Method for improving solubility and emulsibility of walnut protein isolate-epigallocatechin gallate covalent complex

Abstract

The invention discloses a method for improving solubility and emulsibility of a walnut protein isolate-epigallocatechin gallate covalent complex, and belongs to the technical field of food processing. The method comprises the following steps: weighing walnut protein isolate, dispersing the walnut protein isolate in deionized water, and adjusting the pH value to 9-11 to obtain a protein solution; weighing epigallocatechin gallate, dissolving the epigallocatechin gallate in deionized water, and adjusting the pH value to 9-11 to obtain a polyphenol solution; slowly pouring the polyphenol solution into the protein solution, adjusting the pH value to 11 to obtain a mixed solution, carrying out ultrasonic treatment on the mixed solution, adjusting the pH value to 7, dialyzing in a dialysis bag, and drying the dialyzed solution to obtain the walnut protein isolate-epigallocatechin gallate covalent complex. The walnut protein isolate-epigallocatechin gallate covalent complex prepared by the method disclosed by the invention has the characteristics of high solubility and emulsifying property.

Description

Technical Field

[0001] The invention relates to a method for improving the solubility and emulsification of a walnut protein isolate-epigallocatechin gallate covalent complex, belonging to the technical field of food processing. Background Art

[0002] As one of the world's major walnut producing countries, my country has abundant walnut resources. Walnuts contain 15% to 20% protein, and walnut protein contains 18 types of amino acids, including 8 essential amino acids for the human body, with high nutritional value. However, as a by-product of walnut oil production, walnut meal rich in walnut protein is usually used as livestock feed or discarded. Compared with other plant proteins, walnut protein has not been fully developed and utilized, resulting in a great waste of resources. The main reason for the above phenomenon is that walnut protein has a high gluten content, poor water solubility and emulsification ability, and weak technical functionality, resulting in its utilization rate not as high as plant proteins such as soy protein isolate.

[0003] Epigallocatechin gallate (EGCG) is a catechin compound extracted from green tea. It is the main component of tea polyphenols and has many excellent effects such as anti-oxidation and anti-tumor. It is often used in food, medicine and cosmetics processing. A large number of studies have shown that the introduction of polyphenols into protein molecules will affect the electrostatic charge and surface properties of the molecules. The bisphenol group of polyphenols is highly active and can generate o-quinone or semiquinone through enzymatic and molecular oxidation reactions, and further react with the amino or thiol side chains of proteins to form covalent compounds. The covalent complex obtained by modifying proteins with polyphenols has good solubility and emulsification properties, which expands its application possibilities in the food industry.

[0004] At present, the preparation methods of protein-polyphenol covalent complexes mainly include alkaline method, enzyme method and free radical grafting method. In terms of reaction conditions, the alkaline method is relatively mild. It does not require the use of initiators like the free radical grafting method, which makes the reaction easier to control; it does not rely on specific enzymes like the enzyme method, and does not require fine adjustment of conditions. It only needs to react within a certain alkaline pH range. In terms of cost, the alkaline reagent used in the alkaline method is a common chemical reagent with low price. In addition, the alkaline method is applicable to most proteins and polyphenols, and can prepare covalent complexes by catalyzing the reaction between their functional groups (such as phenolic hydroxyl groups, amino groups, etc.); the enzyme method has high specificity and can accurately act on specific substrates, greatly reducing nonspecific reactions, but the enzyme has poor stability and is extremely sensitive to environmental factors such as temperature and pH fluctuations. Slight changes may lead to a significant reduction in enzyme activity or even inactivation, and the preparation cost of the enzyme is high; the free radical grafting method can achieve grafting modification on the surface of a variety of materials, and has good compatibility with the type and morphology of the material, but its initiator is expensive, and the post-processing process of the product after the reaction is complicated. The alkaline method shows significant advantages over the enzyme method and the free radical grafting method. However, the traditional alkaline treatment has a long reaction time, requiring 24-48 hours of reaction at room temperature, high energy consumption, and cannot meet the requirements of shortening the production cycle in industrial production. Summary of the invention

[0005] In order to solve the problems of poor solubility and emulsification of the existing walnut protein isolate-epigallocatechin gallate, the present invention provides a method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex, comprising the following steps:

[0006] (1) Weigh walnut protein isolate and dissolve it in deionized water, and adjust the pH to alkaline to obtain a walnut protein isolate solution.

[0007] (2) Weigh epigallocatechin gallate and dissolve it in deionized water, and adjust the pH to alkaline to obtain an epigallocatechin gallate solution.

[0008] (3) slowly pouring the epigallocatechin gallate solution into the walnut protein isolate solution, adjusting the pH to alkaline, performing ultrasonic treatment, and then stirring in an aerobic environment at room temperature to obtain a walnut protein isolate-epigallocatechin gallate covalent complex solution.

[0009] (4) The pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution obtained in step (3) is adjusted to neutral, dialyzed in a dialysis bag, and the solution in the dialysis bag is freeze-dried to obtain a walnut protein isolate-epigallocatechin gallate covalent complex.

[0010] Preferably, in step (1), the mass ratio of epigallocatechin gallate to walnut protein isolate is 1:50 to 1:12.5.

[0011] Preferably, the concentration of the walnut protein isolate solution in step (1) is 10 mg / mL.

[0012] Preferably, the pH value of the walnut protein isolate solution in step (1) is 9-11.

[0013] Preferably, the concentration of the epigallocatechin gallate solution in step (2) is 10 mg / mL.

[0014] Preferably, the pH value of the epigallocatechin gallate solution in step (2) is 9-11.

[0015] Preferably, in step (3), the mass ratio of the epigallocatechin gallate solution to the walnut protein isolate solution is 1:50 to 1:12.5, and the pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution is 11.

[0016] Preferably, the ultrasonic treatment time in step (3) is 10 to 30 minutes.

[0017] Preferably, the stirring time in the aerobic environment at room temperature in step (3) is 1 to 3 hours.

[0018] Preferably, in step (4), the pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution is 7, the molecular weight of the dialysis bag is 3500Da, and the dialysis time is 48h.

[0019] Beneficial effects of the present invention

[0020] (1) Compared with the traditional alkaline method, the preparation method of the present invention can significantly improve the solubility, emulsification and emulsification stability of the walnut protein isolate-epigallocatechin gallate covalent complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 This is a flow chart of Example 1.

[0022] Attached Figure 2 is the polyphenol binding equivalent of the complex solution prepared in each example and comparative example.

[0023] Attached Figure 3 It is the solubility of the complex solution prepared in each embodiment and comparative example; wherein: WPI represents walnut protein isolate.

[0024] Attached Figure 4The emulsification activity and emulsification stability of the complex solution prepared in each embodiment and comparative example; wherein: WPI represents walnut protein isolate. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0026] Unless otherwise specified, the reagents used in the present invention are reagents well known to those skilled in the art.

[0027] Example 1

[0028] A method for improving the solubility and emulsification of a walnut protein isolate-epigallocatechin gallate covalent complex comprises the following steps:

[0029] (1) Weigh 1 g of walnut protein isolate and dissolve it in 100 mL of deionized water, and adjust the pH to 11 with 0.1 mol / L NaOH to obtain a walnut protein isolate solution.

[0030] (2) Weigh 1 g of epigallocatechin gallate and dissolve it in 100 mL of deionized water, and adjust the pH to 9 with 0.1 mol / L NaOH to obtain an epigallocatechin gallate solution.

[0031] (3) The epigallocatechin gallate solution was slowly poured into the walnut protein isolate solution, the mass ratio of the epigallocatechin gallate solution to the walnut protein isolate solution was 1:50, and the pH was adjusted to 11 with 0.1 mol / L NaOH, and then ultrasonically treated at a power of 450 W for 15 min, and then stirred in an aerobic environment at room temperature for 2 h to obtain a walnut protein isolate-epigallocatechin gallate covalent complex solution.

[0032] (4) The pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution obtained in step (3) was adjusted to 7 with 1 mol / L HCl, and dialyzed in a 3500Da dialysis bag for 48 hours, with the water (dialysis solution) being replaced every 6 hours. The solution in the dialysis bag was freeze-dried for 48 hours to obtain a walnut protein isolate-epigallocatechin gallate covalent complex.

[0033] Example 2

[0034] A method for improving the solubility and emulsification of a walnut protein isolate-epigallocatechin gallate covalent complex comprises the following steps:

[0035] (1) Weigh 1 g of walnut protein isolate and dissolve it in 100 mL of deionized water, and adjust the pH to 9 with 0.1 mol / L NaOH to obtain a walnut protein isolate solution.

[0036] (2) Weigh 1 g of epigallocatechin gallate and dissolve it in 100 mL of deionized water, and adjust the pH to 11 with 0.1 mol / L NaOH to obtain an epigallocatechin gallate solution.

[0037] (3) The epigallocatechin gallate solution was slowly poured into the walnut protein isolate solution, the mass ratio of the epigallocatechin gallate solution to the walnut protein isolate solution was 1:25, and the pH was adjusted to 11 with 0.1 mol / L NaOH, and then ultrasonically treated at a power of 300 W for 10 min, and then stirred in an aerobic environment at room temperature for 3 hours to obtain a walnut protein isolate-epigallocatechin gallate covalent complex solution.

[0038] (4) The pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution obtained in step (3) was adjusted to 7 with 1 mol / L HCl, and dialyzed in a 3500Da dialysis bag for 48 hours, with the water (dialysis solution) being replaced every 6 hours. The solution in the dialysis bag was freeze-dried for 48 hours to obtain a walnut protein isolate-epigallocatechin gallate covalent complex.

[0039] Example 3

[0040] A method for improving the solubility and emulsification of a walnut protein isolate-epigallocatechin gallate covalent complex comprises the following steps:

[0041] (1) Weigh 1 g of walnut protein isolate and dissolve it in 100 mL of deionized water, and adjust the pH to 9 with 0.1 mol / L NaOH to obtain a walnut protein isolate solution.

[0042] (2) Weigh 1 g of epigallocatechin gallate and dissolve it in 100 mL of deionized water, and adjust the pH to 9 with 0.1 mol / L NaOH to obtain an epigallocatechin gallate solution.

[0043] (3) The epigallocatechin gallate solution was slowly poured into the walnut protein isolate solution, the mass ratio of the epigallocatechin gallate solution to the walnut protein isolate solution was 1:12.5, and the pH was adjusted to 11 with 0.1 mol / L NaOH, and then ultrasonically treated at a power of 450 W for 15 min, and then stirred in an aerobic environment at room temperature for 2 h to obtain a walnut protein isolate-epigallocatechin gallate covalent complex solution.

[0044] (4) The pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution obtained in step (3) was adjusted to 7 with 1 mol / L HCl, and dialyzed in a 3500Da dialysis bag for 48 hours, with the water (dialysis solution) being replaced every 6 hours. The solution in the dialysis bag was freeze-dried for 48 hours to obtain a walnut protein isolate-epigallocatechin gallate covalent complex.

[0045] Comparative Example 1

[0046] As a comparison, the difference between this comparative example and Example 1 is that no ultrasonic treatment is performed, and the other steps are the same, and finally a walnut protein isolate-epigallocatechin gallate covalent complex is obtained.

[0047] Comparative Example 2

[0048] As a comparison, the difference between this comparative example and Example 2 is that no ultrasonic treatment is performed, and the other steps are the same, and finally a walnut protein isolate-epigallocatechin gallate covalent complex is obtained.

[0049] Comparative Example 3

[0050] As a comparison, the difference between this comparative example and Example 3 is that no ultrasonic treatment is performed, and the other steps are the same, and finally a walnut protein isolate-epigallocatechin gallate covalent complex is obtained.

[0051] Comparative Example 4

[0052] For comparison, the difference between this comparative example and Example 3 is that only the pH in step (3) needs to be adjusted to 9, and the rest are the same, and finally a walnut protein isolate-epigallocatechin gallate covalent complex is obtained.

[0053] Determination of the properties of the covalent complexes prepared in the examples and comparative examples

[0054] (I) Determination of polyphenol binding equivalents

[0055] The polyphenol content of the covalent complex was determined by the Folin phenol method, and the specific steps were as follows: first, 1 mL of walnut protein isolate-epigallocatechin gallate solution with a concentration of 1 mg / mL was mixed with 5 mL of Folin phenol reagent (10%), and the mixture was reacted in the dark for 5 minutes, and then 4 mL of Na2CO3 solution (the mass concentration of the Na2CO3 solution was 7.5%, that is, 100 mL of the Na2CO3 solution contained 7.5 g of Na2CO3) was added to the mixture, and the mixture was reacted in the dark for 2 hours. After the reaction was completed, the absorbance was measured at 760 nm, and finally expressed as mg / g protein.

[0056] Depend on Figure 2It can be seen that compared with Comparative Examples 1 to 3, the polyphenol binding equivalent of the walnut protein isolate-epigallocatechin gallate covalent complex prepared in Examples 1 to 3 is significantly improved, up to 53.37 ± 0.33 mg / g protein, indicating that ultrasonic treatment can increase the polyphenol binding equivalent, and compared with Comparative Example 4 (38.81 ± 0.21 mg / g protein), the preparation method adopted by the present invention has a higher cross-linking efficiency. This may be because under extremely alkaline conditions, the secondary and tertiary structures of the protein change, and the carboxyl groups of the acidic amino acid residues in the protein molecule will be deprotonated and converted into a negatively charged carboxylate form, so that the protein as a whole is more negatively charged, thereby enhancing the electrostatic interaction with polyphenols. In addition, under extremely alkaline conditions and in the presence of oxygen, the phenolic hydroxyl groups of polyphenols are easily oxidized to strong electrophilic intermediates such as quinones. These intermediates have higher reactivity and can more easily react covalently with nucleophilic groups in proteins, thereby increasing the binding equivalent.

[0057] (II) Determination of solubility

[0058] Accurately weigh 5 mg of the walnut protein isolate-epigallocatechin gallate covalent complex powder obtained in Examples 1 to 3 and Comparative Examples 1 to 4 and disperse it in 1 mL of deionized water. After mixing and dissolving, use a high-speed refrigerated centrifuge to set the speed to 9000 r / min for centrifugation for 15 minutes, and collect the supernatant; take 0.1 mL of the supernatant and add 4.9 mL of deionized water, then add 4 mL of Coomassie G-250 dye solution in a test tube, shake and mix, place it away from light for 5 minutes, and then measure the absorbance at a wavelength of 595 nm. Use the same volume of deionized water as a control. The solubility of walnut protein isolate and walnut protein isolate-epigallocatechin gallate is calculated by the BSA coordinate curve. Calculate the solubility according to the formula.

[0059]

[0060] m0 is the protein content in the sample, mg / g;

[0061] m1 is the protein content in the supernatant, mg / g.

[0062] Depend on Figure 3It can be seen that walnut protein isolate has a low solubility of only 22.22%, while the solubility of Comparative Example 4 is only 65.13%. However, when covalently cross-linked with epigallocatechin gallate under extremely alkaline conditions (Examples 1 to 3), its solubility is 82.35%, 90.34%, and 91.22%, respectively, and the solubility is significantly improved (p>0.05). This may be because the molecular conformational changes of proteins and polyphenols under the defined pH conditions may expose more hydrophilic groups (hydroxyl and carboxyl groups), and the protein-polyphenol covalent complex prepared under extremely alkaline conditions carries more negative charges, and the electrostatic repulsion between molecules is enhanced. This electrostatic repulsion makes it difficult for the complex molecules to aggregate, thereby increasing the degree of dispersion in the solution, that is, the solubility is increased.

[0063] (III) Determination of emulsification activity and emulsification stability

[0064] Weigh the walnut protein isolate-epigallocatechin gallate covalent complex powder obtained in Examples 1 to 3 and Comparative Examples 1 to 4 to prepare 15 mL of a protein solution with a mass fraction of 1%, add 5 mL of soybean oil, use a high-speed homogenizer to stir at a speed of 10000 r / min for 1 min, and repeat three times to prepare an emulsion. Then immediately take 0.1 mL of the emulsion and add it to 10 mL of 0.1% SDS, shake and mix, and immediately use an ultraviolet spectrophotometer to measure its absorbance A0 at 500 nm. After the emulsion is allowed to stand for 30 minutes, take another 0.1 mL of the emulsion and transfer it to 10 mL of 0.1% SDS, shake and mix, and measure the absorbance A 30 (Use SDS to adjust to zero). The calculation formulas for emulsification activity and emulsification stability are as follows

[0065]

[0066] In the formula: DF is the dilution factor;

[0067] C is the protein concentration (g / mL);

[0068] θ is the volume fraction of oil phase (20%);

[0069] Light path (1cm)

[0070] A0 is the absorbance measured at the initial time;

[0071] A 30 The absorbance is measured by the same method after the emulsion has been allowed to stand for 30 minutes.

[0072] Depend on Figure 4 It can be seen that walnut protein isolate (emulsifying activity: 68.17 ± 0.32 m 2 / g; emulsification stability: 36.49%) and Comparative Example 4 (emulsification activity: 66.01±0.65m 2 / g; emulsification stability: 59.44%), the walnut protein isolate-epigallocatechin gallate covalent complex prepared in Examples 1 to 3 has better emulsification activity and emulsification stability. This shows that under extremely alkaline conditions, the high negative charge density of the protein-polyphenol covalent complex enables it to be more effectively adsorbed on the oil-water interface. When the complex is adsorbed on the oil-water interface, its negatively charged part can form a strong hydrogen bond with water molecules. This structure enables the complex to better stabilize the oil-water interface and prevent the aggregation and merging of oil droplets, thereby improving the emulsification. In contrast, when the pH is 9, the charge density and structural characteristics of the complex may not be as effective in reducing the surface tension of the oil-water interface and stabilizing the oil droplets as under extremely alkaline conditions, so the emulsification is not as significant as under extremely alkaline conditions.

Claims

1. A method for improving the solubility and emulsification of a walnut protein isolate-epigallocatechin gallate covalent complex, characterized in that: The following steps are involved: (1) weighing walnut protein isolate and dissolving it in deionized water, and adjusting the pH to alkaline to obtain a walnut protein isolate solution; (2) Weighing epigallocatechin gallate and dissolving it in deionized water, and adjusting the pH to alkaline to obtain an epigallocatechin gallate solution; (3) slowly pouring the epigallocatechin gallate solution into the walnut protein isolate solution, adjusting the pH to alkaline, performing ultrasonic treatment, and then stirring at room temperature in an aerobic environment to obtain a walnut protein isolate-epigallocatechin gallate covalent complex solution; (4) The pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution obtained in step (3) is adjusted to neutral, dialyzed in a dialysis bag, and the solution in the dialysis bag is freeze-dried to obtain a walnut protein isolate-epigallocatechin gallate covalent complex.

2. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: The concentration of the walnut protein isolate solution in step (1) is 10 mg / mL.

3. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: The pH value of the walnut protein isolate solution in step (1) is 9-11.

4. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: The concentration of the epigallocatechin gallate solution in step (2) is 10 mg / mL.

5. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: The pH value of the epigallocatechin gallate solution in step (2) is 9-11.

6. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: In step (3), the mass ratio of the epigallocatechin gallate solution to the walnut protein isolate solution is 1:50 to 1:12.5, and the pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution is 11.

7. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: The ultrasonic treatment time in step (3) is 10 to 30 minutes.

8. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: The stirring time in the aerobic environment at room temperature in step (3) is 1 to 3 hours.

9. The method for improving the solubility and emulsification of the walnut protein isolate-epigallocatechin gallate covalent complex according to claim 1, characterized in that: In step (4), the pH of the walnut protein isolate-epigallocatechin gallate covalent complex solution is 7, the molecular weight of the dialysis bag is selected to be 3500Da, and the dialysis time is 48h.

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