阅读说明：本技术 一种基于深度共熔溶剂从大豆豆脐中提取大豆异黄酮的方法 (Method for extracting soybean isoflavone from soybean navel based on deep eutectic solvent ) 是由 段章群 郭咪咪 李秀娟 杨茜 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种基于深度共熔溶剂从大豆豆脐中提取大豆异黄酮的方法。该方法包括以下步骤：(1)将含水的深度共熔溶剂与脱脂大豆豆脐粉末混合,进行超声处理；(2)用恒温气浴摇床加热进行提取；(3)过滤或离心,得到含大豆异黄酮的提取液；其中,所述深度共熔溶剂为氯化胆碱与氢键供体组成的共熔混合物；所述氯化胆碱与氢键供体的摩尔比为2:1～1:5；所述深度共熔溶剂的含水量为10％～90％(v/v)。本发明的方法具有操作工艺简单、条件温和、环境友好等优点。(The invention discloses a method for extracting soybean isoflavone from soybean hilum based on a deep eutectic solvent. The method comprises the following steps: (1) mixing a water-containing deep eutectic solvent with the defatted soybean navel powder, and carrying out ultrasonic treatment; (2) heating with constant temperature gas bath shaking table for extraction; (3) filtering or centrifuging to obtain extractive solution containing soybean isoflavone; wherein the deep eutectic solvent is a eutectic mixture consisting of choline chloride and a hydrogen bond donor; the mol ratio of the choline chloride to the hydrogen bond donor is 2: 1-1: 5; the water content of the deep eutectic solvent is 10-90% (v/v). The method has the advantages of simple operation process, mild condition, environmental friendliness and the like.)

1. A method for extracting soybean isoflavone from soybean hilum based on deep eutectic solvent is characterized by comprising the following steps:

(1) mixing a water-containing deep eutectic solvent with the defatted soybean navel powder, and carrying out ultrasonic treatment;

(2) heating with constant temperature gas bath shaking table for extraction;

(3) filtering or centrifuging to obtain extractive solution containing soybean isoflavone;

wherein the deep eutectic solvent is a eutectic mixture consisting of choline chloride and a hydrogen bond donor; the mol ratio of the choline chloride to the hydrogen bond donor is 2: 1-1: 5; the water content of the deep eutectic solvent is 10-90% (v/v).

2. The method according to claim 1, wherein the liquid-to-solid ratio of the deep eutectic solvent to the defatted soybean hilum powder is 20 to 100 mL/g.

3. The method according to claim 1, wherein the defatted soybean hilum powder is prepared by pulverizing soybean hilum, sieving with a 40-mesh sieve, and defatting with petroleum ether.

4. The method of claim 3, wherein the degreasing conditions are: weighing 50-100 g of crushed and sieved soybean hilum, adding 200-1000 mL of petroleum ether, performing ultrasonic treatment for 10-50 min, and centrifuging; adding 200-800 mL of petroleum ether into the precipitate, and repeating the steps for 2-3 times to obtain defatted soybean hilum powder; preferably, the soybean hilum is dried in vacuum for 16-24 hours at 30-60 ℃ and then crushed.

5. The method of claim 1, wherein the hydrogen bond donor is one or more of ethylene glycol, propylene glycol, glycerol, butylene glycol, hexylene glycol, malonic acid, tartaric acid, and citric acid.

6. The method according to claim 1, wherein in the step (2), the extraction conditions are that the rotating speed of a shaking table is 100-200 rpm, the temperature is controlled at 40-80 ℃, and the extraction time is 1-6 h.

7. The method of claim 1, further comprising: and (4) separating and purifying the extracting solution containing the soybean isoflavone obtained in the step (3) to prepare refined soybean isoflavone.

8. The method of claim 7, wherein the separation and purification is column chromatography.

9. The method as claimed in claim 8, wherein the filler used for column chromatography is macroporous adsorption resin, the sample loading amount is 5mL, the eluent is 40-80% ethanol water solution, and the elution flow rate is 2-5 mL/min.

10. The method of claim 9, wherein the macroporous adsorbent resin is model PS5, model D101, model AB-8, or model X-5.

Technical Field

The invention relates to the technical field of active substance extraction. More particularly, relates to a method for extracting soybean isoflavone from soybean hilum based on deep eutectic solvent.

Background

The soybean isoflavone is an important physiological active substance, has weak estrogen activity, antioxidant activity, anti-hemolytic activity and antifungal activity, can effectively prevent and inhibit the occurrence of leukemia, hyperosteogeny, colon cancer, gastric cancer, breast cancer, prostatic cancer and other diseases, and particularly has obvious effect on preventing and improving the breast cancer and the prostatic cancer. The soybean contains isoflavone 0.1-0.5% and mainly distributed in cotyledon and navel, wherein isoflavone content in cotyledon is about 0.1-0.3% and isoflavone content in navel is about 1-2%. Therefore, soy bean umbilicus is a better raw material for extracting isoflavone.

The prior extraction process of soybean isoflavone mainly adopts an ultrasonic or microwave-assisted solvent extraction method, but the methods have the defects of easy volatilization of extraction solvent, easy environmental pollution, high production cost and the like.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the present invention aims to provide a method for extracting soybean isoflavone from soybean umbilicus based on Deep Eutectic Solvents (DESS). The method has the advantages of simple operation process, mild condition, environmental protection and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for extracting soybean isoflavone from soybean hilum based on deep eutectic solvent, which comprises the following steps:

(1) mixing a water-containing deep eutectic solvent with the defatted soybean navel powder, and carrying out ultrasonic treatment;

(2) heating with constant temperature gas bath shaking table for extraction;

(3) filtering or centrifuging to obtain extractive solution containing soybean isoflavone;

wherein the deep eutectic solvent is a eutectic mixture consisting of choline chloride and a hydrogen bond donor; the molar ratio of the choline chloride to the hydrogen bond donor is 2: 1-1: 5, such as 2:1, 1:2, 1:3, 1:4, 1:5, and the like; the deep eutectic solvent has a water content of 10% to 90% (v/v), e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, etc., or any range between these volume fractions.

In the method, the soybean isoflavone serving as a target product is fully released due to the violent damage of Deep Eutectic Solvents (DESs) to the cell walls of the soybean hilum, and the extraction rate can reach over 90 percent.

Optionally, the liquid-solid ratio of the deep eutectic solvent to the defatted soybean navel powder is 20-100 mL/g. E.g., 40mL/g, 60mL/g, 80mL/g, etc., or any range between these ratios.

Optionally, the defatted soybean hilum powder is used in the invention, and the defatting can not only avoid emulsification in the soybean isoflavone extraction process, but also obtain the soybean hilum grease with high nutritive value, and a conventional defatting method in the field can be selected for defatting the soybean hilum. According to a specific embodiment of the present invention, the defatted soybean hilum powder is prepared by pulverizing soybean hilum, sieving with a 40-mesh sieve, and then defatting with petroleum ether.

Further, the degreasing conditions may be: weighing 50-100 g of crushed and sieved soybean hilum, adding 200-1000 mL of petroleum ether, performing ultrasonic treatment for 10-50 min, and centrifuging; adding 200-800 mL of petroleum ether into the precipitate, and repeating the steps for 2-3 times to obtain defatted soybean hilum powder; preferably, the soybean hilum is dried in vacuum for 16-24 hours at 30-60 ℃ and then crushed.

In the method, the hydrogen bond donor is one or more of ethylene glycol, propylene glycol, glycerol, butanediol, hexanediol, malonic acid, tartaric acid and citric acid. Preferably, the hydrogen bond donor is malonic acid.

Optionally, in the step (2), the extraction condition is that the rotation speed of a shaking table is 200rpm, the temperature is controlled to be 40-80 ℃, and the extraction time is 1-6 h. Because the extraction of the soybean isoflavone is a solid-liquid heterogeneous system, compared with the conventional ultrasonic and microwave treatment, the shaking table can ensure that solid-liquid substances in the extraction process are in a dynamic uniform distribution state, promote the solid-liquid substances to be better contacted, further improve the mass transfer efficiency, and further be beneficial to improving the extraction rate of the soybean isoflavone.

The above method further comprises: and (4) separating and purifying the extracting solution containing the soybean isoflavone obtained in the step (3) to prepare refined soybean isoflavone.

Optionally, the separation and purification is column chromatography purification.

Optionally, the filler used for column chromatography is macroporous adsorption resin, the sample loading amount is 5mL, the eluent is 40% -80% ethanol water solution, and the elution flow rate is 2-5 mL/min.

Optionally, the model of the macroporous adsorption resin is PS5 model, D101 model, AB-8 model or X-5 model.

The invention has the following beneficial effects:

(1) the deep eutectic solvent used in the invention is simple to prepare, has the advantages of no toxicity, difficult volatilization, high thermal stability, biodegradability and the like, is a novel green solvent, and can reduce the environmental pollution and the personnel injury caused by the traditional extraction solvent to a certain extent.

(2) The method has the advantages of simple operation process, mild condition, environmental protection and the like.

Drawings

FIG. 1 shows the HPLC chromatogram of the soybean isoflavone prepared in example 1.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following embodiments further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments.

Example 1

(1) Preparing a deep eutectic solvent: drying choline chloride (with purity of 99%) in a vacuum drying oven at 50 ℃ for 48 hours, mixing the choline chloride with malonic acid according to a molar ratio of 1:4, placing the mixture in a 500mL reagent bottle with magnetons in advance, plugging the reagent bottle with a plug, sealing the reagent bottle with a sealing film, placing the reagent bottle in an oil bath pan at 150 ℃, and stirring and heating the reagent bottle at a rotating speed of 500rpm for about 2-4 hours until a stable, uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 70 deg.C for 48h, transferring to a dryer, and sealing for storage.

(2) Pretreating the soybean hilum: drying the soybean hilum in a vacuum drying oven at 40 deg.C for 24h, pulverizing, sieving with 40 mesh sieve, and transferring to a dryer for storage. Weighing 50g of soybean umbilicus fine powder, adding 400mL of petroleum ether, performing ultrasonic treatment for 20min, and centrifuging; adding 300mL petroleum ether into the precipitate, and repeating the above steps for 2 times to obtain defatted soybean navel powder.

(3) Extracting soybean isoflavone: adding 50% (v/v) of water into the deep eutectic solvent prepared in the step (1), and stirring to make the mixture uniform. Weighing the defatted soybean navel powder, adding the defatted soybean navel powder into a water-containing deep eutectic solvent according to the liquid-solid ratio of 80mL/g, uniformly stirring, covering a plug, and sealing with a sealing film. Performing ultrasonic treatment for 10min, transferring to a constant temperature gas bath shaking table at 50 deg.C, extracting for 1h, and rotating the shaking table at 200 rpm. After extraction, the extracting solution is centrifuged for 15min at 4000r/min, and the supernatant is diluted by 10% methanol and then is analyzed by HPLC, under the condition, the extraction rate of the soybean isoflavone is 93.3%. The HPLC profile is shown in FIG. 1.

(4) And (3) soybean isoflavone purification: and (4) filtering the supernatant obtained in the step (3) by using a 0.45-micron filter membrane, taking 5mL of the supernatant each time, loading the supernatant into a chromatographic column filled with macroporous adsorption resin, washing the chromatographic column by using deionized water at the flow rate of 2 mL/min. Eluting with 80% ethanol water solution at flow rate of 2mL/min, collecting eluate at elution peak position, and washing for 60 min. And (3) carrying out negative pressure rotary evaporation concentration on the collected ethanol water eluent at 40 ℃, and carrying out vacuum freeze drying on the concentrated solution to obtain the refined soybean isoflavone.

Example 2

(1) Preparing a deep eutectic solvent: drying choline chloride (with purity of 99%) in a vacuum drying oven at 50 ℃ for 48 hours, mixing the choline chloride with propylene glycol according to a molar ratio of 1:5, placing the mixture in a 500mL reagent bottle with magnetons in advance, plugging the reagent bottle with a plug, sealing the reagent bottle with a sealing film, placing the reagent bottle in an oil bath pan at 150 ℃, and stirring and heating the reagent bottle at a rotating speed of 500rpm for about 2-4 hours until a stable, uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 70 deg.C for 48h, transferring to a dryer, and sealing for storage.

(2) Pretreating the soybean hilum: the same as in example 1.

(3) Extracting soybean isoflavone: the same as in example 1. The extraction rate of soybean isoflavone is 90.6%.

Example 3

(1) Preparing a deep eutectic solvent: drying choline chloride (with purity of 99%) in a vacuum drying oven at 50 ℃ for 48 hours, mixing the choline chloride with hexanediol according to a molar ratio of 1:3, placing the mixture in a 500mL reagent bottle with a magneton in advance, plugging the reagent bottle with a plug, sealing the reagent bottle with a sealing film, placing the reagent bottle in an oil bath pan at 150 ℃, and stirring and heating the reagent bottle at the rotating speed of 500rpm for about 2-4 hours until a stable, uniform and transparent solution is formed. Cooling to room temperature, drying in a vacuum drying oven at 70 deg.C for 48h, transferring to a dryer, and sealing for storage.

(2) Pretreating the soybean hilum: the same as in example 1.

(3) Extracting soybean isoflavone: the same as in example 1. The extraction rate of soybean isoflavone was 92.4%.

Comparative example 1

(1) Preparing a deep eutectic solvent: the same as in example 1.

(2) Pretreating the soybean hilum: the same as in example 1.

(3) Extracting soybean isoflavone: adding 50% (v/v) of water into the deep eutectic solvent prepared in the step (1), and stirring to make the mixture uniform. Weighing the defatted soybean navel powder, adding the defatted soybean navel powder into a water-containing deep eutectic solvent according to the liquid-solid ratio of 80mL/g, uniformly stirring, covering a plug, and sealing with a sealing film. Ultrasonic extracting for 1 hr, centrifuging the extractive solution at 4000r/min for 15min, diluting the supernatant with 10% methanol, and performing HPLC analysis, wherein the extraction rate of soybean isoflavone is 53.3%.

Comparative example 2

(1) Preparing a deep eutectic solvent: the same as in example 1.

(2) Pretreating the soybean hilum: the same as in example 1.

(3) Extracting soybean isoflavone: adding 30% (v/v) of water into the deep eutectic solvent prepared in the step (1), and stirring to make the mixture uniform. Weighing the defatted soybean navel powder, adding the defatted soybean navel powder into a water-containing deep eutectic solvent according to the liquid-solid ratio of 60mL/g, uniformly stirring, covering a plug, and sealing with a sealing film. After microwave extraction for 1h, the extract is centrifuged for 15min at 4000r/min, and the supernatant is diluted with 10% methanol for HPLC analysis, under which the extraction rate of soybean isoflavone is 62.1%.

Comparative example 3

(1) Preparing a deep eutectic solvent: drying choline chloride (purity of 99%) in a vacuum drying oven at 50 ℃ for 48h, mixing with pentanediol according to a molar ratio of 1:5, placing in a 500mL reagent bottle containing magnetons in advance, plugging the stopper, sealing with a sealing film, placing in an oil bath pan at 150 ℃, stirring and heating at a rotating speed of 500rpm to form a stable, uniform and transparent solution. Cooling to room temperature, drying in a vacuum drying oven at 70 deg.C for 48h, transferring to a dryer, and sealing for storage.

Comparative example 4

(1) Preparing a deep eutectic solvent: the same as in example 1.

(2) Pretreating the soybean hilum: the same as in example 1.

(3) Extracting soybean isoflavone: and (2) adding 5% (v/v) of water into the deep eutectic solvent prepared in the step (1), and stirring to make the mixture uniform. Weighing the defatted soybean navel powder, adding the defatted soybean navel powder into a water-containing deep eutectic solvent according to the liquid-solid ratio of 80mL/g, uniformly stirring, covering a plug, and sealing with a sealing film. Performing ultrasonic treatment for 10min, transferring to a constant temperature gas bath shaking table at 50 deg.C, extracting for 1h, and rotating the shaking table at 200 rpm. After extraction, the extracting solution is centrifuged for 15min at 4000r/min, and the supernatant is diluted by 10% methanol and then is analyzed by HPLC, under the condition, the extraction rate of the soybean isoflavone is 21.6%.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.