阅读说明：本技术 一种玉米醇溶蛋白-多酚共价复合物及其制备方法 (Zein-polyphenol covalent compound and preparation method thereof ) 是由 魏子淏 徐雅男 薛长湖 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种制备玉米醇溶蛋白–多酚共价复合物的方法,属于食品科学和食品加工技术领域。本发明以玉米醇溶蛋白和植物多酚为原料,采用碳二亚胺介导的偶联法在乙醇-水溶液中构建了玉米醇溶蛋白–多酚共价复合物。所述制备方法具有简单、高效和安全的特点。与对照玉米醇溶蛋白相比,所制备的玉米醇溶蛋白–多酚共价复合物具有更好的热稳定性和溶解性,且共价复合物的抗氧化性得到了显著的提升。本发明产品可以作为功能性食品添加剂,也可以用于构建新型的递送系统。此外,本方法制备的玉米醇溶蛋白–多酚共价复合物仍然保持蛋白原有的淡黄色,更易于被消费者接受,有利于扩大其应用范围。(The invention discloses a method for preparing a zein-polyphenol covalent compound, and belongs to the technical field of food science and food processing. The zein-polyphenol covalent compound is constructed in an ethanol-water solution by taking zein and plant polyphenol as raw materials and adopting a carbodiimide-mediated coupling method. The preparation method has the characteristics of simplicity, high efficiency and safety. Compared with the control zein, the prepared zein-polyphenol covalent compound has better thermal stability and solubility, and the oxidation resistance of the covalent compound is obviously improved. The product of the invention can be used as a functional food additive and can also be used for constructing a novel delivery system. In addition, the zein-polyphenol covalent compound prepared by the method still keeps the original faint yellow color of the protein, is easier to be accepted by consumers, and is beneficial to expanding the application range of the zein-polyphenol covalent compound.)

1. A preparation method of a zein-polyphenol covalent compound is characterized by comprising the following steps:

1) weighing a certain amount of zein, dissolving in 70% (v/v) ethanol-water solution, performing ultrasonic treatment for 5 min, and stirring at 200 rpm for 30 min to completely dissolve.

2) Weighing a certain amount of polyphenol, dissolving in 70% (v/v) ethanol-water solution, performing ultrasonic treatment for 5 min, and stirring at 200 rpm for 30 min to completely dissolve the polyphenol;

3) adding a certain amount of EDC and then a certain amount of NHS into the polyphenol solution obtained in the step 2) at 4 ℃, and continuously stirring (300 rpm) without oxygen for reaction for 0.5-2 h;

4) slowly adding the polyphenol solution obtained in the step 3) into the zein solution obtained in the step 1), adjusting the pH to 5-7, reacting for 1 h at 4 ℃ under the condition of continuous stirring (300 rpm), and then reacting for 24 h at 25 ℃;

5) during the reaction, the pH of the reaction solution was measured every four hours, and the pH was maintained at a fixed value by adding NaOH (0.1M) and HCl (0.1M);

6) after the reaction is finished, the mixture passes through an ultrafiltration device, and the liquid obtained by ultrafiltration is dialyzed in ultrasound.

7) And (3) treating the dialyzed sample by using a nano grinder, pre-freezing, freezing and drying to obtain the zein-polyphenol covalent compound.

2. The method of claim 1, wherein the concentration of zein in the mixing system of step 1) is in the range of 5 mg/mL to 25 mg/mL; the concentration of polyphenol in the step 2) is 2-30 mmol/L; the amount of EDC in the step 3) is 1-5 times of the addition amount of polyphenol, and the addition amount of NHS is 1-3 times of polyphenol.

3. The method of claim 1, wherein the mass ratio of zein to polyphenols in the mixed system of step 4) is 1.5:1 to 10: 1.

4. The method of making a zein-polyphenol covalent complex of claim 1 in which the pH of step 4) is 6.5.

5. The method of claim 1, wherein the polyphenol is selected from the group consisting of chlorogenic acid (CGA), Gallic Acid (GA), caffeic acid, protocatechuic acid, and rosmarinic acid.

6. The method for preparing zein-polyphenol covalent compound as claimed in claim 1, wherein the ultrafiltration membrane of step 6) has a molecular weight of 30 kDa, the dialysis bag has a molecular weight of 3500 Da, the dialysis time is 48 h, and the dialysis temperature is 4 ℃.

7. The method of claim 1, wherein the prefreezing in step 7) is performed at-40 ℃ for 24 hours and the freeze-drying time is 48 hours.

8. The method of claim 2, wherein EDC is added in an amount of 3 times the amount of polyphenol and NHS is added in an amount of 2 times the amount of polyphenol.

9. The method of claim 1, wherein 0.001% (w/v) potassium sorbate is added in step 1).

Technical Field

The patent specifically relates to a preparation method of a zein-polyphenol covalent compound, and belongs to the technical field of food science and food processing.

Background

Zein (zein) is a natural vegetable protein isolated from corn seeds, and is commercially predominantly alpha-zein, having a molecular weight of about 22 kDa. Zein has good biodegradability and biocompatibility, and is often used for constructing a delivery system and delivering bioactive substances. Zein has a large number of nonpolar amino acids and alpha-helical structures in molecules, so that the zein has strong surface hydrophobicity and is hardly dissolved in water. In addition, natural zein is less resistant to oxidation. The low water solubility and antioxidant properties of zein may limit its use in food products. In recent years, there have been a number of studies showing that covalently grafting polyphenols onto proteins can improve the solubility and antioxidant properties of proteins. Plant polyphenols are plant secondary metabolites and are currently the most interesting natural antioxidants. In addition, polyphenol also has a plurality of beneficial effects of anti-inflammation, antivirus, anticancer and the like, so that a proper amount of polyphenol is beneficial to human health. At present, the non-covalent interaction between zein and polyphenols is well studied, but there is little research on the covalent reaction between zein and polyphenols, and the existing method for preparing zein-polyphenol covalent complexes is an alkaline method, the principle is based on the oxidation of polyphenols, which results in products with darker colors

As shown in the following patents: application No.: 202011178964.7, publication No. CN112121178A, entitled "a water-soluble zein-EGCG covalent complex and preparation and application thereof", discloses a method for preparing a zein-epigallocatechin gallate (EGCG) covalent complex, which comprises the following steps: (1) dispersing zein and EGCG in water to form a mixed system, and then adjusting the pH value to 11.0-13.0 to carry out condensation reaction; (2) and after the reaction is finished, separating by using a dialysis bag, and drying the dialyzed solution to obtain the zein-EGCG covalent compound.

The article "A synthetic study of covalent and non-covalent interactions between zein and polyphenols in ethanol-water solution" synthesizes covalent complexes of zein with EGCG, Chlorogenic Acid (CA) and quercetin (Q). The method mainly comprises the following steps: 1 g zein was dissolved in 50 mL 70% (v/v) ethanol-water solution and then the pH was adjusted to 9.0 with 0.1 mol/L NaOH. To prepare the zein-polyphenol covalent complex, 0.2 g of individual polyphenols (EGCG, CA or Q) were dissolved in 50 mL of 70% (v/v) ethanol-water solution and the pH was adjusted to 9.0. The two solutions were then mixed together and exposed to air at 25 ℃ for reaction. After 24 hours, the samples were placed in an ultrasonic bath, exchanged 10 times for 70% (v/v) ethanol-water solution, dialyzed for 24 hours to remove free polyphenols and finally lyophilized to obtain a porous solid. On the basis, the non-covalent zein-polyphenol compound is prepared under the anaerobic condition of pH 7.0.

Disclosure of Invention

The invention provides a novel preparation method of a zein-polyphenol covalent compound, which adopts carbodiimide-mediated coupling reaction to covalently graft polyphenol onto zein.

The preparation method of the zein-polyphenol covalent compound provided by the invention comprises the following steps:

1) weighing a certain amount of zein, dissolving in 70% (v/v) ethanol-water solution, performing ultrasonic treatment for 5 min, and stirring at 200 rpm for 30 min to completely dissolve.

2) Weighing a certain amount of polyphenol, dissolving in 70% (v/v) ethanol-water solution, performing ultrasonic treatment for 5 min, and stirring at 200 rpm for 30 min to completely dissolve the polyphenol;

3) adding a certain amount of EDC and then a certain amount of NHS into the polyphenol solution obtained in the step 2) at 4 ℃, and continuously stirring (300 rpm) without oxygen for reaction for 0.5-2 h;

4) slowly adding the polyphenol solution obtained in the step 3) into the zein solution obtained in the step 1), adjusting the pH to 5-7, reacting for 1 h at 4 ℃ under the condition of continuous stirring (300 rpm), and then reacting for 24 h at 25 ℃;

5) during the reaction, the pH of the reaction solution was measured every four hours, and the pH was maintained at a fixed value by adding NaOH (0.1M) and HCl (0.1M);

6) after the reaction is finished, the mixture passes through an ultrafiltration device, and the liquid obtained by ultrafiltration is dialyzed in ultrasound.

7) And (3) treating the dialyzed sample by using a nano grinder, pre-freezing, freezing and drying to obtain the zein-polyphenol covalent compound.

Preferably, the concentration of the zein in the mixing system in the step 1) is 5 mg/mL-25 mg/mL. The concentration of polyphenol in the step 2) is 2-30 mmol/L. The mass ratio of the zein to the polyphenol in the mixed system in the step 4) is 1.5: 1-10: 1.

By reasonably setting the concentration of the zein and the concentration of the polyphenol, the mass ratio of the zein to the polyphenol can influence the grafting rate of the polyphenol on the protein, if the amount of the polyphenol is too small, the grafting rate is lower, but the amount of the polyphenol capable of being covalently bonded with the zein is limited, and considering economic factors, the amount of the polyphenol cannot be too much, because the cost of the polyphenol is higher compared with that of the protein.

In addition, the amount of EDC also affects the reaction results, and too much EDC may cause crosslinking of the protein itself, and too little EDC may cause low coupling efficiency.

Preferably, the amount of EDC is 1-5 times of the polyphenol, and the amount of NHS is 1-3 times of the polyphenol. Further, the amount of EDC was 3 times the amount of polyphenol added, and the amount of NHS was 2 times the amount of polyphenol added, where the added amount is a molar amount.

The publication No. CN112121178A discloses a water-soluble zein-EGCG covalent compound and preparation and application thereof, and although the zein-EGCG covalent compound is also disclosed, the content of polyphenol in the covalent compound prepared by the method is lower.

There is currently available literature on the EDC/NHS method for the preparation of polysaccharide-polyphenol covalent complexes, for example, the literature published in the prior art under the names "Grafting of collagen acid on to chitosan industries antioxidant activities and alkaline cellulosic properties of the copolymer". This document stirs Chitosan (CS) (0.303 g, 1.85 mmol) with HOBt (0.282 g, 1.85 mmol) in deionized water (30.0 mL) overnight until a clear solution is obtained. To the CS solution was added GA (0.311 g, 1.85 mmol) and then 2.0 mL of EDC.HCl in ethanol (0.355 g, 1.85 mmol) was added dropwise. The reaction is carried out at normal temperature and normal pressure, and the reaction time is 24 h. And pouring the generated liquid into a dialysis tube with the molecular weight of 3500 Da, dialyzing in deionized water for 48 h, replacing the deionized water for 8 times, and freeze-drying the obtained solution to obtain the GA-g-CS solid copolymer.

The method of the above document differs from the present patent mainly in that:

(1) with HOBt (hydroxybenzotriazole), strong ionic and hydrogen bonds exist between the hydroxyl groups of HOBt and the amino groups of proteins, which reduces the covalent interactions between the amino groups of proteins and the polyphenol active groups.

(2) Without the addition of NHS, more side reactions occurred.

(3) The polyphenol has high consumption and high cost.

A similar process is disclosed in the prior art document, Amphiphilic carboxmethyl chitosan-quercetin conjugates with P-gp inhibition properties for oral delivery of paclitaxel, by dissolving 0.4 g of carboxymethyl chitosan (CMCS) in 20 mL of distilled water, dissolving quercetin (Qu) in Dimethylformamide (DMF), and adding it dropwise to the CMCS solution under ice bath and continuous stirring. DMAP (5% of the reaction mass), EDC and NHS (EDC and NHS in a molar ratio of 1: 1.4) were then added to the mixture. After 30 min, the reaction was allowed to warm to room temperature and stirred in the dark for 24 hours. At the end of the reaction, the reaction mixture was precipitated in acetone and filtered. The filtered solid sample was carefully washed with acetone and absolute ethanol in order to remove free quercetin, and the washed sample was dried under vacuum. The dried sample was dissolved in deionized water, dispersed with probe sonication in an ice bath for 20 min, and then centrifuged at 3000 rpm for 10 min. Filtering the supernatant with 0.8 μm water film to remove large particles, and placing into dialysis bag with molecular weight cutoff of 14000 Da to remove small molecular impurities and trace organic solvent.

The method of the above document differs from the present patent mainly in that:

(1) different reaction substrates, dimethylformamide is used in the literature, and the organic reagent cannot be used in food.

(2) Failure to adjust the pH of the reaction may result in a less efficient reaction.

The invention obtains the technical scheme for preparing the protein-polyphenol covalent complex by the EDC method on the basis of referring to the preparation of the polysaccharide-polyphenol covalent complex by the EDC method.

Because of the large difference in the properties of spatial structure, molecular weight, etc. of protein and polysaccharide, the method for preparing polysaccharide-polyphenol covalent complex by EDC method cannot be directly used for preparing protein-polyphenol covalent complex. It was found that the grafting yield of polysaccharide-polyphenol covalent complexes prepared by the EDC process is influenced by the reaction conditions, in relation to the degree of depolymerization of the polysaccharide, the molecular weight, the initial molar ratio of polysaccharide to polyphenol, the reaction pH, the temperature and the time. Because of the large difference in the structure, molecular weight and other properties of different polysaccharides and polyphenols, experimenters usually refer to the molecular ratio of predecessors in EDC experiments. The structure, molecular weight and number of amino groups contained in protein and polysaccharide are greatly different, so that the protein-polyphenol covalent complex cannot be prepared by directly carrying out the carbodiimide method to prepare the polysaccharide-polyphenol. Conditions such as initial molar ratio of protein to polyphenol and reaction pH need to be optimized to allow the reaction to proceed and achieve higher grafting yield.

Preferably, in step 4), the polyphenol solution of step 3) is added to the zein solution of step 1) and the pH is adjusted to 6.5. The main technical considerations are: the reaction of polyphenols with zein is mainly divided into two steps. In the first step, EDC activates the carboxyl groups of the polyphenol to form an intermediate product, which is suitably carried out under acidic conditions (pH 4-6). In the second step, the intermediate is subjected to an amino attack to form amide cross-links, which is suitably carried out under slightly alkaline conditions. The selection of a neutral solution can compromise both.

Preferably, the polyphenol is selected from one or more of chlorogenic acid (CGA) and Gallic Acid (GA). The binding affinities of polyphenols with different molecular weights and structures to proteins differ. In the patent, polyphenol with carboxyl is mainly selected, because the carboxyl of the polyphenol reacts with EDC reagent to generate an intermediate product with higher reaction activity, but the intermediate product easily reacts with water to generate a byproduct, so NHS is added to protect the intermediate product. The intermediate product is then reacted with reactive groups (e.g., amino groups) on the protein.

Other polyphenols of choice are caffeic acid, protocatechuic acid, and rosmarinic acid, among others.

Preferably, 0.001% (w/v) potassium sorbate is also added in the step 1). In the experimental process, the zein solution is found to breed microorganisms, the covalent grafting of the zein and the polyphenol is seriously influenced, and even after the covalent grafting is finished, the covalent grafting structure can be damaged. It is contemplated that the growth of microorganisms may be inhibited by the addition of specific components or special treatments.

It was considered that the addition of sodium azide (0.005, w/w) to the protein solution could well inhibit the growth of microorganisms, but sodium azide is toxic and not suitable.

A large number of experiments prove that the potassium sorbate with the concentration of 0.001% (w/v) is added into the zein solution in the step 1), so that the sterilization effect can be well achieved.

The invention has the beneficial effects that:

the prior art research shows that: the oxidation resistance, surface hydrophobicity, thermal stability and the like of the protein can be improved by covalently grafting the polyphenol to the protein; the related studies of zein-polyphenol complexes have focused mainly on non-covalent complexes; the preparation method of the zein-polyphenol covalent compound is mainly an alkaline method, and the color of the product can be darkened.

The currently used base catalysis methods mainly have the following disadvantages:

(1) under alkaline conditions, polyphenols are first oxidized to the corresponding quinones, which can cause the prepared complexes to turn darker brown or green, affecting their use in food products.

(2) Oxidation of the hydroxyl groups results in a reduction in the antioxidant properties of the polyphenol.

(3) The compound prepared by the existing method has low polyphenol content, and the oxidation resistance is not greatly improved.

The invention utilizes a new method to prepare the zein-polyphenol covalent compound, does not change the color of protein, and obviously improves the oxidation resistance.

The invention adopts carbodiimide-mediated coupling reaction to prepare a covalent compound of zein and two polyphenols, wherein the two polyphenols are chlorogenic acid (CGA) and Gallic Acid (GA). The covalent compound prepared by the method has higher polyphenol content, the oxidation resistance is greatly improved, and the product keeps the original color of protein, thereby being more beneficial to the application of the covalent compound in food.

EDC is a water-soluble carbodiimide reagent, is used as a carboxyl activating reagent in amide synthesis, and is often used together with NHS to improve the coupling efficiency. EDC/NHS is superior to aldehyde crosslinking agent, and the experiment proves that EDC/NHS treatment does not affect the increase of cytotoxicity of the product. EDC/NHS only helps the carboxyl of polyphenol and amino of protein to form amido bond, and does not become a part of crosslinking, and intermediate product (water-soluble urea derivative with low cytotoxicity) can be eliminated and cleaned, and has the characteristics of no toxicity and good biocompatibility.

(1) Compared with the prior art (alkali method), the covalent compound prepared by the carbodiimide crosslinking method does not change the color of the zein and does not destroy the structure of the polyphenol.

(2) SDS-PAGE showed a significant increase in the molecular weight of the complex, confirming the formation of the covalent complex. By measuring the polyphenol content, the polyphenol content and grafting rate of the compound prepared by the invention are found to be higher than those of the prior art.

(3) Through measuring the oxidation resistance, the oxidation resistance of the compound prepared by the invention is found to be greatly improved compared with the original protein, and compared with the prior art, the oxidation resistance of the compound prepared by the invention is improved more.

In conclusion, the zein-polyphenol covalent compound prepared by the invention can obviously improve the oxidation resistance of the zein, and the generated compound still maintains the color of the original protein, thereby being beneficial to the application of the zein-polyphenol covalent compound in food, and particularly being used for constructing a novel delivery system.

Drawings

FIG. 1 shows the electrophoretograms of control zein and zein-polyphenol covalent complexes (bands: 1, standard protein; 2, control zein; 3, zein-CGA covalent complex; 4, zein-GA covalent complex).

FIG. 2. solubility of control zein and zein-polyphenol complexes.

FIG. 3 control zein and zein-polyphenol covalent complexes for antioxidant properties.

FIG. 4 shows the dispersibility of control zein and zein-polyphenol complexes (1, control zein; 2, zein-CGA non-covalent complex; 3, zein-GA non-covalent complex; 4, zein-CGA covalent complex; 5, zein-GA covalent complex).

FIG. 5 scanning electron micrograph of Zein-polyphenol covalent complex (1, Zein-CGA covalent complex; 2, Zein-GA covalent complex).

FIG. 6. carbodiimide-mediated zein-polyphenol covalent complex formation pathway.

FIG. 7 total phenol equivalents and grafting yield of zein-polyphenol covalent complexes.

FIG. 8 controls the free amino content of zein and zein-polyphenol covalent complexes.

FIG. 9 controls the thermal denaturation temperatures of zein and zein-polyphenol complexes.

Detailed Description

The invention is further illustrated by the following examples:

wherein, all the reagents and raw materials used in the invention are common reagents meeting the national raw material standards, and can be purchased from conventional reagent production and sale companies.

Example 1:

1) 200 mg of zein was dissolved in 10 mL of 70% (v/v) ethanol-water solution, sonicated for 5 min, stirred for 30 min to dissolve completely, and 0.001% (w/v) potassium sorbate was added.

2) Dissolving 0.25 mM chlorogenic acid (CGA) in 10 mL 70% (v/v) ethanol-water solution, ultrasonic treating for 5 min, and stirring for 30 min to dissolve completely;

3) adding 0.75 mM EDC and then 0.5 mM NHS into the polyphenol solution in the step 2) at 4 ℃, and stirring for reacting for 1 h;

4) adding the polyphenol solution obtained in the step 3) into the zein solution obtained in the step 1), adjusting the pH to 6.5, reacting at 4 ℃ for 1 h, and reacting at room temperature for 24 h;

5) after the reaction is finished, the mixture passes through an ultrafiltration device, and the liquid obtained by ultrafiltration is dialyzed in ultrasound.

6) And (3) treating the dialyzed sample by using a nano grinder, pre-freezing, freezing and drying to obtain the zein-polyphenol covalent compound.

The specific steps of ultrafiltration and dialysis are as follows:

the molecular weight of the ultrafiltration membrane is 30 kDa, the ultrafiltered sample is put into a dialysis bag, the dialysis bag is placed in distilled water for dialysis for 48 hours, water is changed for 10 times, and the molecular weight of the dialysis bag is 3500 Da. The dialyzed sample was pre-frozen at-40 ℃ for 24 h, followed by freeze-drying for 2 days to obtain a solid sample.

Example 2:

1) 200 mg of zein was dissolved in 10 mL of 70% (v/v) ethanol-water solution, sonicated for 5 min, stirred for 30 min to dissolve completely, and 0.001% (w/v) potassium sorbate was added.

2) Dissolving 0.25 mM Gallic Acid (GA) in 10 mL 70% (v/v) ethanol-water solution, performing ultrasonic treatment for 5 min, and stirring for 30 min to completely dissolve;

3) adding 0.75 mM EDC and then 0.5 mM NHS into the polyphenol solution in the step 2) at 4 ℃, and stirring for reacting for 1 h;

4) adding the polyphenol solution obtained in the step 3) into the zein solution obtained in the step 1), adjusting the pH to 6.5, reacting at 4 ℃ for 1 h, and reacting at room temperature for 24 h;

5) after the reaction is finished, the mixture passes through an ultrafiltration device, and the liquid obtained by ultrafiltration is dialyzed in ultrasound.

6) And (3) treating the dialyzed sample by using a nano grinder, pre-freezing, freezing and drying to obtain the zein-polyphenol covalent compound.

Example 3: determination of the covalent complexes prepared in examples 1 and 2

One, electrophoresis

The zein-chlorogenic acid (zein-CGA) covalent complex and the zein-gallic acid (zein-GA) covalent complex prepared in examples 1 and 2 were assayed.

Experimental methods

The covalent reaction between zein (zein) and polyphenols was assessed by SDS-PAGE. The concentrations of the separation gel and the concentration gel were 12.5% and 5%, respectively. The protein sample was dispersed in distilled water at a concentration of 2 mg/mL, and then mixed with 4 Xloading buffer and heat denatured for 10 min. The loading amount of the standard protein and the sample is 10 mu L, and the electrophoresis buffer solution is Tris-glycine buffer solution. After the electrophoresis was completed, the SDS-PAGE gel was stained with Coomassie Brilliant blue R250 and destained in a solution containing 8% acetic acid.

FIG. 6 is a schematic representation of a carbodiimide-mediated zein-polyphenol covalent complex formation pathway. FIG. 1 is an electrophoretogram of control zein and zein-polyphenol covalent complexes, and it can be seen from FIG. 1 that the zein-polyphenol covalent complex band is elevated compared to the control protein, indicating that the molecular weight of the complex is increased. Since SDS disrupts the non-covalent bonds between proteins and polyphenols, the molecular weight increase of the complex can be attributed to the covalent bonding of the polyphenols to zein.

Secondly, measuring polyphenol content

And (3) measuring the polyphenol content of the covalent compound by a folin phenol method, and calculating the grafting rate. The method comprises the following specific steps: 0.5 mL of zein-polyphenol complex solution (1 mg/mL) was mixed with 2.5 mL of freshly prepared forlin's phenol reagent (200 mmol/L). After 5 min of reaction in the dark, 2 mL Na was added to the mixture₂CO₃The solution (7.5%, w/v) was reacted for 2 h in the dark. The absorbance of the zein-polyphenol complex at 760 nm was then determined, with the final result expressed as millipolyphenol equivalents per gram of complex. The grafting rate was calculated according to the following equation:

graft ratio (%) = (C/C)₀）*100

Wherein C is the polyphenol content of the dialyzed zein-polyphenol compound, C₀Is the addition amount of polyphenol.

As can be seen from FIG. 7, the zein-polyphenol covalent compound prepared by the method has higher total phenol equivalent and higher grafting rate which can reach more than 35%, which shows that the method adopted by the invention has higher coupling efficiency.

Determination of the free amino group

The free amino content of the control protein and zein-polyphenol covalent complex was measured by OPA method. Briefly, 80 mg OPA was dissolved in 2 mL methanol and then 5 mL 2 was added0% (w/v) SDS, 200. mu.L mercaptoethanol, 50 mL sodium tetraborate solution (0.1M), and adding distilled water to a constant volume of 100 mL, thus obtaining the OPA reagent. Then 200. mu.L of the sample solution (2 mg/mL) was mixed with 4 mL of OPA reagent and reacted at 35 ℃ for 2 min, after which its absorbance at 340 nm was measured to_LLeucine is a standard substance.

As can be seen from fig. 8, the content of free amino groups in zein was significantly reduced after covalent reaction with polyphenols, indicating that free amino groups in the protein reacted. The reduction in the free amino group content of the protein further demonstrates the formation of a zein-polyphenol covalent complex, since the OPA reagent contains SDS and mercaptoethanol, which can disrupt the non-covalent bonds between the protein and the polyphenol.

Fourthly, determination of solubility

The experimental method comprises the following steps:

the protein sample was dispersed in distilled water at a concentration of 1 mg/mL, stirred at a constant speed (200 rpm) for 30 min, and then centrifuged for 20 min (8000 rpm). The protein content in the supernatant was determined by Coomassie brilliant blue method, and a standard curve was prepared using bovine serum albumin isolate as a standard. The solubility of the control zein and zein-polyphenol complexes was expressed as the protein concentration in the supernatant as a percentage of the total protein concentration.

Protein solubility is an important property of proteins because it significantly affects the properties of protein such as emulsifiability, gelation, and foaming. As can be seen from FIG. 2, the solubility of the protein increased after reaction with the polyphenol, probably due to the introduction of hydrophilic hydroxyl groups of the polyphenol. The solubility increase of the covalent complexes is more pronounced than that of the non-covalent complexes, which is more advantageous for the use of the complexes in the food industry.

The preparation method of the zein-polyphenol non-covalent compound comprises the following steps:

dispersing polyphenol and protein in water solution, adjusting pH of the solution to 6, adding no EDC and NHS, isolating air, stirring for 24 hr, and freeze drying to obtain non-covalent complex. The reason for using pH 6 is that polyphenols may be oxidized to the corresponding quinones in alkaline and aerobic conditions, the quinones will covalently react with proteins, and the weak acidic conditions will avoid oxidation of polyphenols. The amount of polyphenol added to the non-covalent complex is the same as the polyphenol content of the corresponding covalent complex.

Fifthly, oxidation resistance

The experimental method comprises the following steps:

ABTS stock solution was prepared first, and 10 mg of ABTS was dissolved in 2.6 mL of 2.45 mM potassium persulfate solution and reacted at room temperature with exclusion of light for 16 hours. The stock solution was diluted with deionized water before the experiment to an absorbance of 0.70. + -. 0.02 at a wavelength of 734 nm. Taking a sample solution diluted to a certain concentration, adding 3 mL of ABTS solution, reacting for 60 min at room temperature in a dark place, measuring the light absorption value at the position of 734 nm of wavelength, comparing the ABTS free radical eliminating capacity of the sample with the ABTS free radical eliminating capacity of Trolox, and expressing the ABTS free radical eliminating capacity of the sample by Trolox equivalent (nmol TE/mg sample).

1.7510 is prepared by ethanol^-4 And (3) taking 2 mL of a DPPH solution, diluting the sample solution to a certain concentration, fully mixing the sample solution with the DPPH solution, carrying out a dark reaction at room temperature for 60 min, and detecting the light absorption value of the sample solution at the wavelength of 517 nm. The ability of the sample to scavenge DPPH radicals is expressed in Trolox equivalents (nmol TE per mg of sample).

As can be seen from fig. 3, the oxidation resistance of zein is significantly improved after covalently grafting polyphenol, because the hydroxyl group of polyphenol is introduced. The carbodiimide crosslinking method adopted by the patent is to react the carboxyl of polyphenol with EDC, then link with protein, and can not destroy the hydroxyl of polyphenol, so that the oxidation resistance of polyphenol can be better exerted compared with the common alkali treatment method.

Sixth, thermal stability

The experimental method comprises the following steps:

the thermal stability of the sample was evaluated by measuring the thermal denaturation temperature of the sample with a Differential Scanning Calorimeter (DSC). The experimental method is as follows: weigh 5 mg of sample, seal in an aluminum crucible, and use the sealed empty crucible as a blank control. The heating rate is 10 ℃ per minute, the temperature range is 30-200 ℃, and the nitrogen flow rate is 20 mL per minute. The thermal denaturation temperature (. degree.C.) was obtained by using the analysis software provided in the apparatus.

The results in FIG. 9 show that: after the zein reacts with the polyphenol, the thermal denaturation temperature is increased, which shows that the thermal stability of the zein is improved, wherein the thermal stability of the covalent compound is better than that of the non-covalent compound, and the zein is more beneficial to being applied in the food industry.

Seventhly, dispersibility

The experimental method comprises the following steps:

weighing 10 mg of sample in a glass bottle, adding 10 mL of distilled water, putting the sample in a rotor, stirring for 30 min, taking out the rotor, and taking a picture.

It can be seen from fig. 4 that the dispersibility of the control protein and the non-covalent complex is poor, most of the samples sink to the bottom, and the covalent complex is almost uniformly dispersed in water, indicating that the dispersibility of the protein is significantly improved after the covalent grafting of the polyphenol.

Eighthly, scanning electron microscope

The experimental method comprises the following steps:

and placing a small amount of the freeze-dried sample on conductive gel, spraying gold in vacuum, and observing the microscopic morphology of the sample on a scanning electron microscope instrument.

As can be seen from fig. 5, the zein-polyphenol covalent complex formed uniform spherical particles with a smaller particle size, which may be one of the reasons for the improved dispersibility of the complex.

It should be noted that the patent specification does not limit the ingredient and process of the present invention, and those skilled in the art can make various modifications and improvements based on the basic idea of the invention, but should be within the protection scope of the present invention as long as they do not depart from the basic idea of the invention.