Protein assembly stabilized emulsion construction method under multi-scale regulation strategy

Abstract

According to the construction method of the protein assembly stabilized emulsion under the multi-scale regulation strategy, the emulsifying activity of natural liposome and ovalbumin can reach 20.6 by means of electrostatic force and hydrophobic interaction between phosphate radical groups and hydroxyl, amino and the like in protein, the stability of the double-layer emulsion is improved, the treatment method is simple, and the method is suitable for industrial production. The application of the ovalbumin in an emulsification system is promoted, and the emulsifying capacity and the emulsifying stability of the ovalbumin are improved in a multi-scale manner.

Description

Technical Field

[0001] The present invention relates to the field of protein assembly emulsification, and in particular to a method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy. Background Art

[0002] Phosphorylation is the process of adding phosphate groups to intermediate metabolites or proteins. Its mechanism is that inorganic phosphoric acid forms -CO-Pi or -CN-Pi esterification reaction with specific oxygen atoms or nitrogen atoms on proteins, thereby introducing phosphate groups, increasing the electrostatic repulsion between protein molecules, increasing the electronegativity of proteins, making them easier to disperse, and thus improving the emulsification of proteins. Moreover, phosphorus atoms are an important component of biological membranes and one of the indispensable elements for all organisms. From a toxicological point of view, phosphate ions are necessary for all biological metabolism, but they must be obtained from the diet, so phosphorylation modification of proteins is a practical and effective method;

[0003] Ovalbumin is an important component of egg protein. It is a typical globulin. The primary structure of the protein consists of 385 amino acids, and there is a free water group buried inside the hydrophobic area. Ovalbumin has good foaming and gelling properties. Due to its good foaming and gelling properties, ovalbumin has become an important raw material for food processing, but its lack of emulsifying properties limits its application in emulsified systems.

[0004] At present, the main methods for protein modification are physical, chemical and enzymatic. Physical methods usually use thermal denaturation, freezing, high pressure, mechanical treatment, ultrasound, pulsed electric field and other methods to change the aggregation and higher-order structure of protein molecules. This type of method has the advantages of simple process, no toxic side effects, and little impact on the nutritional value of the product. Chemical methods mainly include phosphorylation, glycosylation, acylation and other methods, which have the characteristics of significant modification effect and wide application range, but there are problems such as complex reactions and easy toxic components to remain. Enzymatic modification has the advantages of strong specificity and fast speed, but it is difficult to screen suitable enzymes and control reaction conditions, and the cost is high, which limits its application. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the shortcomings of the prior art, the present invention provides a method for constructing a stabilized emulsion of a protein assembly under a multi-scale control strategy, which solves the problems in the prior art of being difficult to screen suitable enzymes and control reaction conditions and being costly.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy, characterized in that it comprises the following steps:

[0009] Step 1: Prepare a 20 mg / mL ovalbumin solution, centrifuge it in a low-temperature refrigerated centrifuge for 30 minutes (8000g, 4°C), take the supernatant, and filter it to remove insoluble matter, add 0.005-0.2M sodium tripolyphosphate to the protein solution, adjust the pH of the reaction mixture to 2.0, 5.0, 7.0 and 9.0, heat it in a constant temperature water bath at 55-95°C, and end the water bath heating when the total heating time is 6 hours, dialyze the reaction mixture at 4°C for 48 hours to remove unreacted sodium tripolyphosphate, and self-assemble during the heating process to obtain phosphorylated ovalbumin self-assembled aggregates, and set a blank group, i.e., natural ovalbumin after centrifugal filtration;

[0010] Step 2: Phosphorylated ovalbumin self-assembled aggregates are combined with natural phospholipids, and composite liposomes are prepared by reverse phase evaporation method, and dissolved in water at different concentrations to generate a double-layer emulsion / gel-like emulsion with a shell-core structure. The negatively charged phosphate groups at the double-layer interface increase the electrostatic repulsion, making the emulsion droplets more dispersed;

[0011] Step 3: Add the ovalbumin solution and the liposome dispersion into the stirring container, and after stirring, ultrasonically treat the mixed solution in an ice bath until a uniform emulsion is obtained (no stratification after standing for 3 minutes), and then use a rotary evaporator to remove the organic solvent under reduced pressure at 38° C. until a transparent and uniform suspension is obtained;

[0012] Step 4: The transparent and uniform suspension is fed into a high-pressure homogenizer at a pressure of 9000 psi to obtain a uniform particle size.

[0013] Preferably, the mass concentration of ovalbumin in the ovalbumin solution is 15 mg / mL to 30 mg / mL, and the ovalbumin is derived from egg whites, and fresh eggs without contamination and cracks are selected.

[0014] Preferably, the mass ratio of the ovalbumin to the natural phospholipid is 8:1.

[0015] Preferably, the natural phospholipid is egg yolk lecithin.

[0016] (III) Beneficial effects

[0017] The present invention provides a method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy, which has the following beneficial effects:

[0018] 1. In the present invention, liposomes and ovalbumin are modified by means of electrostatic forces and hydrogen bonds between phosphate groups and sulfhydryl groups, amino groups, etc. in proteins, and the emulsifying activity thereof can reach 20.6 mL / g. Compared with unmodified ovalbumin, the emulsifying property thereof is significantly improved, and the processing method thereof is simple, which is conducive to promoting the application of ovalbumin in emulsification systems. At the same time, the composite modification technology of the interaction between the three significantly improves the emulsifying ability and emulsifying stability of ovalbumin. Sodium tripolyphosphate is a food additive widely used and generally considered safe internationally, and the phospholipid is natural egg yolk lecithin. The formed liposomes can form a stable emulsion with a double-layer shell-core structure.

[0019] 2. In the present invention, the high-pressure homogenizer is used to facilitate control by the staff, has little impact on the nutritional value of the entire product, is safe and reliable, and has no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the HIATNAVLFFGR peptide segment of the present invention.

[0021] Figure 2 This is a schematic diagram of the LYAEERYPILPEYLQCVK peptide segment of the present invention.

[0022] Figure 3 This is a schematic diagram of the ELYRGGLEPINFQTAADQAR peptide segment of the present invention.

[0023] Figure 4 It is a schematic diagram of the structure of the ovalbumin self-assembly of the present invention.

[0024] Figure 5 Schematic diagram of the emulsion structure of the present invention.

[0025] Figure 6 It is a schematic diagram of the structure of the phosphorylated ovalbumin self-assembled aggregate of the present invention.

[0026] Figure 7 It is a schematic diagram of the dispersion structure of the phospholipid complex liposome of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1:

[0029] The embodiment of the present invention provides a method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy, comprising the following steps:

[0030] Step 1: Prepare a 20 mg / mL ovalbumin solution, centrifuge it in a low-temperature refrigerated centrifuge for 30 minutes (8000g, 4°C), take the supernatant, and filter it to remove insoluble matter, add 0.005-0.2M sodium tripolyphosphate to the protein solution, adjust the pH of the reaction mixture to 2.0, 5.0, 7.0 and 9.0, heat it in a constant temperature water bath at 55-95°C, and end the water bath heating when the total heating time is 6 hours, dialyze the reaction mixture at 4°C for 48 hours to remove unreacted sodium tripolyphosphate, and self-assemble during the heating process to obtain phosphorylated ovalbumin self-assembled aggregates, and set a blank group, i.e., natural ovalbumin after centrifugal filtration;

[0031] Step 2: Phosphorylated ovalbumin self-assembled aggregates are combined with natural phospholipids to prepare composite liposomes by reverse phase evaporation method, and dissolved in water at a concentration of 2 mg / mL to generate a double-layer emulsion with a shell-core structure. The negatively charged phosphate groups at the double-layer interface increase the electrostatic repulsion and make the emulsion droplets more dispersed;

[0032] Step 3: Add the ovalbumin solution and the liposome dispersion into the stirring container. After stirring, ultrasonically treat the mixed solution in an ice bath until a uniform emulsion is obtained (no stratification after standing for 3 minutes). Subsequently, use a rotary evaporator to remove the organic solvent under reduced pressure at 38°C until a transparent and uniform suspension is obtained.

[0033] Step 4: The transparent and uniform suspension is fed into a high-pressure homogenizer at a pressure of 9000 psi to obtain a uniform particle size.

[0034] The mass concentration of ovalbumin in the ovalbumin solution is 15 mg / mL to 30 mg / mL, and the ovalbumin is derived from egg whites, and fresh eggs without contamination and cracks are selected.

[0035] The mass ratio of ovalbumin to natural phospholipids is 8:1.

[0036] Natural phospholipids are egg yolk lecithin.

[0037] In steps 3 and 4, after the ovalbumin is first combined with the phosphate group, the phosphate group is negatively charged, which increases the electrostatic repulsion and makes the emulsion more dispersed. In addition, the interaction between macromolecules helps to improve its physical and chemical properties. As a carrier system, liposomes not only have a delivery function, but also their nanoscale size gives them nano effects, such as size effect and surface effect. They can interact with macromolecules such as proteins through hydrogen bonds, hydrophobic effects and other forces, and improve the physical and chemical properties of proteins. Furthermore, the high-pressure homogenization technology has a comprehensive effect of mechanical forces and thermal effects such as high-speed shear, high-frequency oscillation, convection impact, cavitation, etc. on the fluid mixture during the working process, and improves the physical and chemical properties of biological macromolecules. Based on the above reasons, the present invention combines the interaction between macromolecules and the high-pressure homogenization technology to ensure that the obtained liposome dispersion has a small particle size, good stability, and low particle surface tension. At the same time, in the subsequent application process of the liposome dispersion, liposomes of different concentrations are added to water to form an emulsion embedding or a gel emulsion, so as to embed astaxanthin, carotene, etc.

[0038] Embodiment 2:

[0039] The embodiment of the present invention provides a method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy, comprising the following steps:

[0040] Step 1: Prepare a 20 mg / mL ovalbumin solution, centrifuge it in a low-temperature refrigerated centrifuge for 30 minutes (8000g, 4°C), take the supernatant, and filter it to remove insoluble matter, add 0.005-0.2M sodium tripolyphosphate to the protein solution, adjust the pH of the reaction mixture to 2.0, 5.0, 7.0 and 9.0, heat it in a constant temperature water bath at 55-95°C, and end the water bath heating when the total heating time is 6 hours, dialyze the reaction mixture at 4°C for 48 hours to remove unreacted sodium tripolyphosphate, and self-assemble during the heating process to obtain phosphorylated ovalbumin self-assembled aggregates, and set a blank group, i.e., natural ovalbumin after centrifugal filtration;

[0041] Step 2: Phosphorylated ovalbumin self-assembled aggregates are combined with natural phospholipids to prepare composite liposomes by reverse phase evaporation method, and dissolved in water at a concentration of 5 mg / mL to generate a double-layer emulsion with a shell-core structure. The negatively charged phosphate groups at the double-layer interface increase the electrostatic repulsion, making the emulsion droplets more dispersed;

[0042] Step 3: Add the ovalbumin solution and the liposome dispersion into the stirring container. After stirring, ultrasonically treat the mixed solution in an ice bath until a uniform emulsion is obtained (no stratification after standing for 3 minutes). Subsequently, use a rotary evaporator to remove the organic solvent under reduced pressure at 38°C until a transparent and uniform suspension is obtained.

[0043] Step 4: The transparent and uniform suspension is fed into a high-pressure homogenizer at a pressure of 9000 psi to obtain a uniform particle size.

[0044] The mass concentration of ovalbumin in the ovalbumin solution is 15 mg / mL to 30 mg / mL, and the ovalbumin is derived from egg whites, and fresh eggs without contamination and cracks are selected.

[0045] The mass ratio of ovalbumin to natural phospholipids is 8:1.

[0046] Natural phospholipids are egg yolk lecithin.

[0047] Embodiment three:

[0048] The embodiment of the present invention provides a method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy, comprising the following steps:

[0049] Step 1: Prepare a 20 mg / mL ovalbumin solution, centrifuge it in a low-temperature refrigerated centrifuge for 30 minutes (8000g, 4°C), take the supernatant, and filter it to remove insoluble matter, add 0.005-0.2M sodium tripolyphosphate to the protein solution, adjust the pH of the reaction mixture to 2.0, 5.0, 7.0 and 9.0, heat it in a constant temperature water bath at 55-95°C, and end the water bath heating when the total heating time is 6 hours, dialyze the reaction mixture at 4°C for 48 hours to remove unreacted sodium tripolyphosphate, and self-assemble during the heating process to obtain phosphorylated ovalbumin self-assembled aggregates, and set a blank group, i.e., natural ovalbumin after centrifugal filtration;

[0050] Step 2: Phosphorylated ovalbumin self-assembled aggregates are combined with natural phospholipids to prepare composite liposomes by reverse phase evaporation method, and dissolved in water at a concentration of 10 mg / mL to generate a gel emulsion with a core-shell structure. The negatively charged phosphate groups at the double-layer interface increase the electrostatic repulsion, making the emulsion droplets more dispersed;

[0051] Step 3: Add the ovalbumin solution and the liposome dispersion into the stirring container. After stirring, ultrasonically treat the mixed solution in an ice bath until a uniform emulsion is obtained (no stratification after standing for 3 minutes). Subsequently, use a rotary evaporator to remove the organic solvent under reduced pressure at 38°C until a transparent and uniform suspension is obtained.

[0052] Step 4: The transparent and uniform suspension is fed into a high-pressure homogenizer at a pressure of 9000 psi to obtain a uniform particle size.

[0053] The mass concentration of ovalbumin in the ovalbumin solution is 15 mg / mL to 30 mg / mL, and the ovalbumin comes from egg whites, and fresh eggs without pollution and cracks are selected.

[0054] The mass ratio of ovalbumin to natural phospholipids is 12:3.

[0055] Natural phospholipids are egg yolk lecithin.

[0056] Embodiment 4:

[0057] After obtaining the phosphorylated self-assembled aggregates, the phosphorylated peptides and sites were measured by the following methods:

[0058] Mass spectrometry analysis was performed using a Bruker ion mobility-quadrupole-time of flight mass spectrometer timsTOF Pro. Sample injection and separation were performed using a ULtiMate3000RSLCnano liquid chromatograph coupled online to the mass spectrometer. The peptide samples were inhaled by an autosampler and then bound to a Trap column (75 μm × 20 mm, 2 μm particle size, poresize, Thermo), and then eluted to the analytical column (75μm×250mm, 1.6μm particle size, pore size, ionoptics). Two mobile phases (mobile phase A: 0.1% formic acid in H2O and mobile phase B: 0.1% formic acid in ACN) were used to establish the analytical gradient. The flow rate of the liquid phase was set to 300nL / min. The peptides entered the mass spectrometer through the CaptiveSpray nano-ion source for DDA scanning, the TIMS function was turned on, and the PASEF scanning mode was used. Each scan cycle time was 1.1s, consisting of one MS1 scan and 10 PASEF MS / MS scans, and each PASEFMS / MS scan contained 12 MS / MS spectra.

[0059] The protein pH was adjusted to 2.0, 5.0, 7.0 and 9.0, and the water bath heating was terminated when the total heating time was 6 hours. After mass spectrometry identification, the obtained phosphorylated peptides were 17, 13, 10 and 10, respectively, and the number of possible phosphorylation sites was 25, 13, 16 and 16, respectively. Therefore, the phosphorylated aggregates self-assembled under pH 2 conditions have the most possible phosphorylated peptides and sites. In order to verify the above structure, it was placed under a fluorescence microscope for magnification and observation of its structure. Its specific structure is shown in the figure. Figure 4 , Figure 5 As shown: Figure 4 is an ovalbumin self-assembly, and Figure 5 This is a diagram of the structure of the emulsion formed by natural ovalbumin, natural ovalbumin and ovalbumin self-assembly (without a double-layer structure);

[0060] The structure of the phosphorylated ovalbumin self-assembled aggregates / phospholipid complex liposomes after dispersion under fluorescence microscopy (10×): Figure 6 as well as Figure 7 (forming an emulsion with a distinct double-layer structure).

[0061] Embodiment five:

[0062] The modified ovalbumin obtained in Examples 1 to 3 was tested, and the results are shown in Table 1:

[0063]

[0064] As shown in the above table, under the same other emulsification conditions, the emulsification and emulsification stability of ovalbumin modified by the modification method of the present invention and the ovalbumin that has not been treated with a high-pressure homogenizer and has not interacted with liposomes are significantly improved.

[0065]

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy, characterized in that: The following steps are involved: Step 1: Prepare a 20 mg / mL ovalbumin solution, centrifuge it in a low-temperature refrigerated centrifuge for 30 minutes (8000g, 4°C), take the supernatant, and filter it to remove insoluble matter, add 0.005-0.2M sodium tripolyphosphate to the protein solution, adjust the pH of the reaction mixture to 2.0, 5.0, 7.0 and 9.0, heat it in a constant temperature water bath at 55-95°C, and end the water bath heating when the total heating time is 6 hours, dialyze the reaction mixture at 4°C for 48 hours to remove unreacted sodium tripolyphosphate, and self-assemble during the heating process to obtain phosphorylated ovalbumin self-assembled aggregates, and set a blank group, i.e., natural ovalbumin after centrifugal filtration; Step 2: Phosphorylated ovalbumin self-assembled aggregates are combined with natural phospholipids, and composite liposomes are prepared by reverse phase evaporation method, and dissolved in water at different concentrations to generate a double-layer emulsion / gel-like emulsion with a shell-core structure. The negatively charged phosphate groups at the double-layer interface increase the electrostatic repulsion, making the emulsion droplets more dispersed; Step 3: Add the ovalbumin solution and the liposome dispersion into the stirring container, and after stirring, ultrasonically treat the mixed solution in an ice bath until a uniform emulsion is obtained (no stratification after standing for 3 minutes), and then use a rotary evaporator to remove the organic solvent under reduced pressure at 38° C. until a transparent and uniform suspension is obtained; Step 4: The transparent and uniform suspension is fed into a high-pressure homogenizer at a pressure of 9000 psi to obtain a uniform particle size.

2. The method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy according to claim 1, characterized in that: The mass concentration of ovalbumin in the ovalbumin solution is 15 mg / mL to 30 mg / mL, and the ovalbumin comes from egg whites, and fresh eggs without pollution and cracks are selected.

3. The method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy according to claim 1, characterized in that: The mass ratio of the ovalbumin to the natural phospholipid is 8:

1.

4. The method for constructing a protein assembly stabilized emulsion under a multi-scale control strategy according to claim 1, characterized in that: The natural phospholipid is egg yolk lecithin.

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