Method for regulating and controlling rapeseed protein-quercetin binding strength through ultrasonic frequency and application

Abstract

The invention discloses a method for regulating and controlling the binding strength of rapeseed protein-quercetin through ultrasonic frequency and application, and belongs to the technical field of processing of food / agricultural products and byproducts of the food / agricultural products. According to the method, a modified rapeseed protein-quercetin compound is used as a raw material, a compound solution is subjected to multi-frequency plate-type ultrasonic treatment, and the bonding strength of modified rapeseed protein and quercetin in the compound is regulated and controlled by adjusting the ultrasonic frequency and the combination mode thereof. The method disclosed by the invention not only can effectively regulate and control the Gibbs free energy value of the modified rapeseed protein-quercetin compound, but also can effectively change the contribution degree of intermolecular acting force participating in the formation of the compound. The method has the characteristics of simplicity in operation and easiness in amplification, the binding strength of protein-polyphenol in the compound can be effectively regulated and controlled, the application of a multi-frequency ultrasonic technology in preparation of the protein-polyphenol compound is promoted, and meanwhile, the application range of the protein-polyphenol compound in the field of food processing is widened.

Description

Technical Field

[0001] This invention belongs to the field of food / agricultural product and by-product processing technology, and relates to a method and application of ultrasonic frequency regulation of rapeseed protein-quercetin binding strength. Background Technology

[0002] Quercetin, also known as quercetin, is a flavonoid compound widely found in plants, possessing antioxidant, anti-inflammatory, cardiovascular protective, and immunomodulatory effects. In 2010, it was recognized as a safe food ingredient by the FAD (Food and Drug Administration). Although quercetin exhibits various physiological activities, its low water solubility limits its effectiveness and hinders its application in the food industry. Currently, nano-encapsulation technology is primarily used to improve the water solubility and dispersibility of quercetin, thereby enhancing its bioavailability. Proteins, polysaccharides, and glycolipid surfactants can all serve as encapsulation materials for quercetin. Among them, using protein as a wall material to effectively encapsulate quercetin has the following advantages: (1) the digestion of protein in the gastrointestinal tract can be used to regulate the breakdown rate of nanoparticles, thereby achieving gastrointestinal-directed delivery of quercetin; (2) protein can bind to quercetin through hydrogen bonds and hydrophobic interactions to form a protein-polyphenol complex, which can not only improve the bioaccessibility of quercetin, but also improve the functional properties of the protein. However, how to regulate the protein-polyphenol binding strength is very important for quercetin delivery systems using protein as an encapsulation wall material.

[0003] In 2014, rapeseed protein was recognized as a safe food ingredient by the European Commission. It is a complete protein, rich in methionine, lysine, and cysteine, and its amino acid balance is superior to other plant proteins. Rapeseed protein has a casein efficiency ratio of 2.5 and is mainly composed of globulins, prolamins, albumins, and glutenins. Its nutritional value is significantly higher than other plant proteins and comparable to animal proteins. Our research group modified the structure of rapeseed protein using whey protein and zein, significantly improving its water solubility and quercetin encapsulation ability, demonstrating the feasibility of developing a quercetin nanodelivery system.

[0004] Ultrasound is an emerging non-thermal physical processing technology that has been widely used in the preparation of protein-polyphenol complexes. However, current research focuses primarily on the effects of ultrasound duration, temperature, power, and intermittent ratio on the encapsulation capacity of protein-polyphenols, neglecting their influence on the binding strength. Furthermore, ultrasound frequency significantly affects the cavitation effect generated by ultrasound. Generally, higher ultrasound frequencies create a large number of cavitation bubbles with low bursting force, which may affect protein-polyphenol binding. However, the impact of ultrasound frequency on protein-polyphenol interactions has not been fully considered, remaining at the level of optimizing traditional process conditions such as ultrasound duration, power, and temperature. Moreover, although ultrasound technology is widely used in the preparation of protein-polyphenol complexes, it is often aimed at increasing the amount of polyphenols bound to the protein, leading to a focus only on the quantity (loading) of protein-polyphenol binding while neglecting the quality (binding strength). Different types of proteins have different physical and chemical properties, affecting the binding strength between proteins and polyphenols. Different reaction conditions also affect the binding strength and interaction forces between the two. The requirements for the binding strength of protein-polyphenols in the complex also vary significantly across different application scenarios. Generally, complexes with high protein-polyphenol binding strength are suitable for more demanding processing environments such as high temperature, high pressure, and high shear; while complexes with lower binding strength are more prone to structural reorganization with environmental changes, which is beneficial for improving interfacial properties such as emulsification and foaming properties, making them more suitable for use as emulsifiers. Based on this, this invention fully utilizes the frequency effect of ultrasound to establish a method for regulating the binding strength of protein-polyphenols, and proposes this patent. A search revealed no reports on methods and applications for regulating the binding strength of rapeseed protein-quercetin using ultrasonic frequency. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for regulating the binding strength of rapeseed protein-quercetin using ultrasonic frequency, addressing the aforementioned problems. Using modified rapeseed protein and quercetin as raw materials, and relying on a multi-frequency ultrasonic device developed by the Institute of Food Physics and Processing at Jiangsu University, the binding strength between modified rapeseed protein and quercetin is regulated by utilizing the ultrasonic frequency effect. This technology can serve as a method for obtaining high-quality rapeseed protein-quercetin complexes, with the aim of expanding the application of rapeseed protein in the field of quercetin delivery.

[0006] To achieve the above-mentioned objective, the method for regulating the binding strength of rapeseed protein-quercetin using ultrasonic frequency according to the present invention is carried out according to the following steps:

[0007] (1) Modification of rapeseed protein: Rapeseed protein, whey protein and zein were mixed in dry powder at a mass ratio of 1:1:0.2 using the pH-driven method. An appropriate amount of deionized water was added to make the total protein mass concentration 1%. Then, the pH of the protein solution was adjusted to 12 using a 1 mol / L alkaline solution and stirred at 4℃ for 3 h (maintaining the pH of the system at 12). Finally, the pH of the solution was gradually adjusted to 7 using a 0.1 mol / L citric acid solution. After desalting and freeze drying, modified rapeseed protein was obtained.

[0008] (2) Preparation of rapeseed protein-quercetin complex: The modified rapeseed protein from step (1) was dissolved in deionized water at a mass ratio of 0.5%, wherein the volume ratio of modified rapeseed protein-quercetin to quercetin solution was 1:1, and a quercetin solution with a concentration of 5-80 mmol / L was added; the pH was adjusted to 12 with alkaline solution, and the mixture was stirred at 4℃ for 3 h (maintaining the pH of the system at 12), and the pH was adjusted to 7 with citric acid solution. The solution was centrifuged to remove unbound quercetin, and the mixture was desalted and freeze-dried to obtain the modified rapeseed protein-quercetin complex.

[0009] (3) Ultrasonic treatment: The modified rapeseed protein-quercetin complex lyophilized powder from step (2) was dissolved in phosphate buffer at pH 7.5. After full hydration, it was ultrasonically treated for 31 min at 30°C, 300W power, 21W / L power density, and with the ultrasonic frequency mode set to single frequency, synchronous dual frequency and synchronous triple frequency, respectively, to obtain modified rapeseed protein-quercetin complex solutions with different binding strengths.

[0010] (4) Determination of the binding strength between protein and quercetin in the complex: The complex solution from step (3) was incubated in a constant temperature water bath at 298, 303 and 308 K for 30 min in the dark; the fluorescence intensity of the complex was measured using a Card-F98 fluorescence spectrophotometer at an excitation wavelength of 280 nm and an emission scanning range of 300-500 nm; the thermodynamic parameters (Gibbs free energy, enthalpy change and entropy change) of the modified rapeseed protein binding with quercetin were calculated using the Van't Hoff equation to reflect the binding strength between the two.

[0011] The alkaline solution mentioned in step (1) is a mixture of sodium hydroxide and potassium hydroxide in a 1:1 molar ratio.

[0012] The ultrasonic frequencies mentioned in step (3) are: single frequency 20, 40 or 60 kHz, synchronous dual frequency 20 / 40 kHz, 20 / 60 kHz or 40 / 60 kHz, and synchronous tri-frequency 20 / 40 / 60 kHz.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] (1) This invention is the first to use the ultrasonic frequency effect to regulate the binding strength between modified rapeseed protein and quercetin, and proves that modified rapeseed protein-quercetin complexes with different binding strengths can be obtained by controlling the combination mode of ultrasonic frequency, and establishes the regulation technology.

[0015] (2) By changing the combination mode of ultrasonic frequency, the present invention obtains modified rapeseed protein-quercetin complexes with different binding strengths, which can broaden the food processing application scenarios of rapeseed protein-quercetin complexes.

[0016] (3) The present invention can improve the water solubility of rapeseed protein and the encapsulation ability of quercetin by combining ultrasonic frequencies. It is suitable for the modification of proteins with poor water solubility and low ability to encapsulate active substances. At the same time, it can promote the application of ultrasonic frequency combination technology in the preparation of protein-polyphenol complexes. Detailed Implementation

[0017] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below.

[0018] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0019] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention uses a Card-F98 fluorescence spectrophotometer to measure the fluorescence intensity of the complex at an excitation wavelength of 280 nm and an emission scanning range of 300-500 nm; and uses the Van't Hoff equation to calculate the thermodynamic parameters (Gibbs free energy, enthalpy change and entropy change) of the binding of modified rapeseed protein with quercetin.

[0020] The binding constant K of the modified rapeseed protein-quercetin complex a The number of binding sites, n, is calculated using the following formula:

[0021]

[0022] In the formula, F0 and F represent the initial fluorescence intensity of the modified rapeseed protein solution and the fluorescence intensity after the addition of quercetin, respectively; K a is the complex binding constant (L / mol), determined by the slope of the double logarithmic curve; n is the number of binding sites, determined by the intercept of the double logarithmic curve; [Q] represents the concentration of quercetin (mol / L).

[0023] The thermodynamic parameters of the binding between quercetin and modified rapeseed protein were calculated using the van't Hoff equation. The formulas for calculating the Gibbs free energy, enthalpy change, and entropy change are as follows:

[0024]

[0025] ΔG=-RT ln K A (3)

[0026] ΔG=ΔH-TΔS (4)

[0027] In the formula, ΔH, ΔG, and ΔS represent the enthalpy change, free energy change, and entropy change during the bonding process, respectively; T is the solution temperature (K) during the preparation of the complex; and R represents the gas constant 8.314 J·mol⁻¹. -1 ·K -1 ;K A It is the binding constant at the corresponding temperature.

[0028] Example 1

[0029] Whey protein, zein, and rapeseed protein were mixed in a mass ratio of 1:1:0.2, and deionized water was added to make the total protein concentration 1%. The pH of the protein solution was adjusted to 12 using a 1 mol / L alkaline solution (sodium hydroxide and potassium hydroxide were mixed in a 1:1 molar ratio), and stirred at 4°C for 3 h (maintaining the system pH at 12). The pH was gradually adjusted to 7 using a 0.1 mol / L citric acid solution. After desalting and freeze-drying, modified rapeseed protein was obtained. Quercetin at different final concentrations (0, 5, 10, 20, 40, 60, and 80 mmol / L) was added to a 0.5% deionized aqueous solution of modified rapeseed protein. The pH was then adjusted to 12 with the aforementioned alkaline solution, and the mixture was stirred at 4°C for 3 hours (maintaining pH 12). The pH was then adjusted to 7 with citric acid solution. The solution was centrifuged to remove unbound quercetin, and then desalted and freeze-dried to obtain a lyophilized powder of the modified rapeseed protein-quercetin complex. The lyophilized powder was dissolved in phosphate buffer at pH 7.5, fully hydrated, and then sonicated at a single frequency of 20 kHz at 30°C and a power density of 21 W / L for 31 minutes to obtain a single-frequency modified rapeseed protein-quercetin complex at 20 kHz. The complex solution was incubated in a constant temperature water bath at 298, 303, and 308 K for 30 min in the dark. The fluorescence intensity of the complex was measured using a Card-F98 fluorescence spectrophotometer at an excitation wavelength of 280 nm and an emission scanning range of 300-500 nm. The binding constant and binding site of the modified rapeseed protein and quercetin were calculated using formula (1). The Gibbs free energy, enthalpy change, and entropy change of the two were calculated using the Van't Hoff equation according to formulas (2), (3), and (4) to evaluate the binding strength.

[0030] Example 2

[0031] The ultrasonic frequency mode was set to a single frequency of 40 kHz, and ultrasonication was performed at 30°C and a power density of 21 W / L for 31 min to obtain a modified rapeseed protein and quercetin complex with a single frequency of 40 kHz. The remaining steps were the same as in Example 1.

[0032] Example 3

[0033] The ultrasonic frequency mode was set to a single frequency of 60 kHz, and ultrasonication was performed at 30°C and a power density of 21 W / L for 31 min to obtain a modified rapeseed protein and quercetin complex with a single frequency of 60 kHz. The remaining steps were the same as in Example 1.

[0034] Example 4

[0035] The ultrasonic frequency mode was set to synchronous dual-frequency 20 / 40kHz, and ultrasonication was performed at 30℃ and 21W / L power density for 31 min to obtain the modified rapeseed protein and quercetin complex with dual-frequency 20 / 40kHz. The remaining steps were the same as in Example 1.

[0036] Example 5

[0037] The ultrasonic frequency mode was set to synchronous dual-frequency 20 / 60kHz, and ultrasonication was performed at 30℃ and 21W / L power density for 31 min to obtain a modified rapeseed protein and quercetin complex with dual-frequency 20 / 60kHz. The remaining steps were the same as in Example 1.

[0038] Example 6

[0039] The ultrasonic frequency mode was set to synchronous dual-frequency 40 / 60kHz, and ultrasonication was performed at 30℃ and 21W / L power density for 31 min to obtain the modified rapeseed protein and quercetin complex with dual-frequency 40 / 60kHz. The remaining steps were the same as in Example 1.

[0040] Example 7

[0041] The ultrasonic frequency mode was set to synchronous dual-frequency 20 / 40 / 60kHz, and ultrasonication was performed at 30℃ and 21W / L power density for 31 min to obtain a tri-frequency 20 / 40 / 60kHz modified rapeseed protein and quercetin complex. The remaining steps were the same as in Example 1.

[0042] Comparative Examples

[0043] The comparative example is a modified rapeseed protein and quercetin complex prepared without ultrasonic treatment. Except for the absence of ultrasonication, the other steps are the same as in Example 1.

[0044] Table 1 shows the effect of ultrasonic frequency on the binding constant (K) of modified rapeseed protein to quercetin. aThe influence of binding sites (n) on the complexes in Examples 2 and 3 is shown in the table. a Both the K and n values ​​are lower than those of the comparative examples, while the values ​​of the other examples are higher than those of the comparative examples. Among them, the 60kHz example 3 has the lowest K value. a (0.11×10 6 The values ​​of K and n (1.07) are highest for Example 6 at 40 / 60 kHz, while Example 6 has the highest K value. a The values ​​of α and β were approximately 1000 times and 1.66 times those of Example 3, respectively. The changes in the binding constant and binding sites indicate that the ultrasonic frequency can regulate the binding process between the two by exposing or concealing the binding sites in the modified rapeseed protein.

[0045] Table 1. Effects of ultrasonic frequency on the binding constant and binding sites of modified rapeseed protein and quercetin.

[0046] complexes K a (×10 6 )]]> n comparative examples 0.52 1.26 example 1 67.61 1.81 example 2 0.24 1.14 example 3 0.11 1.07 example 4 0.76 1.25 example 5 1.02 1.28 example 6 102.33 1.78 example 7 1.58 1.32

[0047] Table 2 shows the effect of ultrasonic frequency on the thermodynamic parameters of quercetin-modified rapeseed protein, namely Gibbs free energy, enthalpy change, and entropy change.

[0048] Gibbs free energy (ΔG) is a key thermodynamic parameter for determining the spontaneity of the protein-polyphenol binding reaction and the stability of the complex. As shown in Table 2, the ΔG values ​​of all examples are less than 0, indicating that the binding process of modified rapeseed protein and quercetin is spontaneous. The higher the absolute value of the ΔG of the complex, the more complete the binding reaction, the stronger the binding strength between the polyphenol and the protein, and the higher the stability of the complex. Significant differences in the ΔG values ​​of the complexes from different ultrasonic frequency modes indicate that ultrasonic frequency can effectively regulate the binding strength between quercetin and modified rapeseed protein. Specifically, the ΔG value of the complex in Example 3 at 60 kHz is -28.70 kJ·mol⁻¹. -1 Its absolute value was the lowest, significantly lower than the non-ultrasound group, meaning that 60kHz reduces the binding strength between quercetin and modified rapeseed protein in the complex; while the ΔG value of the complex in Example 6 at 40 / 60kHz was -45.70 kJ·mol⁻¹. -1 The absolute value was the highest, approximately 1.59 times that of the complex in Example 3, indicating that 40 / 60 kHz ultrasound can effectively improve the binding strength between quercetin and modified rapeseed protein in the complex. Therefore, the selection and combination of ultrasound frequencies can effectively regulate the binding strength between quercetin and modified rapeseed protein in the complex.

[0049] Enthalpy change (ΔH) is an important indicator for evaluating whether a binding reaction is endothermic or exothermic. ΔH < 0 indicates an exothermic reaction, while ΔH > 0 indicates an endothermic reaction. Table 2 shows that the binding process of quercetin with modified rapeseed protein in the non-ultrasonicated comparative example and Example 1 (20kHz) is an exothermic reaction; while the binding process of quercetin with modified rapeseed protein in the other ultrasonic frequencies and modes is an endothermic reaction, but the ΔH values ​​of the complexes differ significantly between different ultrasonic frequencies. Specifically, the ΔH value of the complex in Example 3 (60kHz) is 56.43 kJ·mol⁻¹. -1 The values ​​were significantly lower than those in the examples; while the complex in Example 4 at 20 / 40kHz had the highest ΔH value (631.25 kJ·mol⁻¹). -1 The frequency of ultrasound was approximately 11.19 times that of the 60kHz single-frequency ultrasound in Example 3. Therefore, the ultrasound frequency can significantly affect the energy changes in the binding process of quercetin and modified rapeseed protein, which is an important reason why the ultrasound frequency regulates the protein-polyphenol binding strength in the complex.

[0050] Entropy change (ΔS) is an important indicator for evaluating the molecular orderliness of a complex during the binding process. ΔS < 0 indicates a decrease in molecular orderliness during binding, while ΔS > 0 indicates an increase in molecular orderliness. As shown in Table 2, the complexes in the comparative examples and Example 1 at 20 kHz exhibit ΔS < 0, indicating an entropy decrease process, meaning that the binding of modified rapeseed protein and quercetin reduces the molecular orderliness of the complex. In contrast, the ΔS values ​​of the complexes in the other ultrasound examples are all > 0, indicating that the binding process of modified rapeseed protein and quercetin is an entropy increase reaction, which can significantly improve the molecular orderliness of the complex. However, significant differences in the ΔS values ​​of the complexes exist in the ultrasound examples with different frequency modes, indicating that ultrasound frequency can significantly affect the molecular orderliness of the complex.

[0051] Table 2. Effects of ultrasonic frequency on the thermodynamic parameters of modified rapeseed protein and quercetin aggregation.

[0052]

[0053]

[0054] Table 3 shows the effect of ultrasonic frequency on the contribution of the interaction between modified rapeseed protein and quercetin. Electrostatic interactions, hydrogen bonds, hydrophobic interactions, and disulfide bonds were disrupted by adding different blocking agents (0.6 mol / L NaCl solution, 2 and 8 mol / L urea solutions, and 0.5 mol / L mercaptoethanol solution), respectively. Based on this, the contribution of the above intermolecular forces was quantitatively calculated according to the intensity change of the intrinsic fluorescence of the complex.

[0055] As shown in Table 3, the contribution of disulfide bonds in the complexes of all examples was higher than that of the comparative examples, while the contribution of hydrophobic interactions decreased. This indicates that ultrasonic treatment can significantly enhance the role of disulfide bonds in the complexes and reduce hydrophobic interactions. The contributions of the four intermolecular forces differed significantly among examples with different ultrasonic frequencies, indicating that ultrasonic frequency can significantly affect the contribution of intermolecular forces in the complex formation process. Among these, Example 6 (40 / 60 kHz) and Example 7 (20 / 40 / 60 kHz) showed the highest contribution of disulfide bonds, at 57.14% and 56.58%, respectively, approximately 30 times that of the comparative examples. It is known that disulfide bonds are covalent bonds and generally have higher bond energies, thus having a greater impact on the stability of the complex. Therefore, ultrasonic frequency can significantly affect the participation of molecular forces in the complex formation process, which is the intrinsic mechanism by which it regulates the binding strength of quercetin and modified rapeseed protein in the complex. By controlling the ultrasonic frequency, complexes with different binding strengths can be obtained.

[0056] Table 3. Effect of ultrasonic frequency on the contribution of quercetin-modified rapeseed protein interaction.

[0057] complexes electrostatic interactions (%) hydrogen bonds (%) hydrophobic interactions (%) disulfide bonds (%) comparative examples 10.68 22.84 64.54 1.94 example 1 12.04 0.84 34.62 52.50 example 2 21.17 20.23 35.16 23.44 example 3 6.89 7.83 37.56 47.72 example 4 26.34 5.22 17.52 50.92 example 5 18.90 21.78 28.43 30.88 example 6 5.94 12.62 24.30 57.14 example 7 3.71 5.77 33.95 56.58

[0058] Ultrasonic frequency can influence the cavitation effect by altering the growth-explosion process of bubbles in a solution. Therefore, the influence of ultrasonic frequency on the cavitation effect is the main reason for its regulation of the binding strength between quercetin and modified rapeseed protein in the complex. Generally, low-frequency ultrasound usually allows bubbles sufficient time to grow into larger bubbles, resulting in a cavitation effect with greater bursting power, but the number and density of bubbles are lower; while high-frequency ultrasound, on the other hand, produces cavitation bubbles with lower bursting power but a larger number. Compared with single-frequency ultrasound, the different frequencies of ultrasound in dual-frequency and tri-frequency ultrasound interfere with each other, producing a completely new cavitation effect. However, it is worth noting that some of the cavitation effect originally produced by single-frequency ultrasound will still remain in multi-frequency ultrasound. The combined effect of multiple cavitation effects is the main reason for its regulation of the binding strength between quercetin and modified rapeseed protein in the complex.

[0059] The invention has been described through embodiments that are currently considered to be the most preferred and practical. It should be understood that the invention is not limited to the disclosed embodiments. Rather, its purpose is to cover various modifications and equivalent arrangements within the spirit and scope of the appended claims, the scope of which is to be interpreted in the broadest possible sense to include all such modifications and equivalent structures permitted under the law.

Claims

1. A method for regulating the binding strength of rapeseed protein-quercetin by ultrasonic frequency, characterized in that The following steps are followed: (1) Modification of rapeseed protein: using pH-driven method, rapeseed protein, whey protein and corn protein are mixed in dry powder according to the mass ratio of 1:1:0.2, and a proper amount of deionized water is added to make the mass concentration of total protein 1%; then, the pH value of the protein solution is adjusted to 12 by using 1 mol / L alkali solution, and stirring is carried out at 4℃ for 3h (maintaining the pH of the system at 12); finally, the pH value of the solution is gradually adjusted to 7 by using 0.1 mol / L citric acid solution, and after desalination, freeze-drying, modified rapeseed protein is obtained; (2) Preparation of rapeseed protein-quercetin complex: the modified rapeseed protein of step (1) is dissolved in deionized water according to the mass ratio of 0.5%, and the volume ratio of modified rapeseed protein-quercetin and quercetin solution is 1:1, and the concentration of quercetin solution is 5-80mmol / L; adjust the pH to 12 by alkali solution, stir at 4℃ for 3h (maintain the pH of the system at 12), adjust the pH to 7 by citric acid solution, centrifuge the solution to remove unbound quercetin, desalt, freeze-dry, and obtain modified rapeseed protein-quercetin complex; (3) Ultrasonic treatment: the modified rapeseed protein-quercetin complex freeze-dried powder in step (2) is dissolved in phosphate buffer solution with pH 7.5, after sufficient hydration, ultrasonic treatment is carried out under the conditions of single frequency, synchronous double frequency and synchronous triple frequency, respectively, for 31min, to obtain modified rapeseed protein-quercetin complex solutions with different binding strengths.

2. The method of claim 1, wherein the ultrasound frequency is in the range of 20- 100 kHz. The ultrasonic conditions are power 300W, power density 21W / L, temperature 30℃, and time 31min.

3. The method of claim 1, wherein the ultrasound frequency is in the range of 20- 100 kHz. The alkali solution in step (1) is a mixed solution of sodium hydroxide and potassium hydroxide with a molar ratio of 1:

1.

4. The method of claim 1, wherein the ultrasound frequency is in the range of 20- 100 kHz. The ultrasonic frequency in step (3) is single frequency of 20, 40 or 60kHz, synchronous double frequency of 20 / 40kHz, 20 / 60kHz or 40 / 60kHz, and synchronous triple frequency of 20 / 40 / 60kHz.

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