A method for preparing a protein-polyphenol covalent complex

Abstract

The application discloses a method for preparing a protein-polyphenol covalent compound. The method comprises the following steps: firstly, treating a protein solution or suspension by using low-temperature plasma; then, adding polyphenol into the treated protein solution and fully reacting; then, dialyzing the mixed solution of the fully reacted polyphenol and protein to remove polyphenol which is not combined with the protein; and finally, obtaining the protein-polyphenol covalent compound by freeze-drying. The method does not add any substance in the whole process, avoids polyphenol oxidation, and can quickly and efficiently prepare the protein-polyphenol covalent compound.

Description

Technical Field

[0001] This invention specifically relates to a method for preparing protein-polyphenol covalent complexes, belonging to the field of food science and processing technology. Background Technology

[0002] In food systems, proteins and polyphenols are two very common types of substances. The nutritional value and specific structural and functional properties of proteins make them widely used in food systems, while polyphenols possess various biological activities and physiological functions and are widely found in plants. Under natural conditions, proteins often bind to polyphenols in non-covalent forms (weak interactions such as hydrophobic interactions, hydrogen bonds, ionic bonds, and π bonds), thereby improving the functional properties of proteins, such as emulsifying and gelling properties, and reducing protein allergenicity. However, non-covalent binding is easily affected by solution and environment, leading to protein-polyphenol separation and unstable effects. Covalent binding refers to the binding of protein and polyphenol molecules through covalent bonds, forming a stable complex. Protein-polyphenol covalent complexes have the following advantages: ① Protein-polyphenol covalent complexes are more likely to form stable network structures, exhibiting better thermal stability and antioxidant properties compared to non-covalent complexes. ② Covalently bound polyphenols can enhance the gelling properties and water retention of proteins. ③ Emulsions prepared from protein-polyphenol covalent compounds can effectively reduce the average particle size of the emulsion and improve the emulsifying properties of the protein. Therefore, the covalent binding of proteins with polyphenols, as a protein modification method, can greatly expand the application scenarios of proteins.

[0003] Currently, the preparation of protein-polyphenol covalent complexes can be categorized into enzymatic and non-enzymatic methods. Enzymatic methods utilize enzyme catalysis and cross-linking to achieve covalent bonding between proteins and polyphenols, forming stable protein-polyphenol covalent complexes. Commonly used enzymes include polyphenol oxidase, laccase, transglutaminase, peroxidase, lipoxygenase, and tyrosinase. The principle is that polyphenols form quinones under enzyme catalysis, which then further cross-link with proteins. Non-enzymatic methods include free radical and alkaline methods. The free radical method utilizes a redox system constructed with ascorbic acid and H₂O₂ to promote the binding of proteins and polyphenols through the oxidative effect mediated by hydroxyl radicals. The principle is that hydroxyl radicals are generated in the ascorbic acid and H₂O₂ redox system, oxidizing polyphenols to form quinones, which then covalently bind to the protein. The alkaline method primarily involves adjusting the pH of the protein-polyphenol complex solution to alkaline conditions. Polyphenols are unstable under alkaline conditions, oxidizing to quinones, which then bind to the nucleophilic groups of the protein.

[0004] Low-temperature plasma technology, as a method for modifying the physical properties of a matrix using high-energy reactive oxygen-nitrogen particles (RONS), has been widely applied in fields such as disinfection, environmental protection, material modification, and medicine due to its unique advantages. In recent years, the application of low-temperature plasma technology in food processing has attracted considerable attention. At room temperature, gas is ionized by a high-energy electric field to generate various reactive particles (RONS). . O3, OH . , HO2, O2 - The plasma (NO and ONOO) forms a complex RONS plasma oxidation system. The oxidation effect mediated by these active particles acts on the food matrix, thereby achieving modification of food macromolecules (proteins, starches, etc.) and sterilization and preservation. Due to its advantages of being green, non-thermal, and leaving no chemical residues, low-temperature plasma technology shows great promise in food processing, especially in modifying proteins and improving their functional properties. However, there are no reports on using plasma treatment to achieve covalent bonding between proteins and polyphenols.

[0005] Currently, the shortcomings of commonly used enzymatic and non-enzymatic methods for covalently binding protein polyphenols include:

[0006] (1) Enzymatic methods are inefficient, have long reaction times (up to 24 hours), and are costly;

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

[0008] (3) Not green and not environmentally friendly;

[0009] (4) Polyphenol oxidation occurs during covalent bonding. Summary of the Invention

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

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

[0012] A method for preparing protein-polyphenol covalent complexes includes the following steps:

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

[0014] (2) Add polyphenols to the protein solution obtained in step (1) and stir to allow it to react fully to obtain a protein-polyphenol covalent complex.

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

[0016] Preferably, the low-temperature plasma treatment time is 15–45 s, and more preferably 15–30 s.

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

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

[0019] Preferably, in step (1), the processing procedure is as follows: a certain amount of sample is placed in a quartz dish, the distance between the two electrodes is adjusted to 8 mm, the insulating dielectric 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 allow it to be fully hydrated.

[0022] Preferably, the polyphenol mentioned in step (2) is one or more of gallic acid, ferulic acid, syringic acid, caffeic acid, ellagic acid, epigallocatechin gallate, epigallocatechin, and epicatechin. Polyphenols with different molecular weights and structures have different binding affinities to 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 the addition of the polyphenol in step (2) is 1 to 100 μmol / mL.

[0025] Preferably, in step (2), the polyphenol and protein mixture solution after sufficient reaction is dialyzed to remove polyphenols that are not bound to the protein, and then freeze-dried to obtain a protein-polyphenol covalent complex.

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

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

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

[0029] This invention utilizes low-temperature plasma technology to achieve rapid and efficient covalent binding of proteins and polyphenols. The mechanism is as follows: First, the gas is ionized by a high-energy electric field to generate various active particles (O... . O3, OH . , HO2, O2 - The RONS plasma oxidation system is formed by the oxidation of proteins by NO and ONOO. The oxidation by RONS disrupts the non-covalent interactions (hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) that maintain protein structure, thereby unfolding the protein structure and fully exposing the binding sites of amino and thiol groups that bind to polyphenols. Simultaneously, the amino (-NH2) and thiol (-SH) groups of the RONS-oxidized protein become amino radicals (-NH2). . ) and sulfur radicals (-S . The polyphenols added after plasma treatment are reacted with protein amino free radicals (-NH). . ) and sulfur radicals (-S . Active intermediates such as protein and polyphenols attack each other, causing free radical chain reactions to form covalent bonds, thereby achieving rapid and efficient covalent binding between proteins and polyphenols.

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

[0031] (1) The method of the present invention is fast (the binding is completed in 10 to 60 seconds by plasma treatment) and efficient (the amount of polyphenols bound reaches 40 mg / g to 100 mg / g).

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

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

[0034] (4) During the covalent binding process of the present invention with the protein, the degree of polyphenol oxidation is extremely low, and no obvious color change caused by oxidation to quinone occurs. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of a low-temperature plasma processing device.

[0036] Figure 2 Electrophoresis diagram of ovalbumin-gallic acid covalent complex.

[0037] Figure 3 Electrophoresis diagram of the ovalbumin-ferulic acid covalent complex.

[0038] Figure 4 Electrophoresis diagram of ovalbumin-syringic acid covalent complex.

[0039] Figure 5 Electrophoresis diagram of ovalbumin-gallic acid covalent complex obtained after long-term treatment.

[0040] Figure 6 This is an image of the ovalbumin-gallic acid complex. Detailed Implementation

[0041] The present invention will be further illustrated below with an example of ovalbumin. However, the embodiments of the present invention are not limited thereto. The present invention can be implemented in many other ways, and those skilled in the art can make similar improvements without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the 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 placed in a quartz dish, which was then placed between the two electrodes of a low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. Treatment was performed for 30 s under the conditions of air as the working gas, 40 kV voltage, and 1.3 kHz frequency. After treatment, gallic acid was added and dispersed in the mixture at a concentration of 20 μmol / mL. The sample was then placed in a 4°C refrigerator for 6 h. Subsequently, dialyzing was performed at 4°C (8–14 kDa molecular weight cutoff) for 48 h. After pre-cooling, the sample was lyophilized to obtain the ovalbumin-gallic acid covalent complex. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined to be 27.59 ± 0.46 mg / g using the Folin-Ciocalteu method.

[0045] Example 2: Ovalbumin-gallic acid covalent complex

[0046] Same as Example 1, except that the plasma treatment time was replaced with 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 placed in a quartz dish, which was then placed between the two electrodes of a low-temperature plasma treatment device. The electrode distance was adjusted to 8 mm. Treatment was performed for 30 s under the conditions of air as the working gas, 40 kV voltage, and 1.3 kHz frequency. After treatment, ferulic acid was added and dispersed in the mixture at a concentration of 20 μmol / mL. The sample was then placed in a 4°C refrigerator for 6 h. Subsequently, dialyzing was performed at 4°C (molecular weight cutoff 8–14 kDa) for 48 h. After pre-cooling, the sample was lyophilized to obtain the ovalbumin-ferulic acid covalent complex. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined to be 57.46 ± 0.19 mg / g using the Folin-Ciocalteu method.

[0049] Example 4: Ovalbumin-Ferulic Acid Covalent Complex

[0050] Same as Example 3, except that the plasma treatment time was replaced with 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 placed in a quartz dish, which was then 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 30 s under the conditions of air as the working gas, 40 kV voltage, and 1.3 kHz frequency. After treatment, syringic acid was added and dispersed in the mixture at a concentration of 20 μmol / mL. The sample was then placed in a 4℃ refrigerator for 6 h. Subsequently, it was dialyzed at 4℃ (8–14 kDa molecular weight cutoff) for 48 h. After pre-cooling, the sample was lyophilized to obtain the ovalbumin-syringic acid covalent complex. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined to be 85.79 ± 0.28 mg / g using the Folin-Ciocalteu method.

[0053] Example 6: Ovalbumin-Syringic Acid Covalent Complex

[0054] Same as Example 5, except that the plasma treatment time was replaced with 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 using alkaline method

[0056] 100 mg of ovalbumin was dissolved in 100 mL of deionized water. The pH was adjusted to 9 with 0.5 M NaOH and stirred overnight. Sodium azide (0.005 wt%) was added to the solution to inhibit microbial growth. Then, 2 mmol of gallic acid was added to the mixture, and the pH was adjusted to 9 with 0.5 M NaOH. The mixture was stirred continuously for 24 h in an open environment at 25 °C, maintaining the pH at 9 with 0.5 M NaOH. Subsequently, the mixture was dialyzed at 4 °C (8–14 kDa molecular weight cutoff) for 48 h. After pre-cooling, the mixture was lyophilized to obtain the ovalbumin-gallic acid covalent complex prepared by the alkaline method. The total phenolic equivalent was determined to be 27.06 ± 0.12 mg / g using the Folin-Ciocalteu method.

[0057] Comparative Example 2: Preparation of ovalbumin-gallic acid covalent complex using the free radical method

[0058] 100 mg of ovalbumin was dissolved in 100 mL of deionized water, then 1.0 mL of H₂O₂ (10 M) and 0.25 g of ascorbic acid were added. The mixture was stirred at 25 °C for 2 h, followed by the addition of 2 mmol of gallic acid. The mixture was stirred at 25 °C for 24 h, and then Trolox (1 mmol / L) was added to terminate the oxidation reaction. The mixture was then dialyzed at 4 °C (8–14 kDa molecular weight cutoff) for 48 h. After pre-cooling, the mixture was lyophilized to obtain the ovalbumin-gallic acid covalent complex prepared by the free radical method. The total phenolic equivalent was determined to be 8.34 ± 0.27 mg / g using the Folin-Ciocalteu method.

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

[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, which was then 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 30 s under the conditions of air as the working gas, 40 kV voltage, and 1.3 kHz frequency. After treatment, the sample was placed in a 4°C refrigerator for 6 h. Subsequently, it was dialyzed at 4°C (8–14 kDa molecular weight cutoff) for 48 h. After pre-cooling, it was lyophilized to obtain the ovalbumin-gallic acid covalent complex. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined to be 15.78 ± 0.58 mg / g using the Folin-Ciocalteu method.

[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 placed in a quartz dish, which was then 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 at 40 kV and 1.3 kHz for 3 min. After treatment, gallic acid was added and dispersed in the mixture at a concentration of 20 μmol / mL. The sample was then placed in a 4℃ refrigerator for 6 h. Subsequently, the mixture was dialyzed at 4℃ (8–14 kDa molecular weight cutoff) for 48 h. After pre-cooling, the mixture was lyophilized to obtain the ovalbumin-gallic acid covalent complex. The treated sample was analyzed by SDS-PAGE gel electrophoresis, and the total phenolic equivalent was determined to be 1.67 ± 0.86 mg / g using the Folin-Ciocalteu method.

[0063] Test Example 1: SDS-PAGE Gel Electrophoresis

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

[0065] The lyophilized ovalbumin-polyphenol was dissolved in deionized water to a concentration of 1 mg / mL. Then, reduced protein loading buffer (4:1, v / v) was added, and the mixture was heated at 90°C for 10 min. Electrophoresis was then performed using a precast gel (4%–20%) (160 V, 50 min), with a loading volume of 10 μL. After electrophoresis, the precast gel was removed, stained with Coomassie Brilliant Blue ultrafast staining solution, and stained on a shaker for 1 h. The staining solution was then discarded, and a suitable amount of distilled water was added for destaining.

[0066] from Figures 2-4 As can be seen, the band of the ovalbumin-polyphenol covalent complex increased compared to the control protein, indicating that the molecular weight of the complex increased. Since SDS can disrupt the non-covalent bonds between the protein and polyphenol, the increase in the molecular weight of the complex proves that the polyphenol and ovalbumin have formed a covalent bond.

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

[0068] Test Example 2: Determination of Total Phenolic Equivalent

[0069] The polyphenol content of the covalent complex was determined using the Folin-Ciocalteu method. The specific steps were as follows: 0.5 mL of ovalbumin-polyphenol complex solution (1 mg / mL) was mixed with 2.5 mL of freshly prepared Folin-Ciocalteu 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 the mixture was reacted in the dark for 2 h. The absorbance of the ovalbumin-polyphenol complex at 760 nm was then measured.

[0070] Weigh out a certain amount of various polyphenols, dilute them to multiple concentrations, and then measure the absorbance values ​​in the same way as described above to prepare a standard curve. Calculate the polyphenol content in the sample according to the standard curve formula. The final result is expressed as milligram polyphenol equivalents per gram of the complex, as shown in Table 1.

[0071] Table 1

[0072]

[0073] As shown in Table 1, the ovalbumin-polyphenol covalent complex prepared in this invention achieved a certain total phenol equivalent and grafting rate under short-time treatment. This indicates that the method used in this invention has high coupling efficiency.

[0074] Test Example 3: Appearance after the reaction

[0075] The appearance and color of the ovalbumin-gallic acid covalent complexes prepared in Examples 1-2 and Comparative Examples 1-3 after dialysis were photographed and recorded.

[0076] Depend on Figure 6 It can be seen that, compared with other methods, the present invention can avoid the oxidation of polyphenols to quinones and the resulting color changes during the preparation of ovalbumin-polyphenol covalent complex.

Claims

1. A method for preparing protein-polyphenol covalent complexes, characterized in that, Includes the following steps: (1) Low-temperature plasma treatment of protein solutions; (2) Add polyphenols to the protein solution obtained in step (1) and stir to allow it to react fully to obtain a protein-polyphenol covalent complex; The protein mentioned in step (1) is ovalbumin; the polyphenol mentioned in step (2) is syringic acid; The duration of the low-temperature plasma treatment is 15-45 seconds.

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

3. The method for preparing a protein-polyphenol covalent complex according to claim 1, characterized in that, The duration of the low-temperature plasma treatment is 15-30 seconds.

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

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

6. A method for preparing a protein-polyphenol covalent complex according to any one of claims 1-3, characterized in that, In step (2), the polyphenol and protein mixture solution after sufficient reaction is dialyzed to remove polyphenols that are not bound to the protein, and then freeze-dried to obtain a protein-polyphenol covalent complex.

7. The method for preparing a protein-polyphenol covalent complex according to claim 6, characterized in that, The dialysis involves placing the sample in a dialysis bag and dialyzing it in deionized water at 4°C for 48 hours. The freeze-drying involves pre-freezing the dialyzed sample at -20°C for 12 hours.

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