as a standard substance, measuring the absorbance value at 260nm, wherein the standard curve is that y is 107.79x-0.0014, R²0.9999. The test results are shown in table 2.

TABLE 2

From experimental example 1, experimental example 2 and example 3, it can be seen that:

in comparison with example 1, no grinding aid was added in comparative example 1, resulting in a low extraction yield and a lower isoflavone content in the soybean extract.

In comparative example 2, no additive and salt were added and no separation was performed after standing, resulting in less separation of protein and soy isoflavone, and thus, the soy extract of comparative example 2 had a smaller proportion of soy isoflavone, a lower purity, and a larger proportion of protein.

In comparative example 3, after stirring at 60 ℃, the temperature was not maintained at a low temperature (1-4 ℃), and in comparative example 3, the ratio of isoflavone to protein was large and the two were not separated.

Comparative example 4 in which an additive was added in an amount of 35% by weight of soybean germ, the amount of the additive was too large, resulting in less separation of protein and soy isoflavone, less flavone content in the soybean extract, and more protein content.

In comparative example 5, no ethanol was added, resulting in failure to form two phases, resulting in low purity of flavone. The soybean extract has a small content of flavone and a large content of protein.

Experimental example 3

The hyaluronidase in vitro inhibition method tests the soybean extracts of examples 1 to 3 and comparative examples 1 to 5 for their great antiallergic effects.

The test method comprises the following steps: the soybean extracts of examples 1 to 3 and comparative examples 1 to 5 were dissolved in water to a concentration of 5 mg/mL;

0.5mL of sample, 0.5mL of 1200U/mL of hyaluronidase and 0.1mL of CaCl₂Mixing the solutions, keeping the temperature for 20min, adding 0.5mL sodium hyaluronate, and keeping the temperature at 37 ℃ for 40 min; adding 0.1mL of sodium hydroxide and 0.5mL of acetylacetone, boiling in a water bath for 15min, carrying out ice bath for 5min, and standing at room temperature for 10 min; 1.0mLP-DAB, adding 3mL of absolute ethyl alcohol, mixing uniformly, and measuring the light absorption value at 530 nm.

The results of the hyaluronidase inhibition ratios of the soybean extracts of examples 1 to 3 and comparative examples 1 to 5 are shown in fig. 8.

Hyaluronidase is a participant in allergic reactions, which breaks down Hyaluronic Acid (HA) in the body, turning it into an acidic stimulus of low molecular weight, causing histamine release, inducing the body to produce allergic symptoms. As can be seen from fig. 9, the soybean extracts provided in examples 1 to 3 had excellent hyaluronidase inhibitory effects.

Experimental example 4

Lipoxygenase inhibition experiments the anti-inflammatory effects of the soybean extracts of examples 1-3, comparative examples 1-5 were tested.

Lipoxygenase (LOX) is a non-heme iron-containing oxidoreductase which specifically catalyzes the dioxygenation reaction of polyunsaturated fatty acids with cis-1, 4-pentadiene structure such as arachidonic acid and linoleic acid to generate conjugated unsaturated fatty acid hydroperoxides such as Leukotrienes (LTs) and hydroxytetracosatetraenoic acid (X-HETE). Therefore, the anti-inflammatory activity of a substance can be reflected to a certain extent by detecting the inhibition rate of the substance on lipoxygenase.

The test method comprises the following steps: the soybean extracts of examples 1 to 3 and comparative examples 1 to 5 were dissolved in water to a concentration of 5 mg/mL. Taking 0.1mL of sample extracting solution and 0.4mL of lipoxygenase 260u/mL, uniformly mixing in a 10mL test tube, carrying out water bath at 30 ℃ for 3min, adding 0.54mL/L of linoleic acid 1.6mL, uniformly mixing in a water bath at 30 ℃ for 3min, then adding 4mL of absolute ethyl alcohol, and detecting the lipoxygenase inhibition rate at 234 nm.

The results of the inhibition ratios of lipoxygenase by the soybean extracts of examples 1 to 3 and comparative examples 1 to 5 are shown in FIG. 10.

As can be seen from fig. 10, the soybean extracts provided in example 1, example 2 and example 3 exhibited a lipoxygenase inhibitory effect of 95.2%, which is far superior to that of the soybean extracts of comparative examples 1 to 5.

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.