Method for optimizing ultrasound-assisted compound enzyme enrichment daidzein based on response surface method

Abstract

The invention provides a method for optimizing ultrasound-assisted compound enzyme enrichment daidzein based on a response surface method. The method comprises the following steps: dissolving soy isoflavone in a phosphate-citric acid buffer solution; the method comprises the following steps: carrying out enzymolysis on soy isoflavone by using ultrasonic-assisted compound enzyme, after the reaction is finished, inactivating the enzyme in a boiling water bath, cooling to room temperature, extracting, separating by using macroporous adsorption resin, eluting by using water and ethanol, concentrating the eluent by using a rotary evaporator, and carrying out vacuum freeze drying to obtain daidzein powder. According to the method, daidzein is enriched by adopting an ultrasonic-assisted enzymolysis method, and soy isoflavone enzymolysis is subjected to process optimization by combining a single-factor experiment with a response surface test. The method provided by the invention realizes higher resource utilization rate and lower environmental pollution risk, and conforms to the concepts of green chemistry and sustainable development; the defect that in the prior art, damage to daidzein is large is overcome, and the enzymolysis rate of soy isoflavone and the purity of daidzein can be remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of daidzein enrichment and extraction technology, specifically to a method for enriching daidzein using ultrasound-assisted complex enzymes based on response surface methodology. Background Technology

[0002] Soy isoflavones are polyphenolic secondary metabolites formed during the growth of soybeans and other legumes. Soybean seeds are the richest source of isoflavones, accounting for about 0.1%-0.5% of soybeans. They are mainly distributed in the seed coat, cotyledons, and hypocotyl. After ingestion, they are eventually metabolized into equol by intestinal flora. Because it can bind to estrogen receptors, it is called "phytoestrogen" and has a variety of biological activities such as anti-oxidation, cancer prevention, immune enhancement, and estrogen-like effects. It has been widely used in food, medicine, and feed research and development.

[0003] Currently, the structure of the parent nucleus of soybean isoflavones has been found to be 3-benzopyranone. There are 12 natural forms of soybean isoflavones, which are mainly divided into two categories based on the differences in their molecular structures: free aglycones and bound glycosides. Free aglycones account for 2%-3% of the total content in soybean seeds, while bound glycosides account for 97%-98%. Free aglycones include diadzein, genistein, and glycitein. Bound glycosides include three types: glucoside, acetylglucosinolate, and malonyl glucoside. Each type has three ligands: genistin, diadzin, and glycitin. The types and functional groups of aglycone and glycoside isoflavones are shown in Table 1.

[0004] Table 1. Types and functional groups of soybean isoflavones

[0005]

[0006] Most isoflavones in soybeans exist in the form of glycosides, with only a few aglycone forms. Free aglycones exhibit significantly higher antioxidant and estrogen-like activities than bound glycosides and are more readily absorbed by the human body. Therefore, to enhance the bioactivity of soybean isoflavones, glycoside-type isoflavones need to be hydrolyzed. Currently, the main methods for hydrolyzing soybean isoflavones include acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis, and microbial fermentation. Hydrochloric acid is typically used as a catalyst in acid hydrolysis of glycoside-type isoflavones. From a practical production perspective, it is generally preferred to use 1-3 mol / L hydrochloric acid at a relatively high temperature (98-100℃) to hydrolyze the glycosides. However, strong acids can affect the stability of the product during hydrolysis. The glycosidic bonds of soybean isoflavones possess both acetal and ester structures; the ester structure can be hydrolyzed in alkaline solutions. In alkaline hydrolysis, the conversion of malonyl glucoside to acetyl glucoside and then to glucoside is relatively easy, while the conversion of glucoside to aglycone is more difficult. Therefore, alkaline hydrolysis of glycoside-type soy isoflavones is inefficient, resulting in poor product stability and easy degradation. Enzymatic hydrolysis uses enzymes such as β-glucosidase to hydrolyze the glycosidic bonds of glycoside-type soy isoflavones, generating soy isoflavone aglycones and glucose. Compared with the previous two methods, enzymatic hydrolysis has milder reaction conditions. Due to the specificity and high efficiency of enzymes, the aglycone conversion rate is high, and the structure of soy aglycones is not destroyed, resulting in good product stability. Microorganisms can produce enzymes that catalyze the hydrolysis of soy isoflavones during fermentation. These enzymes can hydrolyze glycosidic bonds to generate aglycone-type isoflavones. Microbial fermentation offers mild production conditions, low cost, and a simple process. The converted aglycones have high activity and are not easily denatured, effectively improving the bioavailability of soy isoflavones. Summary of the Invention

[0007] The purpose of this application is to provide a response surface methodology-optimized method for the enrichment of daidzein from soybean isoflavones via ultrasound-assisted enzymatic hydrolysis, in order to address the problems existing in the prior art.

[0008] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0009] A method for optimizing ultrasound-assisted enzyme enrichment of daidzein based on response surface methodology, the method comprising:

[0010] S1. Accurately weigh 50 mg of soy isoflavones (Shanghai Maclean Biochemical Technology Co., Ltd.) and add it to 50 mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer solution (Beijing Solarbio Biotechnology Co., Ltd.);

[0011] S2. Add different mass fractions of compound enzymes (β-glucosidase: 10000U / g, Shanghai Yuanye Biotechnology Co., Ltd.; and cellulase: 100U / g, Aladdin Reagent Co., Ltd.) to the above solution in a certain proportion, sonicate the solution (LC-JY92-IIN, China Lichen Technology Co., Ltd.), and then carry out enzymatic hydrolysis at 50℃ to obtain the enzymatic hydrolysate.

[0012] S3. The optimal parameters of the enzymatic hydrolysis process were determined by optimizing the amount of compound enzyme, ultrasonic power, and reaction time using response surface methodology.

[0013] S4. After the reaction is complete, inactivate the enzyme by boiling water bath (DZKW-C, Shanghai Shuli Instrument Co., Ltd.) and cool to room temperature.

[0014] S5. Extract the enzymatically hydrolyzed solution with ethanol (Tianjin Tianli Chemical Reagent Co., Ltd.), take the upper organic phase, remove the ethanol using a rotary evaporator (R206, Shanghai Shensheng Technology Co., Ltd.), and separate it with the pre-treated macroporous adsorption resin (AD-8, Zhengzhou Hecheng New Material Technology Co., Ltd.). First, rinse with water until the eluent is colorless and clear, then continue to elute with ethanol solution and collect the eluent.

[0015] S6. Concentrate using a rotary evaporator and freeze-dry under vacuum (LGJ-10N / A, Beijing Yaxing Instrument Technology Development Co., Ltd.) to obtain daidzein powder.

[0016] Preferably, in step S2, the amount of compound enzyme added is 3%-15%.

[0017] Preferably, in step S2, the ratio of β-glucosidase to cellulase in the complex enzyme is 2:1.

[0018] Preferably, in step S2, the ultrasonic power is 120-360W.

[0019] Preferably, in step S2, the reaction time is 15-55 min.

[0020] Preferably, in step S4, the enzyme inactivation time is 5 minutes.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention uses the amount of compound enzyme added, reaction time, and ultrasonic power as independent variables in the process of ultrasonic-assisted compound enzyme enrichment of daidzein, with the enzymatic hydrolysis rate of soybean isoflavones as the response value. Based on the Box-Behnken response surface methodology, a predictive model of a quadratic polynomial regression equation for the enzymatic hydrolysis rate of soybean isoflavones was simulated, optimizing the process conditions for ultrasonic-assisted compound enzyme hydrolysis enrichment of daidzein and obtaining a green, simple, and efficient extraction method. By studying the technology of ultrasonic-assisted compound enzyme hydrolysis enrichment of daidzein, its application scope and market potential in the fields of medicine, food, and cosmetics can be expanded. In summary, the method provided in this application is simple to operate, operates under mild conditions, has high extraction efficiency, minimizes the loss of daidzein activity, reduces the use of chemical reagents, is environmentally friendly and economical, and produces products with high purity and safety. Attached Figure Description

[0023] The accompanying drawings are provided to further illustrate the present application and form part of the specification. They are used together with the embodiments of the present application to explain the application and do not constitute a limitation thereof. In the drawings:

[0024] Appendix Figure 1 The effect of the amount of compound enzyme added on the enzymatic hydrolysis rate of soybean isoflavones.

[0025] Appendix Figure 2 The effect of ultrasonic power on the enzymatic hydrolysis rate of soybean isoflavones.

[0026] Appendix Figure 3 The effect of reaction time on the enzymatic hydrolysis rate of soybean isoflavones.

[0027] Appendix Figure 4 The response surface plot shows the interaction between the amount of compound enzyme added and the ultrasonic power on the enzymatic hydrolysis rate of soybean isoflavones.

[0028] Appendix Figure 5 The response surface plot shows the interaction between the amount of compound enzyme added and the reaction time on the enzymatic hydrolysis rate of soybean isoflavones.

[0029] Appendix Figure 6 The response surface plot shows the interaction effect of ultrasonic power and reaction time on the enzymatic hydrolysis rate of soybean isoflavones.

[0030] Appendix Figure 7 This is a chromatogram of soybean isoflavones after enzymatic hydrolysis. Detailed Implementation

[0031] The present invention will be further explained and described below with reference to specific embodiments.

[0032] Example 1: Enrichment of daidzein by ultrasound-assisted complex enzyme hydrolysis

[0033] A method for the ultrasonic-assisted enzymatic hydrolysis and enrichment of daidzein, comprising the following steps:

[0034] Step S1: Accurately weigh 50 mg of soy isoflavones and add them to 50 mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer solution.

[0035] Step S2: Add different mass fractions of compound enzymes (β-glucosidase and cellulase) to the above solution at a ratio of 2:1 of β-glucosidase and cellulase. After sonicating the solution, carry out the enzymatic hydrolysis reaction at 50°C to obtain the enzymatic hydrolysate.

[0036] Optionally, the amount of compound enzyme added is 3%-15%, the ultrasonic power is 120-360W, and the enzymatic hydrolysis reaction time is 15-55min.

[0037] Step S3: Use response surface methodology to optimize the amount of compound enzyme added, ultrasonic power, and reaction time to determine the optimal parameters of the enzymatic hydrolysis process.

[0038] Step S4: After the reaction is complete, inactivate the enzyme by boiling in a water bath for 5 minutes, and then cool to room temperature.

[0039] Step S5: Extract the enzymatically hydrolyzed solution with ethanol, collect the upper organic phase, remove the ethanol using a rotary evaporator, and separate it using the pre-treated macroporous adsorption resin. First, rinse with water until the eluent is colorless and clear, then continue eluting with ethanol solution and collect the eluent.

[0040] Step S6: Concentrate using a rotary evaporator and freeze-dry under vacuum to obtain daidzein powder.

[0041] Determination of the purity of daidzein:

[0042] Accurately weigh 0.1 g of daidzein powder, dissolve and dilute it in 80% methanol, filter it through a 0.45 μm filter membrane, and analyze the purity of the prepared daidzein using a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity, Agilent Technologies, USA). A C18 column (Agilent Technologies, USA) was used, with an injection volume of 10 μL, a column temperature of 40℃, and a detection wavelength of 260 nm. The mobile phase was methanol (Tianjin Kemei Chemical Reagent Co., Ltd.) eluted at a gradient rate of 1.0 mL / min. The gradient elution order is shown in Table 2. The calculation formula is:

[0043]

[0044] P is purity, %; C is the mass concentration of daidzein, μg / mL; V is the volume after dilution, mL; m is the mass of daidzein powder, mg.

[0045] Table 2 Gradient Elution

[0046]

[0047] The enzymatic hydrolysis rate of soybean isoflavones was determined by three factors: the amount of compound enzyme added, the reaction time, and the ultrasonic power in step S2.

[0048] (1) With the ratio of β-glucosidase to cellulase fixed at 2:1 and pH at 5, compound enzymes (β-glucosidase and cellulase) were added at mass fractions of 3%, 6%, 9%, 12%, and 15%, respectively. The ultrasonic power was 240W, the reaction time was 35 min, and the reaction temperature was 50℃. After the reaction, the enzyme activity was inactivated by boiling in a water bath for 5 min, and then cooled to room temperature. The effect of the amount of compound enzyme added on the enzymatic hydrolysis rate of soybean isoflavones is shown in the figure below. Figure 1 As shown;

[0049] Depend on Figure 1 It is observed that the enzymatic hydrolysis rate continuously increases with the increase of the amount of compound enzyme added. This is because, initially, the substrate concentration is high, significantly exceeding the concentration of the compound enzyme. Furthermore, with the increase of the compound enzyme, the contact opportunities between the enzyme and the substrate increase. At this point, the decisive factor affecting the conversion of soybean isoflavone glycosides is the enzyme concentration, and the concentration of the compound enzyme is positively correlated with the reaction rate. When the amount of compound enzyme added reaches 9%, the enzymatic hydrolysis rate is the highest, at 84.79%. As the amount of compound enzyme added continues to increase, the change in the enzymatic hydrolysis rate tends to stabilize. This is because the glycoside-type soybean isoflavones available for enzymatic hydrolysis have already fully reacted with the compound enzyme to generate aglycone-type soybean isoflavones, increasing the product concentration. At this point, further increasing the amount of compound enzyme has little effect on the enzymatic hydrolysis of soybean isoflavone glycosides. Adding excessive amounts of compound enzyme increases costs; therefore, to achieve the enzymatic hydrolysis effect more economically, the amount of compound enzyme added is set to 9%.

[0050] (2) The amount of the compound enzyme added was fixed at 9%, the ratio of β-glucosidase to cellulase was 2:1, the pH was 5, and the ultrasonic power was selected as 120W, 180W, 240W, 300W, and 360W, the reaction time was 35 min, and the reaction temperature was 50℃. After the reaction, the enzyme activity was inactivated by boiling in a water bath for 5 min, and then cooled to room temperature. The effect of the ultrasonic power on the enzymatic hydrolysis rate of soybean isoflavones is shown in the figure below. Figure 2 As shown.

[0051] Depend on Figure 2It is observed that the enzymatic hydrolysis rate increases with increasing ultrasonic power, reaching a maximum of 84.79% at an ultrasonic power of 240W. This is because ultrasound activates more enzyme active sites, resulting in a stronger binding between glycoside-type soy isoflavones and the enzyme. The cavitation effect generated by ultrasound causes microbubbles in the liquid to continuously expand and collapse. This cavitation effect not only increases the surface area for mass transfer but also accelerates the mass transfer rate, providing extremely favorable conditions for enzyme-substrate interaction. With further increases in ultrasonic power, the enzymatic hydrolysis rate decreases, possibly because higher ultrasonic power disrupts the enzyme structure, leading to a lower hydrolysis rate. Therefore, to obtain the optimal enzymatic hydrolysis effect, the ultrasonic power should be 240W.

[0052] (3) With a fixed addition amount of 9% of the compound enzyme, a β-glucosidase to cellulase ratio of 2:1, a pH of 5, a reaction temperature of 50℃, and an ultrasonic power of 240W, reaction times of 15 min, 25 min, 35 min, 45 min, and 55 min were selected. After the reaction, the enzyme activity was inactivated by boiling in a water bath for 5 min, and then cooled to room temperature. The effect of ultrasonic power on the enzymatic hydrolysis rate of soybean isoflavones is shown in the figure below. Figure 3 As shown.

[0053] Depend on Figure 3 It can be seen that within the reaction time range of 15-35 min, the enzymatic hydrolysis rate gradually increases, reaching its maximum at 35 min, after which the enzymatic hydrolysis rate curve tends to flatten. This is because in the initial stage of the reaction, the interaction time between the complex enzyme and the substrate is relatively short, at which point the complex enzyme cannot effectively hydrolyze glycoside-type soy isoflavones. With the increase of reaction time and under the action of ultrasound, the enzymatic hydrolysis reaction proceeds in the forward direction, and the enzymatic hydrolysis rate increases rapidly. When the reaction time exceeds 35 min, the complex enzyme and glycoside-type soy isoflavones have fully reacted, the product concentration is high, the reaction reaches equilibrium, and excessively high product concentration has a certain inhibitory effect on the enzymatic hydrolysis reaction, and the enzymatic hydrolysis rate gradually decreases. Therefore, to obtain the optimal enzymatic hydrolysis effect, a reaction time of 35 min is selected.

[0054] Example 2: Response surface methodology optimization of ultrasound-assisted complex enzyme hydrolysis for enrichment of daidzein

[0055] Step S1: Accurately weigh 50 mg of soy isoflavones and add them to 50 mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer solution;

[0056] Step S2: Add different mass fractions of compound enzymes (β-glucosidase and cellulase) to the above solution at a ratio of 2:1 of β-glucosidase and cellulase. After sonicating the solution, carry out the enzymatic hydrolysis reaction at 50°C to obtain the enzymatic hydrolysate.

[0057] Step S3: Using Design Expert software, a Box-Behnken model was established to evaluate the effects of the amount of compound enzyme added (A, 3%-15%), ultrasonic power (B, 120-360W), and reaction time (C, 15-55min) on the enzymatic hydrolysis rate of soybean isoflavones. The optimal parameters of the enzymatic hydrolysis process were determined with the hydrolysis rate as the response variable. The obtained data were analyzed using a quadratic polynomial regression model.

[0058] Step S4: After the reaction is complete, inactivate the enzyme by boiling in a water bath for 5 minutes, and then cool to room temperature.

[0059] Step S5: Extract the enzymatically hydrolyzed solution with ethanol, collect the upper organic phase, remove the ethanol using a rotary evaporator, and separate it using the pre-treated macroporous adsorption resin. First, rinse with water until the eluent is colorless and clear, then continue eluting with ethanol solution and collect the eluent;

[0060] Step S6: Concentrate using a rotary evaporator and freeze-dry under vacuum to obtain daidzein powder.

[0061] In this specific embodiment, response surface methodology was used to optimize the process conditions for enriching daidzein using ultrasound-assisted compound enzyme hydrolysis. The effects of compound enzyme dosage (A), ultrasonic power (B), and ultrasonic time (C) on the enzymatic hydrolysis rate of soybean isoflavones were investigated. Each variable had three different levels, and a total of 17 experiments were conducted. The experimental design and results are shown in Table 3 below:

[0062] Table 3 Response Surface Design and Results

[0063]

[0064] Using Design Expert software, a multiple regression fitting analysis was performed, yielding the regression equation: Y = 84.10 + 2.93A + 2.25B + 1.47C - 0.71AB + 2.12AC + 0.6275BC - 4.33A 2 -9.12B 2 -4.33C 2 (R 2 =0.9913, R 2 Adj = 0.9801, p < 0.0001 (A is the amount of compound enzyme added, B is the ultrasonic power, C is the reaction time, and Y is the enzymatic hydrolysis rate).

[0065] Analysis of variance and significance tests were performed on the regression equation. Table 4 shows that the regression model's p < 0.01, indicating that the model is highly significant at the 0.01 level. The p = 0.7408 > 0.05 for the lack-of-fit term, indicating that the lack-of-fit term is not significant. This proves that the model selection is correct and can be used to optimize the experimental conditions for ultrasound-assisted enzymatic hydrolysis of soybean isoflavones. A, B, AC, A 2 B2 C 2 The p-values ​​for all values ​​were less than 0.01, indicating that they had a highly significant impact on the enzymatic hydrolysis rate. The p-value for C was less than 0.05, indicating that it had a significant impact on the enzymatic hydrolysis rate. The most important parameter affecting enzymatic hydrolysis was the amount of compound enzyme added (A), followed by the ultrasonic power (B) and the reaction time (C). The R-value of the regression model was... 2 R is 0.9913. 2 An Adj value of 0.9801 indicates that 99.13% of the data in the model can be explained by the model, demonstrating a good fit and a high correlation between the predicted and actual values. In summary, the model exhibits high fit, small error, and high reliability, making it suitable for optimizing the process of ultrasonic-assisted enzymatic hydrolysis and enrichment of daidzein.

[0066] Table 4. Analysis of Variance for Regression Models

[0067]

[0068] Note: * indicates that the p < 0.05 level is significant, and ** indicates that the p < 0.01 level is highly significant.

[0069] Figures 4-6 Two-dimensional (2D) contour plots and three-dimensional (3D) response surface plots based on a regression model are shown to better understand the interaction of independent parameters. The interaction is positively correlated with the steepness of the 3D surface plot. Therefore, the steep distribution of the 3D surface plot indicates a stronger correlation between the two factors. Specifically, Figure 4 The diagram shows the contour plot and response surface plot of the effect of the amount of compound enzyme added and the ultrasonic power on the enzymatic hydrolysis rate of soybean isoflavones in a specific embodiment of the method for optimizing the ultrasonic-assisted compound enzyme hydrolysis and enrichment of daidzein based on response surface methodology. Figure 5 The diagram shows the contour plot and response surface plot of the effect of the amount of compound enzyme added and the reaction time on the hydrolysis rate of soybean isoflavones in a specific embodiment of the method for enriching daidzein by ultrasonic-assisted hydrolysis based on response optimization provided in this application. Figure 6 The contour plot and response surface plot show the effect of ultrasonic power and reaction time on the enzymatic hydrolysis rate of soybean isoflavones in a specific embodiment of the method for optimizing ultrasonic-assisted complex enzyme hydrolysis and enrichment of daidzein based on response surface methodology.

[0070] Figures 4-6 For contour plots and response surface plots, Figure 4 This indicates the effect of the interaction between the amount of compound enzyme added and the ultrasonic power on the hydrolysis rate at the mid-reaction time level. As the amount of compound enzyme added and the ultrasonic power increase, the hydrolysis rate first increases and then decreases. The increased amount of compound enzyme increases the opportunity for enzyme to contact the substrate, and the ultrasonic action activates the enzyme's active sites. Figure 5This indicates the effect of the interaction between the amount of compound enzyme added and the reaction time on the enzymatic hydrolysis rate when the ultrasonic power is at a central level. With increasing amounts of compound enzyme added and reaction time, the enzymatic hydrolysis rate first increases and then decreases, with a low response surface tilt and p = 0.0028 < 0.05, indicating a significant interaction between the amount of compound enzyme added and the reaction time. Figure 6 This indicates the effect of the interaction between ultrasonic power and reaction time on the hydrolysis rate when the amount of compound enzyme added is at the median level. With increasing ultrasonic power and reaction time, hydrolysis initially increases and then decreases. Excessively high or low temperatures will denature and inactivate the enzyme, and excessive ultrasonic power will also alter the enzyme structure, thereby reducing the hydrolysis rate.

[0071] The optimal process parameters for the enzymatic hydrolysis of glycosidic soybean isoflavones by ultrasound-assisted compound enzymes (β-glucosidase and cellulase) are as follows: compound enzyme addition amount 10.1852%, ultrasound power 247.06W, reaction time 37.7487min, compound enzyme ratio 2:1, reaction temperature 50℃, and enzymatic hydrolysis rate 85.0157%.

[0072] Example 3: Enzymatic hydrolysis of glycoside-type soybean isoflavones using actual process parameters optimized by the response surface model of Example 2. The specific steps are as follows:

[0073] S1. Accurately weigh 50 mg of soy isoflavones and add them to 50 mL of pH 5.0 disodium hydrogen phosphate-citric acid buffer solution.

[0074] S2. Add 10% of a compound enzyme (β-glucosidase and cellulase) to the above solution at a ratio of 2:1 of β-glucosidase and cellulase. After sonicating the solution at 250W, carry out the enzymatic hydrolysis reaction at 50℃ for 38 minutes to obtain the enzymatic hydrolysate.

[0075] S3. After the reaction is complete, inactivate the enzyme by boiling in a water bath for 5 minutes, and then cool to room temperature.

[0076] S3. Extract the enzymatically hydrolyzed solution with ethanol, collect the upper organic phase, remove the ethanol using a rotary evaporator, and separate it using the pre-treated macroporous adsorption resin. First, rinse with water until the eluent is colorless and clear, then continue eluting with ethanol solution and collect the eluent.

[0077] S5. Concentrate using a rotary evaporator and freeze-dry under vacuum to obtain daidzein powder.

[0078] The enzymatic hydrolysis rate of soy isoflavones obtained in this embodiment was 84.17%, which is close to the theoretical value (85.0157%), indicating that the enzymatic hydrolysis condition parameter model is stable and reliable.

[0079] Example 4: Determination of the content and purity of daidzein

[0080] The content of aglycone-type soybean isoflavones after enzymatic hydrolysis is as follows: Figure 7 As shown. Figure 7 Compared with the peak time of the daidzein standard, the absorption peak of daidzein was observed at 27.3 min. Based on the peak area of ​​daidzein, the content of daidzein after enzymatic hydrolysis was calculated to be 21.5 mg / g. The purity of daidzein after resin separation and purification was 93.16%.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the technical solution of the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for enriching daidzein using ultrasound-assisted complex enzymes based on response surface methodology, characterized in that, The method includes: S1. Accurately weigh 50 mg of soy isoflavones and add them to 50 mL of disodium hydrogen phosphate-citric acid buffer solution (pH 5.0); S2. Add different mass fractions of compound enzymes (β-glucosidase: 10000U / g and cellulase: 100U / g) to the above solution in a certain proportion, sonicate the solution, and then carry out enzymatic hydrolysis at 50℃ to obtain the enzymatic hydrolysate. S3. The optimal parameters of the enzymatic hydrolysis process were determined by optimizing the amount of compound enzyme, ultrasonic power, and reaction time using response surface methodology. S4. After the reaction is complete, inactivate the enzyme by boiling water bath and cool to room temperature. S5. Extract the enzymatically hydrolyzed solution with ethanol, take the upper organic phase, remove the ethanol using a rotary evaporator, separate it with the pre-treated macroporous adsorption resin, first wash with water until the eluent is colorless and clear, continue to elute with ethanol solution, and collect the eluent. S6. Concentrate using a rotary evaporator and freeze-dry under vacuum to obtain daidzein powder.

2. The method for enriching daidzein based on response surface methodology using ultrasound-assisted complex enzymes according to claim 1, characterized in that: The amount of compound enzyme added in step S2 is 3%-15%.

3. The method for enriching daidzein based on response surface methodology using ultrasound-assisted complex enzymes according to claim 1, characterized in that: In step S2, the ratio of β-glucosidase to cellulase in the complex enzyme is 2:

1.

4. The method for enriching daidzein based on response surface methodology using ultrasound-assisted complex enzymes according to claim 1, characterized in that: The ultrasonic power in step S2 is 120-360W.

5. The method for enriching daidzein based on response surface methodology using ultrasound-assisted complex enzymes according to claim 1, characterized in that: The reaction time in step S2 is 15-55 min.

6. The method for enriching daidzein based on response surface methodology using ultrasound-assisted complex enzymes according to claim 1, characterized in that: The enzyme inactivation time in step S4 is 5 minutes.

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