Method for preparing protein-polyphenol covalent complex

Abstract

The invention discloses a method for preparing a protein-polyphenol covalent complex, which comprises the following steps: firstly, treating a protein solution or turbid liquid by using low-temperature plasma, adding polyphenol into the treated protein solution to fully react, dialyzing the fully reacted polyphenol and protein mixed solution to remove the polyphenol which is not combined with the protein, and drying to obtain the protein-polyphenol covalent complex. And carrying out freeze drying to obtain the protein-polyphenol covalent complex. According to the method, no additive is added in the whole process, polyphenol oxidation is avoided, and the protein-polyphenol covalent binding compound can be rapidly and efficiently prepared.

Description

Technical Field

[0001] The invention specifically relates to a method for preparing a protein-polyphenol covalent complex, and belongs to the field of food science and processing technology. Background Art

[0002] In the food system, proteins and polyphenols are two very common substances. The nutritional value of proteins and their specific structural and functional properties make them widely used in food systems, while polyphenols have a variety of biological activities and physiological functions, and are widely sourced in plants. Under natural conditions, proteins often bind to polyphenols in a non-covalent form (non-covalent bonds formed by weak interactions such as hydrophobic interactions, hydrogen bonds, ionic bonds, and π bonds), thereby improving the functional properties of proteins, such as emulsification, gelation, etc., and reducing protein allergenicity. However, the non-covalent binding method is easily affected by the solution and the environment, resulting in separation of proteins and polyphenols, resulting in unstable effects. Covalent binding refers to the binding of protein and polyphenol molecules in the form of covalent bonds, and the two form a stable complex. The covalent binding complex of protein and polyphenol has the following advantages: ① Protein-polyphenol covalent conjugates are easier to form a stable network structure, and compared with non-covalent conjugates, their thermal stability and antioxidant properties are better. ② Covalently bound polyphenols can improve the gelation of proteins and enhance the water retention of proteins. ③ The emulsion prepared by protein-polyphenol covalent conjugate can effectively reduce the average particle size of the emulsion and improve the emulsification of protein. Therefore, the covalent conjugation of protein and polyphenol as a protein modification method can greatly expand the application scenarios of protein.

[0003] At present, there are two types of preparation of protein-polyphenol covalent complexes: enzymatic method and non-enzymatic method. The enzymatic method is to react protein and polyphenol through enzyme catalysis and cross-linking to achieve covalent bonding between the two to form a stable protein-polyphenol covalent bonding complex. Commonly used enzymes include polyphenol oxidase, laccase, transglutaminase, peroxidase, lipoxygenase, tyrosinase, etc. The principle is that polyphenol forms quinone under enzyme catalysis, and then further cross-links with protein. Non-enzymatic methods include free radical method and alkaline method. The free radical method promotes the binding of protein and polyphenol through the oxidation effect mediated by hydroxyl radicals generated by the redox system constructed by ascorbic acid and H2O2. The principle is that hydroxyl radicals are generated in the redox system of ascorbic acid and H2O2, oxidizing polyphenol to generate quinone, and quinone is covalently bonded to protein. The main principle of the alkaline method is to adjust the pH of the protein-polyphenol complex solution to alkaline. Polyphenol is unstable under alkaline conditions, oxidized to quinone, and then combined with the nucleophilic group of protein.

[0004] As a method of modifying the physical properties of substrates using high-energy reactive oxygen and nitrogen particles (RONS), low-temperature plasma technology has been widely used in disinfection, environmental protection, material modification, and medicine due to its unique advantages. In recent years, the application of low-temperature plasma technology in the field of food processing has attracted great attention. Under room temperature conditions, gases are ionized by high-energy electric fields to produce a variety of reactive particles (O . 、O3、OH . , HO2, O2 - , NO and ONOO), forming a complex RONS plasma oxidation system. The oxidation effect mediated by these active particles acts on the food matrix, thereby achieving the modification of food macromolecules (protein, starch, etc.) and the sterilization and preservation of food. Due to its advantages of being green, non-thermal, and free of chemical residues, low-temperature plasma technology has good application prospects in the field of food processing, especially in the modification of proteins and the improvement of their functional properties. However, there are no related reports on the use of plasma treatment to achieve covalent bonding of proteins and polyphenols.

[0005] At present, the commonly used enzymatic and non-enzymatic methods for covalently binding protein polyphenols have the following shortcomings:

[0006] (1) The enzymatic method has low efficiency, long reaction time (up to 24 hours), and high cost;

[0007] (2) Non-enzymatic methods (free radical method and alkaline method) involve the addition of several chemical reagents and the reaction time is long;

[0008] (3) Not green or environmentally friendly;

[0009] (4) Polyphenol oxidation occurs during the covalent binding process. Summary of the invention

[0010] In order to overcome the shortcomings of the prior art, the present invention provides a method for preparing a protein-polyphenol covalent complex, which uses a low-temperature plasma-mediated method to covalently bind polyphenols to proteins.

[0011] The scheme of the present invention is as follows.

[0012] A method for preparing a protein-polyphenol covalent complex comprises the following steps:

[0013] (1) Low-temperature plasma treatment of protein solution;

[0014] (2) adding polyphenols to the protein solution obtained in step (1), stirring to allow the solution to react fully, and obtaining a protein-polyphenol covalent complex.

[0015] Preferably, during the low-temperature plasma treatment in step (1), the working gas is air, nitrogen, helium, or argon, the treatment voltage is 10 to 50 kV, the treatment frequency is 0.5 to 5 kHz, and the treatment time is 10 to 60 s.

[0016] Preferably, the low-temperature plasma treatment time is 15 to 45 seconds, preferably 15 to 30 seconds.

[0017] Preferably, the voltage of the low-temperature plasma treatment is 40 kV and the treatment frequency is 1.3 kHz.

[0018] Preferably, the low-temperature plasma treatment equipment used in step (1) consists of a reaction high-voltage power supply, a reactor and a sample container, wherein the high-voltage power supply can achieve a voltage of 1 to 100 kV and a frequency of 0.1 to 20 kHz; the reactor consists of upper and lower electrodes with a size of φ50 mm, the insulating medium is a quartz plate with a size of φ102 mm×1 mm, the spacing between the electrodes is adjustable from 0 to 10 mm, and the sample container is a quartz dish with a size of φ50 mm×5 mm.

[0019] Preferably, in step (1), the processing process is: a certain amount of sample is placed in a quartz dish, the distance between the two electrodes is adjusted to 8 mm, the insulating medium plate is fixed to the upper electrode, and air is used as the ionization medium.

[0020] Preferably, the protein in step (1) is one or more of ovalbumin, β-lactoglobulin, pea protein isolate, and soy protein isolate.

[0021] Preferably, the protein solution in step (1) is obtained by weighing a certain amount of protein, dissolving it in deionized water, and stirring it for a period of time to fully hydrate it.

[0022] Preferably, the polyphenols in step (2) are one or more of gallic acid, ferulic acid, syringic acid, caffeic acid, ellagic acid, epigallocatechin gallate, epigallocatechin, and epicatechin. Polyphenols of different molecular weights and structures have different binding affinities with proteins.

[0023] Preferably, the concentration of protein in the protein solution in step (1) is 1 to 20 mg / mL.

[0024] Preferably, the concentration of the polyphenol in the mixed system after adding the polyphenol in step (2) is 1 to 100 μmol / mL.

[0025] Preferably, in step (2), the fully reacted mixed solution of polyphenol and protein is dialyzed to remove polyphenol not bound to the protein, and then freeze-dried to obtain a protein-polyphenol covalent complex.

[0026] Preferably, the dialysis is performed by placing the sample in a dialysis bag in deionized water at 4°C for 48 hours, and the freeze-drying is performed by pre-freezing the dialyzed sample at -20°C for 12 hours.

[0027] Preferably, the molecular cutoff of the dialysis bag selected for the dialysis is 1 kDa.

[0028] Preferably, the water is changed more than 10 times during the dialysis, and the free polyphenols in the sample solution that are not bound to proteins are ensured to be completely dialyzed.

[0029] The present invention uses low-temperature plasma technology to achieve rapid and efficient covalent bonding of proteins and polyphenols. The mechanism is as follows: First, the gas is ionized by a high-energy electric field to generate a variety of active particles (O . 、O3、OH . , HO2, O2 - , NO and ONOO) form a RONS plasma oxidation system; the RONS oxidation disrupts the non-covalent interactions that maintain the protein structure (hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.), thereby unfolding the protein structure and fully exposing the amino and sulfhydryl binding sites that bind to polyphenols; at the same time, RONS oxidizes the amino (-NH2) and sulfhydryl (-SH) groups of proteins into ammonia free radicals (-NH . ) and thiol radicals (-S . After plasma treatment, the added polyphenols are converted into protein amine free radicals (-NH . ) and thiol radicals (-S . ) and other active intermediates to attack and produce a free radical chain reaction to form a covalent bond, thereby achieving rapid and efficient covalent bonding of protein and polyphenols.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The method of the present invention is fast (plasma treatment takes 10 to 60 seconds to complete binding) and efficient (the polyphenol binding amount reaches 40 mg / g-100 mg / g).

[0032] (2) The equipment used in the present invention is simple, stable and low-cost.

[0033] (3) The reaction process of the present invention does not require the addition of chemical reagents, is suitable for the development of high value-added protein products, and is in line with the theme of low-carbon green.

[0034] (4) The degree of polyphenol oxidation during the covalent binding process between the present invention and the protein is extremely low, and no obvious color change caused by oxidation to quinone occurs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of a low-temperature plasma processing device.

[0036] Figure 2 This is the electrophoresis diagram of ovalbumin-gallic acid covalent complex.

[0037] Figure 3 This is the electrophoresis diagram of ovalbumin-ferulic acid covalent complex.

[0038] Figure 4 This is the electrophoresis diagram of ovalbumin-syringic acid covalent complex.

[0039] Figure 5 This is the electrophoresis diagram of the ovalbumin-gallic acid covalent complex obtained after long-term treatment.

[0040] Figure 6 This is the appearance of the ovalbumin-gallic acid complex. DETAILED DESCRIPTION

[0041] The present invention is further described below with reference to an implementation case using ovalbumin as an example, but the implementation mode of the present invention is not limited thereto. However, the present invention can be implemented in many other ways different from this, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific implementation disclosed below.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0043] Example 1 Ovalbumin-Gallic Acid Covalent Complex

[0044] Ovalbumin was dispersed in deionized water at a concentration of 1 mg / mL. 5 mL of the sample was taken in a quartz dish. The quartz dish was placed between the two electrodes of the low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. The working gas was air, the voltage was 40 kV, and the frequency was 1.3 kHz. The treatment was performed for 30 seconds. After the treatment, gallic acid was added to the mixed system at a concentration of 20 μmol / mL. The sample was placed in a 4°C refrigerator for reaction for 6 hours. It was then dialyzed in a 4°C refrigerator (8-14 kDa molecular weight cutoff) for 48 hours. After precooling and freeze-drying, an ovalbumin-gallic acid covalent complex was obtained. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined by the Folin-Ciocalteu method to be 27.59±0.46 mg / g.

[0045] Example 2 Ovalbumin-Gallic Acid Covalent Complex

[0046] Same as Example 1, except that the plasma treatment time was changed to 15 s. The total phenol equivalent was measured to be 35.22±0.56 mg / g.

[0047] Example 3 Ovalbumin-ferulic acid covalent complex

[0048] Ovalbumin was dispersed in deionized water at a concentration of 1 mg / mL. 5 mL of the sample was taken into a quartz dish. The quartz dish was placed between two electrodes of a low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. The treatment was performed for 30 seconds under the conditions of air as the working gas, 40 kV as the voltage, and 1.3 kHz as the frequency. After the treatment, ferulic acid was added to the mixed system at a concentration of 20 μmol / mL. The sample was placed in a 4°C refrigerator for reaction for 6 hours. It was then dialyzed (8-14 kDa molecular weight cutoff) in a 4°C refrigerator for 48 hours. After precooling and freeze-drying, an ovalbumin-ferulic acid covalent complex was obtained. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined by the Folin-Ciocalteu method to be 57.46±0.19 mg / g.

[0049] Example 4 Ovalbumin-ferulic acid covalent complex

[0050] Same as Example 3, except that the plasma treatment time was changed to 15 s. The total phenol equivalent was measured to be 40.92±0.26 mg / g.

[0051] Example 5 Ovalbumin-syringic acid covalent complex

[0052] Ovalbumin was dispersed in deionized water at a concentration of 1 mg / mL. 5 mL of the sample was taken in a quartz dish. The quartz dish was placed between the two electrodes of the low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. The treatment was carried out for 30 seconds under the conditions of air as the working gas, 40 kV as the voltage, and 1.3 kHz as the frequency. After the treatment, syringic acid was added and dispersed in the mixed system at a concentration of 20 μmol / mL. The sample was placed in a 4°C refrigerator for reaction for 6 hours. It was then dialyzed (8-14 kDa molecular weight cutoff) in a 4°C refrigerator for 48 hours. After precooling and freeze-drying, an ovalbumin-syringic acid covalent complex was obtained. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined by the Folin-Ciocalteu method to be 85.79±0.28 mg / g.

[0053] Example 6 Ovalbumin-syringic acid covalent complex

[0054] Same as Example 5, except that the plasma treatment time was changed to 15 s. The total phenol equivalent was measured to be 61.05±0.39 mg / g.

[0055] Comparative Example 1 Preparation of Ovalbumin-Gallic Acid Covalent Complex by Alkaline Method

[0056] Weigh 100 mg of ovalbumin and dissolve it in 100 mL of deionized water. Adjust the pH to 9 with 0.5 M NaOH and stir overnight. Add sodium azide (0.005 wt%) to the solution to inhibit microbial growth. Then add 2 mmol of gallic acid to the mixed system, adjust the pH to 9 with 0.5 M NaOH, and continue stirring for 24 hours in an open environment at 25 ° C. During this period, use 0.5 M NaOH to maintain the pH of the solution at 9. Then dialyze (8-14 kDa molecular weight cutoff) in a 4 ° C refrigerator for 48 hours. After precooling and freeze-drying, obtain the ovalbumin-gallic acid covalent complex prepared by the alkaline method. The total phenolic equivalent was determined by the Folin-Ciocalteu method to be 27.06 ± 0.12 mg / g.

[0057] Comparative Example 2 Preparation of Ovalbumin-Gallic Acid Covalent Complex by Free Radical Method

[0058] Dissolve 100 mg of ovalbumin in 100 ml of deionized water, then add 1.0 mL of H2O2 (10 M) and 0.25 g of ascorbic acid, stir at 25 ° C for 2 h, then add 2 mmol of gallic acid to the mixed system, stir at 25 ° C for 24 h, and then add Trolox (1 mmol / L) to terminate the oxidation reaction. Then dialyze (8-14 kDa molecular weight cutoff) in a 4 ° C refrigerator for 48 h. After precooling and freeze-drying, the ovalbumin-gallic acid covalent complex prepared by the free radical method was obtained. The total phenolic equivalent was determined by the Folin-Ciocalteu method to be 8.34±0.27 mg / g.

[0059] Comparative Example 3: Preparation of ovalbumin-gallic acid covalent complex by simultaneous treatment of protein polyphenols with low temperature plasma

[0060] Gallic acid was dispersed in deionized water at a concentration of 20 μmol / mL, and ovalbumin was dispersed in the mixed system at a concentration of 1 mg / mL. 5 mL of the sample was placed in a quartz dish, and the quartz dish was placed between the two electrodes of the low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. The working gas was air, the voltage was 40 kV, and the frequency was 1.3 kHz. The sample was placed in a 4 ° C refrigerator for 6 hours after the treatment. Then it was dialyzed (8-14 kDa molecular weight cutoff) in a 4 ° C refrigerator for 48 hours. After precooling and freeze-drying, the ovalbumin-gallic acid covalent complex was obtained. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined by the Folin-Ciocalteu method to be 15.78 ± 0.58 mg / g.

[0061] Comparative Example 4 Preparation of ovalbumin-gallic acid covalent complex by long-term low-temperature plasma treatment

[0062] Ovalbumin was dispersed in deionized water at a concentration of 1 mg / mL. 5 mL of the sample was taken in a quartz dish. The quartz dish was placed between the two electrodes of a low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. The treatment was carried out for 3 minutes at a voltage of 40 kV and a frequency of 1.3 kHz. After the treatment, gallic acid was added to the mixed system at a concentration of 20 μmol / mL. The sample was placed in a 4°C refrigerator for reaction for 6 hours. It was then dialyzed (8-14 kDa molecular weight cutoff) in a 4°C refrigerator for 48 hours. After precooling and freeze-drying, an ovalbumin-gallic acid covalent complex was obtained. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined by the Folin-Ciocalteu method to be 1.67±0.86 mg / g.

[0063] Test Example 1 SDS-PAGE gel electrophoresis

[0064] The ovalbumin-polyphenol covalent complexes prepared in Examples 1 to 6 and Comparative Example 4 were measured.

[0065] The lyophilized ovalbumin-polyphenol was dissolved in deionized water and the concentration was adjusted to 1 mg / mL. Then, the reduced protein loading buffer (4:1, v / v) was added, mixed and heated at 90°C for 10 min. Then, a precast gel (4% to 20%) was used for electrophoresis (160V, 50 min), and the sample volume was 10 μL. After the electrophoresis was completed, the precast gel was removed, and Coomassie Brilliant Blue Ultrafast Staining Solution was used and placed on a shaker for staining for 1 hour. Then, the staining solution was poured off and an appropriate amount of distilled water was added for decolorization.

[0066] from Figures 2 to 4 It can be seen that the band of the ovalbumin-polyphenol covalent complex has increased compared to the control protein, indicating that the molecular weight of the complex has increased. Since SDS can destroy the non-covalent bond between protein and polyphenol, the increase in the molecular weight of the complex proves that the polyphenol and ovalbumin have covalently bound.

[0067] from Figure 5 It can be seen that long-term low-temperature plasma treatment will destroy the primary structure of ovalbumin, which is not conducive to the combination with polyphenols. Therefore, treatment less than or equal to 1 min is more suitable for the preparation of protein-polyphenol covalent complexes.

[0068] Test Example 2 Determination of total phenol equivalent

[0069] The polyphenol content of the covalent complex was determined by the Folin-Ciocalteu method. The specific steps are as follows: 0.5 mL of ovalbumin-polyphenol complex solution (1 mg / mL) was mixed with 2.5 mL of freshly prepared Folin phenol reagent (200 mmol / L). After reacting in the dark for 5 min, 2 mL of Na2CO3 solution (7.5%, w / v) was added to the mixture and reacted in the dark for 2 h. The absorbance of the ovalbumin-polyphenol complex at 760 nm was then measured.

[0070] Weigh a certain amount of various polyphenols, dilute to multiple concentrations, and then measure the absorbance value in the same way as above to make a standard curve. Calculate the content of polyphenols in the sample according to the standard curve formula, and the final result is expressed in milligrams of polyphenol equivalents per gram of complex, see Table 1.

[0071] Table 1

[0072]

[0073] It can be seen from Table 1 that the ovalbumin-polyphenol covalent complex prepared by the present invention reaches a certain total phenol equivalent and grafting rate under short-time treatment, which indicates that the method adopted by the present invention has a high coupling efficiency.

[0074] Test Example 3 Appearance after Binding Reaction

[0075] Take photos to record the appearance colors of the ovalbumin-gallic acid covalent complexes prepared in Examples 1 to 2 and Comparative Examples 1 to 3 after dialysis.

[0076] Depend on Figure 6 It can be seen that, in the process of preparing the ovalbumin-polyphenol covalent complex, the present invention can avoid the oxidation of polyphenols to quinones and the color change caused by it compared with other methods.

Claims

1. A method for preparing a protein-polyphenol covalent complex, characterized in that: The following steps are involved: (1) Low-temperature plasma treatment of protein solution; (2) adding polyphenols to the protein solution obtained in step (1), stirring to allow the solution to react fully, and obtaining a protein-polyphenol covalent complex.

2. A method for preparing a protein-polyphenol covalent complex according to claim 1, characterized in that: In the low-temperature plasma treatment process of step (1), the working gas is air, nitrogen, helium, or argon, the treatment voltage is 10 to 50 kV, the treatment frequency is 0.5 to 5 kHz, and the treatment time is 10 to 60 s.

3. A method for preparing a protein-polyphenol covalent complex according to claim 2, characterized in that: The low temperature plasma treatment time is 15 to 45 seconds, preferably 15 to 30 seconds.

4. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1 to 3, characterized in that: The low-temperature plasma treatment equipment used in step (1) consists of a high-voltage power supply, a reactor and a sample container, wherein the high-voltage power supply can achieve a voltage of 1 to 100 kV and a frequency of 0.1 to 20 kHz; the reactor consists of upper and lower electrodes with a size of φ50 mm, the insulating medium is a quartz plate with a size of φ102 mm×1 mm, the spacing between the electrodes is adjustable from 0 to 10 mm, and the sample container is a quartz dish with a size of φ50 mm×5 mm.

5. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1 to 3, characterized in that: The protein in step (1) is one or more of ovalbumin, β-lactoglobulin, pea protein isolate, and soy protein isolate.

6. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1 to 3, characterized in that: The polyphenols described in step (2) are one or more of gallic acid, ferulic acid, syringic acid, caffeic acid, ellagic acid, epigallocatechin gallate, epigallocatechin, and epicatechin.

7. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1 to 3, characterized in that: The protein concentration in the protein solution of step (1) is 1 to 20 mg / mL.

8. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1 to 3, characterized in that: The concentration of the polyphenol in the mixed system after adding the polyphenol in step (2) is 1 to 100 μmol / mL.

9. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1 to 3, characterized in that: In step (2), the fully reacted mixed solution of polyphenol and protein is dialyzed to remove the polyphenol not bound to the protein, and then freeze-dried to obtain a protein-polyphenol covalent complex.

10. A method for preparing a protein-polyphenol covalent complex according to claim 9, characterized in that: The dialysis is performed by placing the sample in a dialysis bag in deionized water at 4° C. for 48 hours, and the freeze-drying is performed by pre-freezing the dialyzed sample at -20° C. for 12 hours.

Images