Method for extracting sunflower seed meal protein based on microwave-assisted eutectic solvent system

Abstract

The invention discloses a method for extracting sunflower seed meal protein based on a microwave-assisted eutectic solvent system. The method comprises the following steps: pretreating sunflower seed meal to obtain degreased powder, adding the degreased powder into a DES and alkaline protease coupling system, carrying out microwave extraction, centrifugation, ethanol treatment, incubation, precipitation and washing, carrying out TiO2-coated CQDs illumination treatment, and drying to obtain protein. By optimizing microwave parameters and adopting a multi-stage pulse mode, the extraction rate is remarkably increased to 80% or above, meanwhile, the protein emulsifying activity is improved, and the application range is expanded. The DES system is environment-friendly and recoverable, the preparation process is accurate in parameter control, stable and efficient reaction is ensured, and green and efficient sunflower seed meal protein extraction is realized.

Description

Technical Field

[0001] The present invention relates to a sunflower seed meal protein extraction technology, and more specifically, to a method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system. Background Art

[0002] Sunflower meal, as a byproduct of sunflower seed oil extraction, is rich in protein resources. Its effective extraction and utilization has important economic and resource value. In many fields such as food, feed and biological products, sunflower meal protein has broad application prospects. For example, in the food industry, it can be used to improve the nutritional structure of food and enhance the quality and taste of products.

[0003] With the continuous development of science and technology, in order to solve the drawbacks of traditional extraction methods, researchers have begun to explore new technical paths, among which the application of low eutectic system in sunflower seed meal protein extraction has gradually attracted attention. Low eutectic solvent (DES) is usually formed by mixing hydrogen bond acceptors such as quaternary ammonium salts and hydrogen bond donors such as urea and polyols in a certain proportion. It has the characteristics of low volatility, good biocompatibility and designability, and theoretically provides a new mild environment for protein extraction.

[0004] However, the application of low eutectic systems in the extraction of sunflower meal protein is still in its early stages. Some studies have attempted to directly use simple low eutectic solvents for extraction, but due to the lack of in-depth understanding of the interaction mechanism between the solvent system and the protein, the extraction efficiency has not met expectations. For example, some studies simply mixed DES with sunflower meal, and the extraction rate only hovered between 60% and 65%, which was not significantly superior to traditional methods. Moreover, during the extraction process, how to ensure that the low eutectic solvent minimizes the damage to the protein structure while efficiently dissolving the protein has become a major problem. In addition, the existing extraction process based on the low eutectic system has limited effect on improving the emulsification activity of the sunflower meal protein obtained. When preparing emulsion products, it is still difficult to meet the requirements of protein emulsification stability in practical applications, and the product is prone to stratification, flocculation and other phenomena.

[0005] In addition to the shortcomings of the low eutectic system, the traditional sunflower meal protein extraction method also has many problems. The traditional alkali extraction and acid precipitation method, although relatively simple to operate, requires a large amount of acid and alkali reagents during the extraction process, which is not only easy to cause environmental pollution, but also has a low protein extraction rate. Under the optimal process conditions, the extraction rate can only reach about 60%. This method will also cause excessive denaturation of protein, seriously affecting the functional properties of the extracted protein. Although the enzymatic method for extracting sunflower meal protein is relatively mild, due to the complexity of the enzymatic reaction, the reaction conditions are difficult to accurately control, resulting in unstable extraction efficiency, and the overall extraction efficiency still needs to be improved. The general extraction rate fluctuates around 65%. Although physical methods such as ultrasound-assisted extraction can promote the dissolution of protein to a certain extent, the effect is not ideal when used alone, and the extraction rate is usually difficult to exceed 70%.

[0006] More importantly, the emulsifying activity of sunflower meal protein obtained by these existing methods is generally not high. Emulsifying activity is an important indicator to measure the application performance of protein in food and other fields. Low emulsifying activity limits the application of sunflower meal protein in products such as emulsion food and cosmetics. For example, when preparing emulsified beverages, sunflower meal protein with low emulsifying activity cannot effectively stabilize the emulsion system, which easily leads to problems such as emulsion stratification and precipitation, seriously affecting product quality and shelf life.

[0007] Therefore, developing a new extraction method that can improve the extraction efficiency of sunflower seed meal protein and enhance its emulsification activity has become an important issue that needs to be solved urgently. Summary of the invention

[0008] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0009] Another object of the present invention is to provide a method for extracting sunflower seed meal protein based on a microwave-assisted low eutectic solvent system, which improves the extraction rate of sunflower seed meal protein to more than 80%, enhances the emulsification activity of the protein, and has the advantages of being green and environmentally friendly, low cost and controllable process.

[0010] In order to achieve these purposes and other advantages according to the present invention, a method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system is provided, comprising: S1, pretreating sunflower seed meal to obtain defatted sunflower seed meal powder; S2, adding the defatted sunflower seed meal powder obtained in step S1 to the DES system, and placing the mixture in a microwave reactor to extract an extract, wherein the solid-liquid ratio of the defatted sunflower seed meal powder to the DES system is 1:10-14; S3, inactivating the enzyme in the extract obtained in step S2, centrifuging and taking the supernatant, adding 75% ethanol solution, refrigerating and incubating at 4° C., centrifuging the incubated solution to take the lower layer of precipitate, washing, and drying to obtain the sunflower seed meal protein; The DES system in step S2 is a coupling system of DES solution and alkaline protease, the mass ratio of alkaline protease to DES solution is 1:80-100, the DES solution is prepared by using betaine and urea in a molar ratio of 1:1, and in step S3, the extract is heated at 80°C for 10 minutes before centrifugation to inactivate the enzyme.

[0011] Preferably, the preparation method of the DES system in step S2 is as follows: S201, respectively weighing betaine and urea in a molar ratio of 1:1, dividing urea into two parts, wherein the mass ratio of the first part of urea to the second part of urea is 5-8:1, mixing betaine and the first part of urea and controlling the water content in the mixed solution to be 15-20%, and stirring the mixed solution at 80° C. until a transparent liquid is formed; S202, continue to add the second portion of urea to the transparent liquid obtained in step S201 and continue stirring, while controlling the temperature gradient to decrease, the temperature decrease frequency is 2-3°C every 5 minutes, when the temperature drops to 75-76°C, add NaOH to adjust the system pH to 8.5; S203, dissolve CaCl2﹒2H2O in deionized water to prepare 0.1mMCa 2+ When the system temperature of step S202 drops to 70-72°C, add the prepared Ca 2+ Stock solution, Ca 2+ The amount of stock solution added was set to maintain the Ca 2+ The effective concentration is 0.08~0.12mM; S204. When the system temperature of step S203 drops to 60-64°C, add alkaline protease into the system and continue stirring at 60°C until the system forms a transparent liquid again.

[0012] Preferably, after adding 75% ethanol solution to the supernatant in step S3, at least one temperature cycle is first performed to induce β-fold rearrangement, and the temperature change from 4°C→25°C→4°C is one temperature cycle.

[0013] Preferably, after washing the lower precipitate obtained in step S3, a visible light-responsive sugar-based catalyst TiO2@CQDs is added, and after irradiation at 50°C for 10-15 minutes, a drying operation is performed.

[0014] Preferably, the specific operation of pre-treating the sunflower seed meal in step S1 is: firstly crush the sunflower seed meal with a crusher and then pass it through a 60-mesh sieve, take the sieve material and defat it by Soxhlet extraction, extract it for 6 to 10 hours, and then naturally dry the sieve material after extraction to obtain the defatted sunflower seed meal powder.

[0015] Preferably, in step S2, the microwave power of the microwave extraction is 300-600 W, the extraction time is 5-15 min, and the extraction temperature is 50-60° C.

[0016] Preferably, in step S2, the microwave reactor is set to a multi-stage pulse microwave mode for extraction, and the multi-stage pulse microwave mode is set to: In the initial stage, 600W continuous microwave was used to quickly raise the temperature to 60°C, and the duration was 1-3 minutes; The pulse mode was used in the middle stage, with the microwave power set to 400 W, a duty cycle of 30 s on / 15 s off, and a duration of 4 to 6 min; In the later stage, 300 W microwave power was used to maintain the temperature at 55 ± 1 °C for 4 to 6 min.

[0017] Preferably, after the solution incubated in step S3 is centrifuged, the supernatant is taken and sequentially passed through a 5kDa ultrafiltration membrane and a reverse osmosis membrane to recover the DES solution, and the recovered DES is supplemented with betaine and then recycled.

[0018] Preferably, Ca in step S203 2+ Stock solution available Ca 2+ -Betaine chelate replacement, Ca 2+ - Betaine chelate Ca 2+ The molar ratio of betaine is 1:2.

[0019] The present invention has at least the following beneficial effects: First, the present invention adopts a microwave-assisted DES system to extract sunflower seed meal protein. The DES system is a coupling system of a low eutectic solvent (DES) and alkaline protease. The thermal effect and non-thermal effect of microwaves are used to accelerate the dissolution of sunflower seed meal protein. Combined with the unique dissolution performance of the DES system, the extraction efficiency of sunflower seed meal protein is greatly improved. Compared with the extraction rate of about 60% by the traditional alkali extraction and acid precipitation method, the extraction rate of about 65% by the enzymatic hydrolysis method, and the extraction rate of 70% that is difficult to exceed by ultrasound-assisted extraction, the method for extracting sunflower seed meal protein based on a microwave-assisted low eutectic solvent system provided by the present invention can achieve an extraction rate of more than 80% for sunflower seed meal protein, significantly improving the extraction rate of sunflower seed meal protein and greatly improving the utilization rate of sunflower seed meal protein resources. Second, the present invention further optimizes the microwave parameters and adopts a multi-stage pulse microwave mode. In the initial stage, 600W continuous microwave is used to quickly heat up to 60°C, a 400W pulse mode (30s on / 15s off) is used in the middle stage, and a 300W mode is used to maintain the temperature at 55±1°C in the later stage, which effectively promotes the interaction between the protein and the DES system and further improves the extraction rate of the sunflower seed meal protein. Third, in step S3, the present invention adds 75% ethanol solution to the supernatant and then performs temperature cycling to induce β-folding rearrangement, and after washing the lower precipitate, adds visible light responsive sugar-based catalyst TiO2@CQDs and treats it with light, which effectively improves the structure of the protein, thereby enhancing its emulsifying activity. Compared with the low emulsifying activity sunflower meal protein obtained by the existing method, the protein extracted by the present invention can more effectively stabilize the emulsion system in the preparation of emulsified beverages and other products, reduce the occurrence of emulsion stratification and precipitation, significantly extend the shelf life of the product, and expand the application scope of sunflower meal protein in food, cosmetics and other fields with high requirements for emulsifying performance; Fourthly, the DES solution of the present invention is prepared by using betaine and urea in a molar ratio of 1:1, and the raw materials are widely available and environmentally friendly. Unlike the traditional alkali extraction and acid precipitation method that uses a large amount of acid and alkali reagents to cause environmental pollution, the present invention reduces the use of chemical reagents. At the same time, after the solution incubated in step S3 is centrifuged, the supernatant is taken and the DES solution is recovered through a 5kDa ultrafiltration membrane box reverse osmosis membrane. The recovered DES can be recycled after supplementing with betaine, which reduces production costs, realizes the effective use of resources, and conforms to the concept of green chemistry and sustainable development; Fifth, in the preparation process of the DES system, by strictly controlling the parameters of each step, the urea is divided into two parts, the water content of the mixed solution, the temperature gradient drop, the pH value and the Ca 2+ The composition and performance of the DES system are precisely regulated by adjusting the concentration, etc., so that the DES system and alkaline protease can work better together, providing a stable and suitable reaction environment for the efficient extraction of sunflower seed meal protein. Compared with the enzymatic method, which is difficult to accurately control the reaction conditions, the present invention has obvious advantages in process control and ensures the stability and reliability of the extraction process.

[0020] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The Fourier transform infrared spectra of the sunflower seed meal protein obtained in Example 1 of the present invention and Comparative Example 1 are shown; Figure 2 The secondary structure analysis diagram of the sunflower seed meal protein obtained in Example 1 of the present invention and Comparative Example 1; Figure 3The scanning electron microscope images of the sunflower seed meal protein obtained in Example 1 of the present invention and Comparative Example 1 are shown; Figure 4 This is a thermogravimetric analysis diagram of the sunflower seed meal protein obtained in Example 1 of the present invention; Figure 5 This is a thermogravimetric analysis diagram of the sunflower seed meal protein obtained in Comparative Example 1 of the present invention; Figure 6 This is a solubility analysis chart of the sunflower seed meal protein obtained in Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can implement the invention with reference to the description.

[0023] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0024] Example 1 S1. First, crush the sunflower seed meal with a grinder and pass it through a 60-mesh sieve. Take the sieve material and defat it by Soxhlet extraction. After extraction for 10 hours, dry the sieve material naturally to obtain defatted sunflower seed meal powder; S2. Preparation of DES system: S201, respectively weighing betaine and urea in a molar ratio of 1:1, dividing urea into two parts, the mass ratio of the first part of urea to the second part of urea is 5:1, mixing betaine and the first part of urea and controlling the water content in the mixed solution to be 20%, and stirring the mixed solution at 80° C. until a transparent liquid is formed; S202, continue to add the second portion of urea to the transparent liquid obtained in step S201 and continue stirring, while controlling the temperature gradient to decrease, the temperature decrease frequency is 2°C every 5 minutes, when the temperature drops to 76°C, add NaOH to adjust the system pH to 8.5; S203, dissolve CaCl2﹒2H2O in deionized water to prepare 0.1mMCa 2+ When the system temperature of step S202 drops to 72°C, add the prepared Ca 2+ Stock solution, Ca 2+ The amount of stock solution added was set to maintain the Ca 2+ The effective concentration is 0.12mM; S204, when the system temperature in step S203 drops to 64°C, add alkaline protease to the system and continue stirring at 60°C until the system forms a transparent liquid again; The defatted sunflower seed meal powder obtained in step S1 is added to the prepared DES system, and the mixture is placed in a microwave reactor to extract an extract, wherein the solid-liquid ratio of the defatted sunflower seed meal powder to the DES system is 1:12, the microwave power of the microwave extraction is 600 W, the extraction time is 15 min, and the extraction temperature is 60° C.; S3, heating the extract obtained in step S2 at 80° C. for 10 min to inactivate the enzyme, centrifuging to obtain the supernatant, adding 75% ethanol solution, and refrigerating and incubating at 4° C., centrifuging the incubated solution to obtain the lower precipitate, washing, and drying to obtain the sunflower seed meal protein.

[0025] Example 2 S1, same as in Example 1; S2. Preparation of DES system: same as in Example 1; The defatted sunflower seed meal powder obtained in step S1 is added to the prepared DES system, and placed in a microwave reactor to extract an extract, wherein the solid-liquid ratio of the defatted sunflower seed meal powder to the DES system is 1:12, and the microwave reactor is set to a multi-stage pulse microwave mode for extraction, and the multi-stage pulse microwave mode is set to: In the initial stage, 600W continuous microwave was used to quickly raise the temperature to 60°C for 3 min; The pulse mode was used in the middle stage, with the microwave power set to 400 W, a duty cycle of 30 s on / 15 s off, and a duration of 6 min; In the later stage, 300 W microwave power was used to maintain the temperature at 55 ± 1 °C for 6 min; S3. Same as Example 1.

[0026] Example 3 S1, same as Example 2; S2, same as in Example 2; S3, heating the extract obtained in step S2 at 80°C for 10 minutes to inactivate the enzyme, centrifuging and taking the supernatant, adding 75% ethanol solution, first performing a temperature cycle to induce β-folding rearrangement, the temperature change from 4°C → 25°C → 4°C as one temperature cycle, and then refrigerating and incubating at 4°C, centrifuging the incubated solution to take the lower precipitate, washing, and drying to obtain the sunflower seed meal protein.

[0027] Example 4 S1, same as Example 2; S2, same as in Example 2; S3. The extract obtained in step S2 is heated at 80°C for 10 min to inactivate the enzyme, and the supernatant is centrifuged and added with 75% ethanol solution, and first subjected to a temperature cycle to induce β-folding rearrangement, wherein the temperature changes from 4°C → 25°C → 4°C as one temperature cycle, and then refrigerated and incubated at 4°C, the incubated solution is centrifuged to remove the lower precipitate, washed, and a visible light responsive sugar-based catalyst TiO2@CQDs is added, and the sunflower seed meal protein is obtained after being illuminated at 50°C for 15 min and then dried.

[0028] Example 5 S1, same as Example 2; S2, same as in Example 2; S3. The extract obtained in step S2 is heated at 80°C for 10 min to inactivate the enzyme, and after centrifugation to obtain the supernatant, a 75% ethanol solution is added, and a temperature cycle is first performed to induce β-folding rearrangement, and the temperature changes from 4°C→25°C→4°C as one temperature cycle, and then refrigerated incubation is performed at 4°C, the incubated solution is centrifuged to remove the lower layer of precipitate, washed, and a visible light responsive sugar-based catalyst TiO2@CQDs is added, and the sunflower seed meal protein is obtained after being illuminated at 50°C for 15 min, and then dried to obtain the sunflower seed meal protein. The incubated solution is centrifuged and the supernatant is successively passed through a 5kDa ultrafiltration membrane and a reverse osmosis membrane to recover the DES solution, and the recovered DES is supplemented with betaine and recycled, and can be recycled 8 times.

[0029] Example 6 S1, same as Example 2; S2. Preparation of DES system: S201, respectively weighing betaine and urea in a molar ratio of 1:1, dividing urea into two parts, the mass ratio of the first part of urea to the second part of urea is 5:1, mixing betaine and the first part of urea and controlling the water content in the mixed solution to be 20%, and stirring the mixed solution at 80° C. until a transparent liquid is formed; S202, continue to add the second portion of urea to the transparent liquid obtained in step S201 and continue stirring, while controlling the temperature gradient to decrease, the temperature decrease frequency is 2°C every 5 minutes, when the temperature drops to 76°C, add NaOH to adjust the system pH to 8.5; S203, when the system temperature of step S202 drops to 72°C, add Ca to the system of step S202 2+ -Betaine chelate, Ca 2+ - Betaine chelate Ca 2+ The molar ratio of Ca to betaine is 1:2. 2+ - The amount of betaine chelate added is set to maintain the Ca 2+ The effective concentration is 0.12mM; S204, when the system temperature in step S203 drops to 64°C, add alkaline protease to the system and continue stirring at 60°C until the system forms a transparent liquid again; The remaining steps are the same as in Example 2; S3. Same as Example 2.

[0030] Comparative Example 1 (Alkali Extraction and Acid Precipitation) S1, same as in Example 1; S2. The obtained defatted sunflower seed meal powder is placed in a container, NaOH is added to adjust the pH to 9-10, and the mixture is stirred and extracted at 40° C. for 1.5 hours to obtain an extract.

[0031] S3, centrifuge the extract and collect the supernatant. Slowly add hydrochloric acid solution to the supernatant to adjust the pH to 4-5 to precipitate the protein. Centrifuge again, collect the precipitate, wash it with deionized water for several times, and dry it at 50°C to obtain sunflower seed meal protein.

[0032] Comparative Example 2 S1, same as in Example 1; S2. Add the obtained defatted sunflower seed meal powder to the buffer solution of alkaline protease, control the mass ratio of alkaline protease to substrate at 1:80, adjust the pH to 8.5, and perform enzymolysis at 50° C. for 2 h. After the enzymolysis is completed, heat the reaction solution at 80° C. for 10 min to inactivate the enzyme; and obtain an extract.

[0033] S3. Same as Example 1.

[0034] Comparative Example 3 S1, same as in Example 1; S2, preparing a DES solution composed of betaine and urea in a molar ratio of 1:1, adding the obtained defatted sunflower seed meal powder to the DES solution, and placing the mixture in a microwave reactor to extract an extract, wherein the solid-liquid ratio of the defatted sunflower seed meal powder to the DES system is 1:12, the microwave power of the microwave extraction is 600 W, the extraction time is 15 min, and the extraction temperature is 60° C.; S3. Same as Example 1.

[0035] The extraction rate of the sunflower meal protein obtained in Examples 1 to 6 and Comparative Examples 1 to 3 was calculated, the emulsification stability index ESI of the sunflower meal protein obtained in Examples 1 to 6 and Comparative Examples 1 to 3 was determined, and the solubility of the sunflower meal protein obtained in Examples 1 to 6 and Comparative Examples 1 to 3 was determined (measured by the amount of protein dissolved in a unit mass of solvent), and the protein content was determined by the Kjeldahl method. Accurately weigh a certain amount of sunflower meal protein samples obtained in each embodiment and comparative example, add concentrated sulfuric acid for digestion, and convert the nitrogen in the protein into ammonium sulfate. Then add excess sodium hydroxide, convert the ammonium salt into ammonia and distill it out, absorb it with a boric acid solution, and finally titrate it with a standard hydrochloric acid solution, and calculate the protein content according to the amount of hydrochloric acid consumed. This method is a classic method for determining protein content with high accuracy. The results obtained are shown in Table 1.

[0036] Table 1 Extraction rate, emulsifying activity and solubility of sunflower seed meal protein As shown in Table 1, the extraction rates of Examples 1 to 6 range from 82 to 85%, which are much higher than those of Comparative Example 1 (alkali extraction and acid precipitation method, 60%), Comparative Example 2 (enzymatic method, 64%), and Comparative Example 3 (ordinary low eutectic solvent microwave extraction method, 63%). The embodiment adopts a microwave-assisted DES system, wherein the DES system is a coupling system of a DES solution and an alkaline protease, the mass ratio of the alkaline protease to the DES solution is 1:80 to 100, and the DES solution is prepared by using betaine and urea with a molar ratio of 1:1. The thermal effect of microwaves causes the temperature of the system to rise rapidly, accelerates the molecular movement, and strongly promotes the dissolution of the protein. The coupling system of the DES solution and the alkaline protease adjusts the pH value of the DES system to 8.5 by NaOH, ensuring that the catalytic active center of the enzyme is in the optimal ionized state, thereby maintaining efficient hydrolysis ability. At the same time, controlling the pH of the DES system in a weakly alkaline environment can reduce the decomposition of DES components (such as urea) (such as the formation of cyanate), thereby avoiding the inhibition of the activity of the alkaline protease by byproducts. Furthermore, betaine, as an osmotic protectant, stabilizes the natural conformation of alkaline protease through preferential exclusion, reduces the risk of denaturation of alkaline protease under high temperature or high viscosity environment, and partially immobilizes urea in the DES hydrogen bond network, and its free concentration is significantly lower than that in the pure water system, thereby reducing the damage of the chaotropic effect to the alkaline protease, thereby optimizing the activity and stability of the alkaline protease. The DES solution (betaine-urea) dissolves the hemicellulose and pectin in the sunflower seed meal through penetration and hydrogen bond breaking, destroys the integrity of the cell wall, and makes the internal protein more easily exposed. Microwave-assisted heating further accelerates the rupture of the cell wall, forming nano-scale pores (SEM shows that the pore size is significantly enlarged), promoting the contact between the enzyme and the substrate. At the same time, the strong polar environment of the DES solution (betaine-urea) causes the hydrophobic protein to undergo reversible denaturation, exposing more enzyme cleavage sites (such as the peptide bonds of the hydrophobic core), and improving the hydrolysis efficiency of the alkaline protease. More importantly, alkaline protease selectively hydrolyzes alcohol-soluble proteins in sunflower seed meal, retaining the target water-soluble / salt-soluble proteins, reducing the intensity of the impurity protein band (20~30kDa) by 60%~70%; enzymatic hydrolysis of polyphenol-protein complexes reduces the interference of anti-nutritional factors such as tannins on the target protein, and the free phenol content decreases by about 40%, further improving the nutritional utilization of the final product. Furthermore, the non-thermal effect of microwaves (high-frequency oscillation of polar molecules) reduces the viscosity of the system, accelerates the frequency of enzyme-substrate diffusion collisions, and the pulsed microwave mode avoids local overheating and maintains the stability of enzyme activity. Finally, 0.1mMCa was added 2+ The active center conformation of the enzyme was locked by coordination, which prolonged its half-life in the DES environment. Adding alkaline protease to the DES system and controlling the pH at 8.5 formed a synergistic process of dissolution-targeted hydrolysis-stabilization, thereby improving the extraction rate of sunflower seed meal protein.

[0037] It can also be seen from Table 1 that the ESI of Examples 1 to 6 is between 65 and 90 min, while the ESI of Comparative Example 1 is only 30 min, the ESI of Comparative Example 2 is 40 min, and the ESI of Comparative Example 3 is 45 min. There are four reasons for this. First, alkaline protease enzymatic hydrolysis induces protein structure remodeling, which is specifically manifested in the moderate hydrolysis of multiple chains and the optimization of protein spatial conformation. Alkaline protease selectively cuts the peptide bonds near the hydrophobic amino acids in the protein peptide chain to generate small molecule peptide segments. Small molecule peptides have higher surface activity and are more easily adsorbed on the oil-water interface to form a stable membrane structure, thereby regulating the molecular weight; hydrolysis exposes the hydrophobic region and hydrophilic groups originally embedded in the protein, exposes the functional domain, and enhances amphiphilicity. Alkaline protease enzymatic hydrolysis reduces the α-helix content, increases the β-folding and random coiling ratio, improves the flexibility of the molecular chain, and promotes interface spreading; at the same time, during the alkaline protease enzymatic hydrolysis, some disulfide bonds are broken and rearranged to form a looser but stable three-dimensional network, which optimizes the protein spatial conformation. The second is the synergistic effect of DES solution. Betaine-urea destroys hemicellulose and pectin through hydrogen bonds, releasing bound proteins and providing sufficient substrate for enzymatic hydrolysis. Betaine, as a compatible solute, reduces the denaturing effect of DES on the enzyme. Urea is fixed by hydrogen bonds in DES, reducing the free concentration and alleviating the chaotropic effect. At the same time, the weak alkaline environment of DES solution increases the negative charge on the protein surface, thereby enhancing the electrostatic repulsion between emulsion droplets and inhibiting aggregation. The third is the optimization of interfacial adsorption kinetics. The diffusion coefficient of small molecule peptides is higher than that of intact proteins, which improves the diffusion efficiency. Enzymatic peptides form a dense interfacial film through intermolecular hydrogen bonds and hydrophobic effects. The fourth is the coordinated regulation of process parameters. The assistance of pulsed microwaves promotes enzyme-substrate binding through local thermal effects, while avoiding enzyme inactivation caused by continuous high temperature. The control of temperature gradient balances enzyme activity and protein denaturation rate. Adding 0.1mMCa 2+ Coordinates with the Asp residue in the active center of the enzyme to fix the conformation of the catalytic triad, Ca 2+ The repulsion between peptide chains is reduced through electrostatic shielding, and hydrophobic interactions are promoted. In summary, the microwave-assisted DES-alkaline protease synergistic system significantly improves the emulsification performance of sunflower seed meal protein through the triple mechanism of targeted enzymolysis-structural remodeling-interface modification. Example 3 induces β-folding rearrangement through temperature cycling, changes the secondary structure of the protein, changes the intermolecular interaction, forms a structure that is more conducive to emulsification, and then improves the emulsification stability; in Examples 4 and 5, the visible light responsive sugar-based catalyst TiO2@CQDs reacts specifically with the protein under light, introduces functional groups on the protein surface, or changes the charge distribution on the protein surface, enhances the dispersibility and interfacial activity of the protein in the emulsion system, and greatly improves the emulsification activity. Compared with the traditional alkali extraction and acid precipitation method, which easily causes excessive denaturation of the protein, the extraction process of the present invention is mild, has little damage to the natural structure and functional groups of the protein, retains the structure related to the emulsification activity of the protein, and makes the extracted protein have better emulsification performance.

[0038] It can be seen from the data in Table 1 that the protein solubility of Examples 1 to 6 is 12 to 18 g / 100 mL, the solubility of Comparative Example 1 is only 8 g / 100 mL, the solubility of Comparative Example 2 is 10 g / 100 mL, and the solubility of Comparative Example 3 is 11 g / 100 mL. Similar to the improvement of emulsification activity, the microwave-assisted DES-alkaline protease synergistic system forms a three-step mechanism of wall breaking release-targeted enzymolysis-solvent stability to improve the solubility of sunflower seed meal protein through the destruction of protein structure, reduction of molecular weight, optimization of surface properties, efficient cell wall breaking and optimization of process parameters. Temperature cycle-induced β-folding rearrangement changes the protein structure with TiO2@CQDs light treatment, so that the hydrophilic groups of protein molecules are more exposed on the surface, increasing the interaction between protein and solvent molecules, thereby improving solubility. During the extraction process, the microwave-assisted DES system modifies the protein surface and removes impurities that affect protein dissolution, making the protein more soluble in the solvent. At the same time, precisely controlled reaction conditions help maintain the stable structure of the protein, avoid protein aggregation or denaturation due to improper reaction conditions, and improve protein solubility.

[0039] It can be seen from the data in Table 1 that the method for extracting sunflower seed meal protein by the microwave-assisted DES-alkaline protease synergistic system provided by the present invention has a DES solution with a number of cycles greater than or equal to 8 times, and can even reach 15 times, but the ordinary low eutectic solvent extraction method extracts sunflower seed meal protein (Comparative Example 3), and its DES can be circulated less times. This is because the ordinary low eutectic solvent undergoes some irreversible reactions with the components in the sunflower seed meal during the recycling process, and on the other hand, it is affected by external factors such as temperature and pH during the extraction process, resulting in changes in the composition and properties of the solvent, which reduces its extraction ability. In this case, too many cycles may make the extraction effect worse and worse, so a smaller number of cycles is selected to ensure a certain extraction effect.

[0040] In order to further compare the difference between the microwave-assisted DES-alkaline protease synergistic system of the present invention and the traditional alkali extraction and acid precipitation method, the Fourier transform infrared spectra of the sunflower seed meal protein obtained in Example 1 and Comparative Example 1 were measured (eg Figure 1 ) and secondary structure analysis (as shown Figure 2 Fourier transform infrared spectroscopy was used to analyze the -1 The infrared spectrum of the protein sample was measured in the range of 1700-1600 cm. By identifying the different peaks in the FTIR spectrum, the information of the functional groups in the protein was obtained. In addition, by analyzing the amide I region of the spectrum (1700-1600 cm -1) to obtain information about the secondary structure of the protein. The results are as follows: Fourier transform infrared spectroscopy (FT-IR) was used to analyze the changes in the secondary structure of the protein after alkali extraction, acid precipitation and alkaline protease synergistic system extraction. One of the unique FT-IR bands of sunflower meal protein is located at 1600-1700cm -1 The changes in the amide region are mainly caused by the stretching vibration of the amide CO bond, reflecting the changes in the secondary structure of the protein molecule. The different amide I band wavelength positions correspond to different secondary structures of the protein molecule. These structures include α-helix (1650-1660cm -1 )、β-fold(1610-1642cm -1 )、β-angle(1660-1680cm -1 ), β-reverse folding (1680-1700cm -1 ) and random curl (1642-1650cm -1 ). Figure 2 The proportion of secondary structure content of β-fold and β-turn is the ratio of the total area of ​​amide I band to the equivalent area. According to the figure, β-fold is the main component of protein secondary structure. Compared with the protein extracted by alkali extraction and acid precipitation and the untreated powder, DES extraction has more β-fold. The hydrogen bonds formed between -C=O and -NH2 groups of the same or adjacent peptides help stabilize the β-fold structure. The increase of β-fold indicates that the normal and stable structure of the protein has been replaced by an elastic and disordered structure. At the same time, the increase of β-fold is accompanied by the exposure of hydrophobic groups, which is conducive to protein aggregation and leads to changes in protein function. However, the good elasticity and ductility of β-fold can be used in jelly, jam, biomedicine and other fields.

[0041] In order to further compare the difference between the microwave-assisted DES-alkaline protease synergistic system of the present invention and the traditional alkali extraction and acid precipitation method, the present invention further measured the scanning electron microscopy images of the sunflower seed meal protein obtained in Example 1 and Comparative Example 1 (as shown in FIG. Figure 3 As shown in Figure 2, sunflower meal protein was dried in an oven at 120°C to constant weight. The dried protein samples were attached to short stakes with double-sided adhesive and sprayed with gold before being examined under a scanning electron microscope. All samples were tested at an accelerating voltage of 7 kV. The results are as follows: The microstructure of the protein extracted by alkali extraction and acid precipitation and DES was observed by scanning electron microscopy. The results are shown in Figure 2. Figure 3As shown in the figure, the proteins (a) and (b) extracted by the alkali extraction and acid precipitation method are irregular and rough particles, which may be because the alkali extraction and acid precipitation lead to the exposure of more hydrophobic groups and protein aggregation. Compared with the alkali extraction and acid precipitation method, the proteins (c) and (d) extracted by the microwave-assisted DES-alkaline protease synergistic system are smooth, which may be due to the good solubility of the extracted protein. And they appear in the form of aggregation because the hydrogen bonds in the protein molecules are destroyed during the extraction, and hydrogen bonds are formed between the proteins, which aggregate the proteins. This protein can be widely used in the food field, such as baked products and pasta products, to increase the crisp effect, improve the rheological properties of the dough, and have a better taste.

[0042] In order to further compare the difference between the microwave-assisted DES-alkaline protease synergistic system of the present invention and the traditional alkali extraction and acid precipitation method, the present invention also measured the thermogravimetric analysis of the sunflower seed meal protein obtained in Example 1 and Comparative Example 1. The thermogravimetric analysis of the sunflower seed meal protein obtained in Example 1 is as follows: Figure 4 As shown, the thermogravimetric analysis of the sunflower seed meal protein obtained in Comparative Example 1 is as follows Figure 5 As shown. The thermal properties of the protein extracts were determined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Thermogravimetric analysis is performed by measuring the change in mass when a sample is heated under controlled conditions. Initially, 10 mg of freeze-dried protein sample was placed in a high-temperature resistant platinum pan. The sample was then heated from 25 to 1000°C at a rate of 10°C / min while continuously flushing with 90 mL / min of nitrogen, and the mass was recorded. DSC analysis is performed by measuring the heat flow of the sample and reference when heated under controlled conditions. First, 10 mg of freeze-dried protein sample was placed in a platinum pan and sealed, while an empty sealed platinum pan was used as a reference. Then, the two pans were heated from 30°C to 1000°C at a rate of 10°C / min while flushing with nitrogen at a flow rate of 100 mL / min, and the heat flow was recorded. The results are as follows: Thermogravimetric analysis (TG) and differential thermogravimetric analysis (DTG) are often used to explore the relationship between the weight and temperature of high molecular weight polymers. As Figure 4 and 5As shown in the figure, the mass loss curves of proteins extracted by microwave-assisted DES-alkaline protease synergistic system and alkali extraction and acid precipitation method can be divided into three cycles between 30℃ and 1000℃. In the first stage of 30~200℃, the mass loss of proteins extracted by microwave-assisted DES is 10.51g / 100g, and the mass loss of proteins extracted by alkali extraction and acid precipitation method is 6.45g / 100g, which is due to the loss of free water and bound water in the sample. At 200℃, the masses of proteins extracted by alkali extraction and acid precipitation method and microwave-assisted DES are 93.55 and 89.49g / 100g, respectively. Among them, the mass loss of proteins extracted by microwave-assisted DES-alkaline protease synergistic system is the largest, followed by the mass loss of proteins extracted by alkali extraction and acid precipitation method. The mass loss is large and the water content is high. The second mass loss zone usually occurs at 200~400℃, which is attributed to the thermal decomposition of proteins. At such high temperatures, covalent bonds are cleaved, resulting in the formation of more volatile substances, which are lost from the sample. The maximum mass loss rate of the protein extracted by the microwave-assisted DES-alkaline protease synergistic system occurred at 265.12°C, and the maximum mass loss rate of the protein extracted by the alkali extraction and acid precipitation method occurred at 314.61°C. Among them, the protein extracted by the alkali extraction and acid precipitation method has the highest thermal stability, which may be related to the presence of hydrophobic groups on the surface and cross-linked hydrogen bonds inside. This structure improves the thermal stability of the protein. The third stage is when the temperature exceeds 400°C, representing the degradation of the components and the carbonization of the residue. The mass loss of the protein extracted by the alkali extraction and acid precipitation method is significantly lower than that of the protein extracted by the microwave-assisted DES-alkaline protease synergistic system. This may be related to the extraction method and the purity of the protein in the extract, or it may be because there are hydrophobic groups on the surface and cross-linked hydrogen bonds inside, which improves the thermal stability of the protein.

[0043] In order to further compare the difference between the microwave-assisted DES-alkaline protease synergistic system of the present invention and the traditional alkali extraction and acid precipitation method, the present invention also measured the solubility of the sunflower seed meal protein obtained in Example 1 and Comparative Example 1 (e.g. Figure 6 As shown), the solubility determination method is as follows: Mix 1g of sunflower seed meal protein with 100 mL of water, shake vigorously for 1h at room temperature, and centrifuge at 8000rpm for 10min. Weigh the precipitate and determine the nitrogen content by Kjeldahl nitrogen determination, solubility = (m1-m2) / m1, where m1 is the total nitrogen content in 1g, in g; m2 is the nitrogen content that is not dissolved in water, in g. The results are as follows: Good solubility is related to the various functional properties of protein and is the key to the processing and application of food protein. As shown Figure 6As shown in the figure, the solubility of the protein extracted by DES is the highest, followed by the protein extracted by alkali extraction and acid precipitation. This may be related to the extraction system. The protein unfolds in DES under alkaline conditions, and the electrostatic repulsion between proteins is strong enough to prevent excessive aggregation of proteins, resulting in high solubility. However, the principle of alkali extraction and acid precipitation is electrostatic repulsion at high pH, ​​which promotes the exposure of hydrophobic groups and the aggregation of proteins through hydrophobic interactions. At the isoelectric point, the hydrophobic groups are further enhanced by electrostatic forces, thereby forming a compact protein structure, which also leads to poor functional properties of proteins extracted by alkali extraction and acid precipitation, showing lower solubility.

[0044] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the specific embodiments shown and described herein.

Claims

1. A method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system, characterized in that: include: S1, pretreating sunflower seed meal to obtain defatted sunflower seed meal powder; S2, adding the defatted sunflower seed meal powder obtained in step S1 to the DES system, and placing the mixture in a microwave reactor to extract an extract, wherein the solid-liquid ratio of the defatted sunflower seed meal powder to the DES system is 1:10-14; S3, inactivating the enzyme in the extract obtained in step S2, centrifuging and taking the supernatant, adding 75% ethanol solution, refrigerating and incubating at 4° C., centrifuging the incubated solution to take the lower layer of precipitate, washing, and drying to obtain the sunflower seed meal protein; The DES system in step S2 is a coupling system of a DES solution and alkaline protease, the mass ratio of alkaline protease to the DES solution is 1:80-100, and the DES solution is prepared by using betaine and urea in a molar ratio of 1:

1.

2. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 1, characterized in that: The preparation method of the DES system in step S2 is as follows: S201, respectively weighing betaine and urea in a molar ratio of 1:1, dividing urea into two parts, wherein the mass ratio of the first part of urea to the second part of urea is 5-8:1, mixing betaine and the first part of urea and controlling the water content in the mixed solution to be 15-20%, and stirring the mixed solution at 80° C. until a transparent liquid is formed; S202, continue to add the second portion of urea to the transparent liquid obtained in step S201 and continue stirring, while controlling the temperature gradient to decrease, the temperature decrease frequency is 2-3°C every 5 minutes, when the temperature drops to 75-76°C, add NaOH to adjust the system pH to 8.5; S203, dissolve CaCl2﹒2H2O in deionized water to prepare 0.1mMCa 2+ When the system temperature of step S202 drops to 70-72°C, add the prepared Ca 2+ Stock solution, Ca 2+ The amount of stock solution added was set to maintain the Ca 2+ The effective concentration is 0.08~0.12mM; S204. When the system temperature of step S203 drops to 60-64°C, add alkaline protease into the system and continue stirring at 60°C until the system forms a transparent liquid again.

3. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 1, characterized in that: After adding 75% ethanol solution to the supernatant in step S3, at least one temperature cycle is performed to induce β-fold rearrangement, and the temperature changes from 4°C → 25°C → 4°C as one temperature cycle.

4. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 1, characterized in that: After washing the lower precipitate obtained in step S3, a visible light-responsive sugar-based catalyst TiO2@CQDs is added, and after irradiation at 50°C for 10-15 minutes, a drying operation is performed.

5. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 1, characterized in that: The specific operation of pre-treating the sunflower seed meal in step S1 is: firstly crush the sunflower seed meal with a crusher and then pass it through a 60-mesh sieve, take the sieve material and defat it by Soxhlet extraction, extract it for 6 to 10 hours, and then naturally dry the sieve material after extraction to obtain the defatted sunflower seed meal powder.

6. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 1, characterized in that: In step S2, the microwave power of the microwave extraction is 300-600 W, the extraction time is 5-15 min, and the extraction temperature is 50-60° C.

7. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 6, characterized in that: In step S2, the microwave reactor is set to a multi-stage pulse microwave mode for extraction, and the multi-stage pulse microwave mode is set to: In the initial stage, 600W continuous microwave was used to quickly raise the temperature to 60°C, and the duration was 1-3 minutes; The pulse mode was used in the middle stage, with the microwave power set to 400 W, a duty cycle of 30 s on / 15 s off, and a duration of 4 to 6 min; In the later stage, 300 W microwave power was used to maintain the temperature at 55 ± 1 °C for 4 to 6 min.

8. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 1, characterized in that: Step S3: After the incubated solution is centrifuged, the supernatant is taken and sequentially passed through a 5 kDa ultrafiltration membrane and a reverse osmosis membrane to recover the DES solution. The recovered DES is supplemented with betaine and then recycled.

9. The method for extracting sunflower seed meal protein based on a microwave-assisted deep eutectic solvent system according to claim 2, characterized in that: Ca in step S203 2+ Stock solution available Ca 2+ -Betaine chelate replacement, Ca 2+ - Betaine chelate Ca 2+ The molar ratio of betaine to 1:2.

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