Egg white protein thermal stability regulation and control method based on microenvironment reconstruction and application

Abstract

The invention discloses an egg white protein thermal stability regulation and control method based on microenvironment reconstruction and application, and relates to the technical field of food processing. The method comprises the following steps: pre-treating egg white protein to remove water-insoluble protein so as to obtain water-soluble egg white protein; the preparation method comprises the following steps: diluting water-soluble egg white protein until the protein concentration is 0.5%-3% (w / v), then adjusting the pH value to 10.1-10.5, and heating for 20-40 minutes under the condition of 90-100 DEG C; cooling the solution, adjusting the pH to be neutral, and performing ultrafiltration concentration to obtain a neutral protein concentrated solution; or cooling the solution, adjusting the pH value to 4-6, centrifugally collecting the protein, adding a proper amount of pure water, adjusting the pH value to obtain a neutral protein concentrated solution, and carrying out freeze drying or spray drying to obtain the heat-stable nano-scale recombinant egg white protein with a food industry full bacteriocidal spectrum (70-135 DEG C). The microenvironment reconstruction treatment process is simple, low in cost and suitable for large-scale production, the heat stability of the egg white protein is still maintained at high temperature and high pressure (120 DEG C / 0.1 MPa), and the advantages in the aspects of cost reduction and environmental influence reduction are outstanding.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and in particular relates to a method and application of regulating the thermal stability of egg white protein based on microenvironment reconstruction. Background Art

[0002] Solid or liquid high-protein beverages have become a research hotspot in the global health food field because of their rich protein and easy portability. The thermosensitive properties of egg white protein lead to a key bottleneck in its industrial application: under mainstream high-temperature and high-pressure sterilization (121°C / 20min, 0.1MPa) and other processes, egg white protein will inevitably undergo irreversible disordered agglutination when the concentration is ≥3%, and the existing technical system is difficult to simultaneously meet the requirements of high concentration, high thermal stability and fluid transparency. This defect causes the solid content of egg-based transparent beverages to be unable to meet the market demand for liquid high-protein beverages (≥5%), greatly limiting its application in the field of protein sports nutrition foods and reducing consumers' choices. Therefore, it is extremely urgent to break through the thermal stability of bulk egg liquid base materials, withstand the thermal environment of conventional industrial protein food processing, and resolve the contradiction between restricting the development of the poultry and egg industry and the strong consumer demand in multiple scenarios.

[0003] At present, the strategies for improving the thermal stability of proteins mainly include physical modification, chemical modification and biological regulation, but their effects are often limited, especially under extremely high temperatures, the stability of proteins is still not ideal. For example, the patent publication number CN118383492A is a method for preparing an egg white liquid with high thermal stability, which inhibits the thermal denaturation of egg white protein by combining enzymatic hydrolysis and high-pressure homogenization, but can only ensure that the egg white remains stable under pasteurization conditions of heating at 70°C for 3min, and still cannot meet the conditions for long-term storage at room temperature. The patent publication number CN105660983B is a method for preparing an insoluble egg protein aggregate particle and its application, which adopts heat treatment-mechanical shearing technology to prepare egg protein aggregate particles, and requires the additional introduction of complex and expensive high-speed homogenization equipment, and the energy consumption is 3-5 times that of conventional heat treatment. And the average particle size range of the egg white protein aggregate particles prepared by this method is between 100-1500nm, the light transmittance is low, the whiteness coefficient is high, and it cannot be applied to the development of high-protein transparent beverages. Patent publication number CN108669474A discloses a processing method for improving the heat resistance of whole egg liquid and the whole egg liquid thereof. The study prepares whole egg liquid capable of withstanding heat treatment at 100°C by adjusting pH and performing step-by-step enzymatic hydrolysis. However, the steps are complicated and time-consuming (4-9h), and some bacteria can still reproduce at an enzymatic hydrolysis temperature of 53-58°C.

[0004] Recent studies have found that the synergistic effect of pH and heat can bypass these limitations to regulate the aggregation pathway of soy protein isolate. However, existing strategies based on pH mainly focus on the gelation of egg white protein or single plant-based protein systems, such as the method of preparing heat-stable soy protein by preheating and high pH treatment in patent publication number CN115104664A, which ignores the complexity of multi-component proteins. Egg white protein is a multi-component system (ovalbumin, ovotransferrin, lysozyme, etc.), and the interaction between protein components makes the system more sensitive to temperature changes. The most critical thing is that egg white protein is a blank for the application of liquid high-protein beverages as a high-nutrition bulk food base.

[0005] The present invention develops an egg powder base material with high concentration and extreme heat treatment resistance, breaks through the technical bottleneck of poor thermal stability of egg white protein under high concentration conditions (6%-10%), and scientifically evaluates its heat resistance and rheological properties under various sterilization conditions. The present invention overcomes the limited effect of the prior art in improving thermal stability by adjusting the pH value and heating in a simple and easy way. The flexible unfolding and re-aggregation behavior of egg white protein under the treatment of microenvironment reconstruction is revealed, the structure-activity relationship between its structural characteristics and heat resistance is clarified, and a protein nano-aggregate with high thermal stability is constructed. The treatment of microenvironment reconstruction optimizes the solubility and turbidity of the protein, avoiding the common transparency reduction problem in traditional heat treatment and cross-linking methods. At the same time, the technology can effectively avoid the inconsistency of structural changes and ensure the stability of protein function by controlling pH, protein concentration and heat treatment conditions. Compared with methods such as high-pressure homogenization, chemical cross-linking and enzymatic hydrolysis, the treatment process of microenvironment reconstruction is simple and low-cost, more suitable for large-scale production, and meets the requirements of sustainable development, reducing the negative impact on the environment. Therefore, the microenvironment reconstruction processing technology solution has significant advantages in improving the thermal stability of egg white protein, optimizing the process, reducing costs and environmental impact. Summary of the invention

[0006] The purpose of the present invention is to provide a method for regulating the thermal stability of egg white protein based on microenvironment reconstruction. By directional regulation of the microenvironment parameters of egg white protein (pH 10.1-10.5, concentration 1%-3%, temperature 90-100°C, time 20-40min), the structure of egg white protein is optimized and its thermal sensitivity problem during thermal processing is solved, providing a solution with both thermal stability and green production compatibility for the industrialization of high-protein transparent beverages.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention proposes a method for regulating the thermal stability of egg white protein based on microenvironment reconstruction, comprising the following steps:

[0009] S1, pre-treating egg white protein to remove water-insoluble protein to obtain water-soluble egg white protein;

[0010] S2, diluting the water-soluble egg white protein to a protein concentration of 0.5%-3% (w / v), adjusting the pH to 10.1-10.5, and heating at 90-100° C. for 20-40 min;

[0011] S3, cooling the heated solution and collecting the protein by centrifugation or concentrating by ultrafiltration to obtain a neutral protein concentrate;

[0012] S4. The neutral protein concentrate is freeze-dried or spray-dried to obtain a heat-stable nano-scale recombinant egg white protein having a full sterilization spectrum (70-135° C.) for the food industry.

[0013] The egg white protein obtained by the microenvironment reconstruction treatment of the present invention is a nano-scale aggregate. The protein concentration of 6-10% after redissolution can maintain good fluidity and transparency under high temperature and high pressure conditions and ultra-high temperature instantaneous sterilization conditions, and has better application prospects in the production of high-protein beverages at room temperature.

[0014] Preferably, the pretreatment in S1 is specifically as follows: mixing and diluting egg white protein with pure water in a volume ratio of 1:0 to 2, adjusting the pH to neutral, mixing the egg white liquid and centrifuging to remove water-insoluble protein to obtain water-soluble egg white protein.

[0015] Furthermore, in S1, egg white protein and pure water are mixed and diluted in a volume ratio of 1:1.

[0016] Preferably, in S2, the water-soluble egg white protein is diluted to a protein concentration of 1%-3%.

[0017] Preferably, the heating in S2 is carried out at 90° C. for 30 min.

[0018] Preferably, the heating in S2 is performed at 100° C. for 20 min.

[0019] Preferably, the centrifugal collection of protein in S3 is specifically: cooling the heated solution and adjusting the pH value to 4-6, centrifugally collecting the protein and adding pure water to re-dissolve it, and adjusting the pH to neutral again to obtain a neutral protein concentrate.

[0020] Preferably, the ultrafiltration concentration in S3 is specifically as follows: cooling the heated solution, adjusting the pH to neutral, and performing ultrafiltration concentration using a 5-30 kDa filter membrane to obtain a neutral protein concentrate.

[0021] Preferably, the freeze-drying technology in S4 is specifically: cold trap temperature -50°C, vacuum degree 1Pa, freeze-drying for 36 hours.

[0022] Preferably, the spray drying technology in S4 is specifically as follows: inlet temperature 180° C., flow rate 600 mL / h.

[0023] Preferably, the nanoscale recombinant egg white protein is a nanoscale aggregate, and the average hydrodynamic diameter measured by dynamic light scattering is 20-100 nm.

[0024] Preferably, the heat-stable nano-scale recombinant egg white protein with a full sterilization spectrum of the food industry is specifically: the nano-scale recombinant egg white protein is re-dissolved and then heat-treated at 70-135° C. at a protein concentration of 6-10%, and still has transparency and its rheological properties conform to typical shear-thinning behavior.

[0025] Furthermore, after the nano-scale recombinant egg white protein is re-dissolved, it is heat-treated at 100-135° C. at a protein concentration of 6-10%, and still maintains good fluidity and solubility, and its rheological properties conform to typical shear-thinning behavior.

[0026] Furthermore, after the nano-scale recombinant egg white protein is re-dissolved, it is heat-treated at 120-135° C. at a protein concentration of 6-10%, and still maintains good fluidity and solubility, and its rheological properties conform to typical shear-thinning behavior.

[0027] Furthermore, after the nano-scale recombinant egg white protein was re-dissolved, it was subjected to high temperature and high pressure at a temperature of 120°C and a pressure of 0.1 MPa at a protein concentration of 6-10%, and still maintained good fluidity and solubility, and its rheological properties were consistent with typical shear thinning behavior; the thermal stability of egg white protein was maintained from room temperature to high temperature and from normal pressure to high pressure.

[0028] In a second aspect, the present invention provides a nano-scale recombinant egg white protein with thermal stability prepared according to the above method.

[0029] The present invention proposes, in a third aspect, the application of the heat-stable nano-scale recombinant egg white protein in food.

[0030] Preferably, the heat-stable nano-scale recombinant egg white protein is used in high-protein transparent beverages.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The method of the present invention effectively improves the thermal stability of egg white protein. The present invention regulates the thermal stability of egg white protein by microenvironment reconstruction, and the microenvironment reconstruction is achieved by coordinated regulation of pH, concentration and temperature. Mainly by optimizing the pH value and concentration, the structural changes of the protein are effectively controlled, its thermal stability and fluidity are improved, and the problem of protein aggregation or gelation caused by traditional heat treatment methods is solved. The treated egg white protein tolerates a concentration of 6-10% under high temperature and high pressure (120°C / 20min, 0.1MPa) and extreme heat treatment (135°C / 1min), and the solution remains transparent and clear with good fluidity. Compared with the low-temperature pasteurized egg white liquid that relies on refrigerated transportation and has a short shelf life, the method of the present invention breaks through the linear constraint of concentration-thermal stability, solves the industry problem of easy aggregation and instability during high-temperature sterilization and thermal processing of egg-based high-protein beverages, and significantly improves the appearance quality and functionality of the product.

[0033] (2) The method of the present invention reduces production costs. The method of the present invention effectively saves energy consumption and raw material consumption. Traditional methods such as high-pressure homogenization or enzymatic hydrolysis require additional energy input (high-speed homogenization, shearing) or exogenous components (enzyme preparations, polysaccharides), while the method of the present invention mainly relies on simple pH control and heating to achieve the improvement of the stability of thermosensitive proteins, reduces dependence on external additives, reduces raw material costs, and significantly saves energy consumption.

[0034] (3) The method of the present invention simplifies the process. The post-processing steps such as enzyme inactivation and polysaccharide separation required in the traditional process are simplified to a "one-step" process. Not only does it reduce the production steps, but it also improves the processing efficiency. Moreover, the method of the present invention does not require a complex control system, but only requires a conventional heating tank and a pH meter, which is easy to industrialize and mass produce.

[0035] (4) The method of the present invention improves environmental friendliness. From the perspective of environmental pollution control, the treatment method created by the present invention reduces the use of chemicals and additional additives compared to traditional methods, reduces pollution to the environment, and meets the requirements of green and sustainable development.

[0036] In general, the microenvironment reconstruction regulation method of the present invention not only improves the functionality and quality of egg white protein, but also has obvious advantages in saving costs, improving efficiency and reducing environmental impact. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a comparison diagram of the differences in appearance and fluidity of egg white proteins before and after pasteurization in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0038] Figure 2It is a comparison diagram of the differences in appearance and fluidity of the egg white proteins in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 before and after being sterilized under normal pressure and moist heat conditions;

[0039] Figure 3 It is a comparison diagram of the differences in appearance and fluidity of egg white protein before and after high temperature and high pressure sterilization in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0040] Figure 4 It is a comparison diagram of the differences in appearance and fluidity of egg white protein before and after ultra-high temperature instantaneous sterilization in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3;

[0041] Figure 5 This is a graph showing the particle size change of egg white protein under different sterilization conditions in Example 1 of the present invention;

[0042] Figure 6 This is a graph showing the particle size change of egg white protein under different sterilization conditions in Example 2 of the present invention;

[0043] Figure 7 This is a graph showing the particle size change of egg white protein under different sterilization conditions in Example 3 of the present invention;

[0044] Figure 8 The graph is a change graph of the viscosity and storage modulus of the egg white protein before and after being sterilized under normal pressure and moist heat conditions in Examples 1 to 3 of the present invention and Comparative Example 3;

[0045] Fig. 9 The graph is a graph showing changes in viscosity and storage modulus of egg white protein before and after high temperature and high pressure sterilization in Examples 1 to 3 of the present invention and Comparative Example 3;

[0046] Fig.10 The graph is a graph showing changes in viscosity and storage modulus of egg white protein before and after ultra-high temperature instantaneous sterilization in Examples 1 to 3 of the present invention and Comparative Example 3;

[0047] Fig.11 TEM image of the heat-stable egg white protein before and after being sterilized under normal pressure and moist heat in Example 2 of the present invention;

[0048] Fig.12 It is a comparison diagram of the ζ-potential changes of egg white proteins in Example 1, Example 2 and Comparative Example 3 of the present invention;

[0049] Fig.13 It is a comparison chart of the changes in the sulfhydryl content of egg white protein in Example 1, Example 2 and Comparative Example 3 of the present invention;

[0050] Fig.14It is a comparison diagram of the changes in the surface hydrophobicity of egg white proteins in Example 1, Example 2, and Comparative Example 3 of the present invention;

[0051] Fig.15 It is a comparison diagram of the changes in the intermolecular forces of egg white proteins in Example 1, Example 2, and Comparative Example 3 of the present invention;

[0052] Fig.16 It is a comparison diagram of the changes in the secondary structure of egg white protein in Example 1, Example 2 and Comparative Example 3 of the present invention;

[0053] Fig.17 It is a comparison diagram of the turbidity change of egg white protein before and after heating in Example 1, Example 2 and Comparative Example 3 of the present invention;

[0054] Fig.18 It is a comparison chart of the solubility change of egg white protein before and after heating in Example 1, Example 2 and Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0055] The following will be combined with the drawings in 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.

[0056] The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction is as follows:

[0057] Separate the egg white and yolk from fresh eggs, dilute the egg white protein with water at a volume ratio of 1:0-2, mix thoroughly and adjust the pH to 7, and centrifuge to obtain water-soluble egg white protein. Subsequently, dilute the sample to 0.5%-3%, adjust the pH to 10.1-10.5, and heat at 90-100°C for 20-40 minutes to change the protein structure and improve its thermal stability. Adjust the pH of the egg white protein to between 4-6, collect the precipitate by centrifugation and add appropriate pure water to re-dissolve it, adjust the treated protein back to a neutral pH of 7, and obtain thermally stable egg white protein powder by freeze-drying technology.

[0058] The present invention carries out the following preferred embodiments and designs comparative examples to observe the changes in the characteristics of the egg white proteins obtained in the embodiments and comparative examples before and after being sterilized under different conditions.

[0059] Embodiment 1:

[0060] Add 1 volume of water to the egg white, adjust the pH to 7, mix well, and centrifuge to remove water-insoluble protein. Dilute the egg white solution to 1% protein concentration, adjust the pH to 10.1, and heat at 90°C for 30 minutes; adjust the pH of the egg white protein to 5.3, collect the precipitate by centrifugation and add appropriate amount of pure water to reconstitute, adjust the pH to neutral, and freeze-dry. Reconstitute into protein solutions of different concentrations.

[0061] Embodiment 2:

[0062] Add 1 volume of water to the egg white, adjust the pH to 7, mix well, and centrifuge to remove water-insoluble protein. Dilute the egg white solution to 1% protein concentration, adjust the pH to 10.5, and heat at 90°C for 30 minutes; adjust the pH of the egg white protein to 4.7, collect the precipitate by centrifugation and add appropriate amount of pure water to re-dissolve, adjust the pH to neutral, and freeze-dry. Re-dissolve into protein solutions of different concentrations.

[0063] Embodiment 3:

[0064] Add 1 volume of water to the egg white, adjust the pH to 7, mix well, and centrifuge to remove water-insoluble protein. Dilute the egg white solution to 1% protein concentration, adjust the pH to 10.5, and heat at 100°C for 20 minutes; adjust the pH of the egg white protein to 5, collect the precipitate by centrifugation and add appropriate amount of pure water to re-dissolve, adjust the pH to neutral, and freeze-dry. Re-dissolve into protein solutions of different concentrations.

[0065] Comparative Example 1:

[0066] Reference patent CN105660983B discloses a method for preparing insoluble egg protein aggregate particles and its application. Specifically:

[0067] The egg white was diluted with water to a final protein content of 7.5%, the pH was adjusted to 3.5 with acid, and the mixture was heated in a 90°C water bath for 30 min. The formed gel was stored at 4°C for 24 h and then sheared at 10,000 rpm for 10 min. The concentration of the resulting protein microgel was then adjusted to 6% and the pH was 7.

[0068] Comparative Example 2:

[0069] Reference patent CN118383492A is a method for preparing egg white liquid with high thermal stability. Specifically:

[0070] The thick egg white and the thin egg white in the egg white were stirred at a low speed to mix evenly, 0.3% alkaline protease and neutral protease were added, and the mixture was incubated at 50°C for 30 minutes. The egg white solution after enzymatic hydrolysis was homogenized at a high pressure (50 MPa), and then the protein concentration was diluted to 6% and the pH was 7.

[0071] Comparative Example 3:

[0072] Add 1 volume of water to the egg white, adjust the pH to 7, mix well, and centrifuge to remove water-insoluble protein. Freeze-dry to obtain egg white powder, and reconstitute it into protein solutions of different concentrations.

[0073] The above-mentioned Examples 1 to 3 are methods for preparing egg white protein based on microenvironment reconstruction in the present invention. Comparative Example 1 and Comparative Example 2 are respectively methods for improving the thermal stability of egg white protein in the prior art, wherein Comparative Example 1 adopts a combination of heat treatment and mechanical shearing technology to prepare protein microgels, and Comparative Example 2 adopts enzymatic hydrolysis and high-pressure homogenization to prepare protein. Comparative Example 3, based on Examples 1 to 3, does not adopt microenvironment reconstruction, and directly prepares egg white protein. The present invention adopts the egg white protein prepared in Examples 1 to 3 and Comparative Examples 1 to 3, and observes the changes of the obtained egg white protein before and after different sterilization conditions. The details are as follows:

[0074] See also Figure 1-Figure 4 The obtained egg white proteins were sterilized under pasteurization, normal pressure and moist heat sterilization, high temperature and high pressure sterilization and ultra-high temperature instantaneous sterilization conditions, and the changes before and after of different egg white proteins were compared.

[0075] Figure 1 The difference in appearance and fluidity of egg white protein before and after pasteurization (70°C, 30 min) (Figure (a) is before heating and Figure (b) is after heating). The protein concentration of Comparative Example 1 and Comparative Example 2 is 6%, and the protein concentration of Comparative Example 3 and Examples 1 to 3 is 10%.

[0076] Figure 2 The difference in appearance and fluidity of egg white protein before and after normal pressure moist heat sterilization (100°C, 30 minutes) (Figure (a) is before heating and Figure (b) is after heating). Among them, the protein concentration of Comparative Example 1 and Comparative Example 2 is 6%, and the protein concentration of Comparative Example 3 and Examples 1 to 3 is 8%.

[0077] Figure 3 The difference in appearance and fluidity of egg white protein before and after high temperature and high pressure sterilization (120℃, 30min, 0.1MPa) (Figure (a) is before heating and Figure (b) is after heating). The protein concentration is 6%.

[0078] Figure 4 The difference in appearance and fluidity of egg white protein before and after ultra-high temperature instantaneous sterilization (135°C, 1 min) (Figure (a) is before heating and Figure (b) is after heating). Among them, the protein concentration of Comparative Example 1 and Comparative Example 2 is 6%, and the protein concentration of Comparative Example 3 and Examples 1 to 3 is 10%.

[0079] Depend on Figure 1-Figure 4It can be seen that the egg white protein prepared in Examples 1 to 3 has the characteristics of transparency and good fluidity before and after treatment under different sterilization conditions, and still maintains this characteristic after extreme heat treatment at a concentration of 10%. The egg white protein obtained in Comparative Example 1 has a higher whiteness coefficient before heat treatment, and there is no obvious difference in appearance between Comparative Example 3 and Example 3. Comparative Examples 1 and 3 both have gelation problems under three different sterilization conditions. Although Comparative Example 2 has a certain fluidity under pasteurization conditions (70°C, 30min), white aggregates with uneven particle sizes can be clearly observed and the transparency is reduced. It can be seen that the egg white protein preparation method in the prior art and the egg white protein preparation method that does not use the microenvironment reconstruction treatment in the present invention are still not ideal in terms of protein stability at high temperatures.

[0080] See also Figure 5-Figure 7 , the particle size changes of Examples 1 to 3 under different sterilization conditions. Figure 5-Figure 7 It can be seen that the average particle size of the egg white protein obtained in Examples 1 to 3 before heating is in the range of 20-40 nm, and the average particle size remains in the range of 30-50 nm after being treated under three different sterilization conditions of 100° C., 121° C., and 135° C. Therefore, it can be proved that the method of the present invention can effectively control the structural changes of the protein and improve its thermal stability by optimizing the pH value and temperature.

[0081] See also Figure 8-Figure 10 The obtained egg white protein was sterilized under normal pressure and moist heat sterilization, high temperature and high pressure sterilization and ultra-high temperature instantaneous sterilization conditions, and the changes in viscosity and storage modulus of the egg white protein before and after were compared.

[0082] Figure 8 The viscosity and storage modulus of egg white protein before and after normal pressure moist heat sterilization (100°C, 30 min) are shown in Figures (a) and (b) respectively show the viscosity and storage modulus of egg white protein before heating, and Figures (c) and (d) show the viscosity and storage modulus of egg white protein after heating. The protein concentration is 8%.

[0083] Fig. 9 The viscosity and storage modulus of egg white protein before and after high temperature and high pressure sterilization (120℃, 30min, 0.1MPa) change (Figure (a) and Figure (b) are the changes in viscosity and storage modulus of egg white protein before heating, and Figure (c) and Figure (d) are the changes in viscosity and storage modulus of egg white protein after heating). Among them, the protein concentration is 6%.

[0084] Fig.10The viscosity and storage modulus of egg white protein before and after ultra-high temperature instantaneous sterilization (135°C, 1 min) change (Figure (a) and Figure (b) are the changes in viscosity and storage modulus of egg white protein before heating, and Figure (c) and Figure (d) are the changes in viscosity and storage modulus of egg white protein after heating). The protein concentration is 10%.

[0085] Depend on Figure 8-Figure 10 It can be seen that before sterilization, there is no significant difference in viscosity and storage modulus between the heat-stable egg white protein prepared in Examples 1 to 3 and the natural egg white protein in Comparative Example 3, indicating that the egg white protein obtained by this method has good fluidity. After heat sterilization under different conditions, the viscosity and storage modulus of the natural egg white protein in Comparative Example 3, i.e., the natural egg white protein prepared without microenvironment reconstruction treatment, increased significantly, indicating that heating promoted the disordered aggregation of egg white protein and formed a gel-like structure. The proteins in Examples 1-3 showed good thermal stability, and maintained low viscosity and storage modulus after sterilization, close to the values ​​before sterilization.

[0086] See also Fig.11 TEM images of heat-stable egg white protein before and after normal pressure moist heat sterilization (100°C, 30 min) (Figure (a) is before heating and Figure (b) is after heating). Fig.11 It can be seen that the protein obtained in Example 2 is a small particle with a dense structure, showing clear boundaries and uniformly distributed nanoparticles. During the pretreatment process, pH and high temperature may have greatly destroyed the original high-order structure of the protein, causing it to tend to a uniform small particle state. The disulfide bond and electrostatic repulsion work together with the protein to form a stable particle core, and the covalent cross-linking locks the protein structure so that it can still maintain its original form in a reheating environment, showing good thermal stability.

[0087] The present invention further analyzes the essential reason why microenvironment reconstruction regulates the structure of egg white protein, and the present invention uses the egg white protein obtained in Example 1, Example 2, and Comparative Example 3 for observation. The details are as follows:

[0088] 1) detecting the ζ-potential of the obtained egg white protein;

[0089] ζ-potential is crucial in understanding protein self-assembly, aggregation behavior, and structural changes after thermal treatment. Fig.12As shown, the potentials of Example 1 and Example 2 are both negative, and their absolute values ​​are significantly greater than that of Comparative Example 3 (11.57 ± 0.49 mV) (P < 0.05), indicating that the microenvironment reconstruction process enhances the negative charge on the protein surface and improves the electrostatic stability of the solution system. In addition, as the pH increases, the absolute value of the ζ-potential gradually increases. Specifically, the absolute values ​​of the potentials of Example 1 and Example 2 are 26.33 ± 0.91 mV and 29.83 ± 0.40 mV, respectively, indicating that higher pH treatment results in more negative charges on the protein surface.

[0090] This phenomenon is mainly attributed to the effect of the alkaline environment on the functional groups on the protein surface. As the pH increases, the concentration of hydrogen ions in the solution decreases, and the carboxyl groups (-COOH) on the protein surface are deprotonated and converted into negatively charged carboxyl groups (-COO - ), thereby increasing the negative charge density on the particle surface. This increase in charge also promotes the unfolding of the egg white protein structure. In contrast, under lower pH treatment, some -COOH may still be in a protonated state, resulting in a relatively small total negative charge of the protein and a low absolute value of the potential. Heat treatment of proteins in a strong alkaline environment will form a more stable negatively charged conformation, enhance the electrostatic repulsion between molecules, thereby improving solubility and reducing nonspecific aggregation.

[0091] 2) detecting the sulfhydryl content in the obtained egg white protein;

[0092] The thiol content in egg white protein is an important indicator reflecting the oxidation state and structural stability of the protein. Its changes reveal the rearrangement of chemical bonds within the protein molecules during microenvironment reconstruction.

[0093] like Fig.13 As shown, after the microenvironment reconstruction treatment, the mean values ​​of free-SH and surface-SH of the protein nanoaggregates in Examples 1 and 2 were significantly higher than those in Control Example 3 (P<0.05). The -SH in natural egg white protein is usually buried inside the molecular structure and cannot bind to the indicator DTNB. During the microenvironment reconstruction process, the protein denatures and loses its original secondary and tertiary structures, thereby causing -SH to be exposed. As the pH in the egg white protein microenvironment increases, the levels of free-SH and surface-SH that can bind to DTNB in ​​the sample decrease significantly (P<0.05). In an alkaline environment, the increase in pH will also lead to partial deprotonation of the sulfhydryl groups of cysteine ​​residues (i.e., from *SH to -S-). When the sulfhydryl groups of two cysteine ​​residues approach each other, an oxidation reaction occurs to generate -SS-disulfide bonds. When the pH rises to 10.5, the oxidation of -SH and the formation of -SS-bonds reduce the level of surface -SH, and the protein forms a more compact aggregate under the action of electrostatic repulsion, hydrophobic interaction and -SS-bond rearrangement.

[0094] This phenomenon shows that in the process of microenvironment reconstruction, the intermolecular interaction of proteins is transformed from a loose non-covalent interaction to a covalent aggregation dominated by -SS-bond cross-linking, resulting in a significant reduction in free -SH and surface -SH. After adding the reducing agent β-mercaptoethanol, the total content of -SH inside the protein molecule (such as -SH in the disulfide bond) and exposed on the surface can be effectively evaluated. The changes in the microenvironment of Examples 1 and 2 lead to the conversion of a large amount of free -SH into -SS-bonds, and the total -SH content is lower than that of Control Example 3. In an alkaline environment, the unfolded protein is "locked" by the -SS-bond, forming a nanoscale protein dispersion, while electrostatic repulsion prevents the particles from merging.

[0095] 3) detecting the surface hydrophobicity of the obtained egg white protein;

[0096] Surface hydrophobicity can reflect the exposure degree of hydrophobic regions in protein molecules and the adjustment of overall surface properties caused by conformational changes, which is of great significance for understanding the structural stability of egg white protein.

[0097] like Fig.14 As shown, after the combined microenvironment regulation of pH, heat and concentration, the surface hydrophobicity of Example 1 and Example 2 is significantly higher than that of Control Example 3 (P<0.05). This is because under the dual effects of alkali and heat, part of the folded region of the protein is exposed, causing the hydrophobic groups wrapped inside the protein to be exposed in the solution and bind to the ANS fluorescent probe. The surface hydrophobicity of Example 1 is significantly higher than that of Example 2. This shows that the tertiary structure of the protein has changed significantly under alkaline conditions, resulting in different degrees of exposure of the hydrophobic groups. The egg white protein was heat treated under the conditions of 1% diluted protein concentration and pH 10.1 in egg white solution. At this time, the electrostatic repulsion was enhanced, and the alkaline environment prompted the protein structure to unfold more thoroughly, resulting in the complete exposure of the hydrophobic region originally located inside the protein. Under the treatment conditions of pH 10.5, the surface hydrophobicity of Example 2 was lower than that of the protein nanoaggregates of Example 1.

[0098] It is speculated that the reason for the decrease in surface hydrophobicity may be that the protein undergoes structural reorganization (such as intramolecular disulfide cross-linking or refolding) in a more alkaline environment, causing the originally exposed hydrophobic area to be rewrapped. Under stronger alkaline conditions, the electrostatic repulsion is further enhanced, and the protein tends to form a more stable folded conformation. At the same time, the -SH groups in the protein in the alkaline environment are converted into disulfide bonds (-SS-). This process makes the protein structure fold more tightly, further reducing the exposure of the hydrophobic area, thereby reducing the surface hydrophobicity.

[0099] 4) detecting the intermolecular forces of the obtained egg white protein;

[0100] The dynamic balance of intermolecular forces constitutes the regulatory hub of egg white protein thermal stability, and the deconstruction of its synergistic network provides a key entry point for revealing the mechanism of heat-induced aggregation. Fig.15 The synergistic growth and decline of the interaction between the microenvironment and the egg white protein molecules was quantified: the strength of the ionic bond decreased significantly with the increase of pH (10.1-10.5) (P<0.05), and this result was negatively correlated with the trend that the absolute value of the surface potential increased with the increase of pH. With the increase of pH, positively charged amino groups (such as lysine and arginine) and negatively charged carboxyl groups (such as aspartic acid and glutamic acid) may dissociate or neutralize. The negative charge density on the surface of protein nanoaggregates increased, and the electrostatic repulsion between molecules was enhanced, which weakened the effect of ionic bonds. In addition, under high pH conditions, the degree of ionization of amino acids increased, which may cause the originally stable ionic bonds in proteins to break, resulting in the unfolding or instability of protein structure. Hydrogen bonds play a vital role in the stability of protein secondary structures (such as α-helix and β-fold). Hydrogen bonds help maintain the three-dimensional structure of proteins by enhancing the interaction between molecules. Heating intensifies the movement of protein molecules, and thermal energy provides sufficient energy to break the hydrogen bonds in proteins. With the increase of pH, the contribution of hydrogen bonds gradually decreases (P<0.05). This is because the alkaline environment causes hydrogen bond donors (such as -NHa + , -OH), and at the same time, the changes in the higher-order structure of the protein destroy the local ordered structure.

[0101] The synergistic evolution of hydrophobic interaction and disulfide bonds constitutes the key to regulating the thermal stability of proteins, and its nonlinear response characteristics reveal the pH-induced conformational rearrangement mechanism. It is worth noting that the hydrophobic bond content of Example 1 is higher than that of Example 2. This shows that the protein is fully stretched under this condition, exposing more hydrophobic sites. However, due to the strong electrostatic repulsion between proteins in Example 2, the proportion of hydrophobic bonds decreases, so it can be inferred that the protein may self-aggregate into a more compact folded conformation, re-wrap the hydrophobic groups, and the number of hydrophobic bonds decreases accordingly. The pH change in the environment is positively correlated with the level of disulfide bond content in the protein. This indicates that more free thiol groups (-SH) may be involved in the formation of -SS- in the system. Under alkaline conditions, -SH groups are more easily oxidized to -SS-, and this transition is particularly prone to occur in a heated environment. The increase in disulfide bonds is usually associated with the thermal stability of proteins, because these covalent bonds can help proteins maintain their three-dimensional structures in extreme environments and reduce heat-induced structural unfolding and aggregation.

[0102] In summary, by systematically evaluating the dynamic changes of ionic bonds, hydrogen bonds, hydrophobic interactions and disulfide bonds in protein nanoaggregates during microenvironment reconstruction, the regulation of protein aggregation behavior by the microenvironment under the combined action of pH, concentration and heat can be revealed. Overall, at pH 10.1, the main forces are electrostatic repulsion and hydrophobic interactions. The alkaline environment promotes the unfolding of proteins, exposing the hydrophobic core region of the protein. However, the environment far away from the isoelectric point prevents large-scale aggregation of proteins, which is one of the reasons why the particle size is smaller than that of control example 3 under pH 10.1. Hydrophobic interactions drive proteins to form linear aggregations, while electrostatic repulsion inhibits lateral cross-linking. As the pH increases further, the main forces of the protein system at this time turn to electrostatic repulsion and disulfide bonds. The increase in disulfide bonds indicates that more -SH groups are oxidized to form intramolecular -SS- bonds, and the further reduction in particle size suggests that the protein may form dense particles under this condition. Overall, under alkaline and high temperature treatment conditions, egg white protein aggregation behavior and molecular weight distribution change significantly due to changes in intermolecular interactions, structural flexibility unfolding and refolding, and dynamic conversion of thiol and disulfide bonds. This intrinsic connection provides important clues for understanding the function and stability of proteins under different treatment conditions, and reveals the regulatory mechanism of protein aggregation and thermal stability.

[0103] The present invention verifies the stable structure of egg white protein in combination with the above analysis. The details are as follows:

[0104] 1) analyzing the obtained egg white protein secondary structure;

[0105] By analyzing the changes in the secondary structure of a protein, it can be determined whether the protein has undergone deconstruction, rearrangement, or formed a new stable structure. Fig.16 It was observed that the β-fold content of Example 2 increased from 26.8±5.74% of Control Example 3 to 50.27±6.96% (P<0.05), while the α-helix ratio decreased significantly (from 23.53±0.59% to 12.4±1.87%, P<0.05). During the heat treatment process, the β-fold gradually became the main conformational unit, because the heating destroyed the local hydrogen bond structure in the α-helix, causing the peptide chain to unfold. At the same time, the alkaline environment increased the hydrophobic interaction between proteins, prompting the protein to re-form a more stable lamellar structure, which is consistent with the results of surface hydrophobicity. This structure gives egg white protein higher rigidity and stability, helps to maintain the overall conformation of the protein, and enhances its ability to resist changes in the external environment. The increase in β-fold structure drives the orderly aggregation of proteins, because the rigidity of the β-fold layer limits the flexibility of the protein chain, promotes the aggregation of hydrophobic residues (such as Trp, Phe), and forms small-sized, compact aggregates, which is consistent with the results of particle size.

[0106] In summary, the change of the microenvironment induces the unfolding of α-helix and the reconstruction of β-sheet through electrostatic repulsion, and the degree is co-regulated by pH-concentration. Among them, the high β-sheet proportion (50.27±6.96%), high surface charge (-29.83±0.40mV), and high -S-S- bond content under Example 2 endow the protein with a smaller particle size and potentially excellent stability.

[0107] 2) Detect the degree of light scattering or absorption of protein aggregates in the egg white protein solution;

[0108] By measuring the degree of light scattering or absorption of protein aggregates in the solution, the aggregation degree of egg white protein under different treatment conditions can be indirectly reflected, which is crucial for evaluating the transparency and stability of the solution. As Fig.17 shown, the turbidity of natural egg white protein (Comparative Example 3) (pH 7.0) is 53.76±0.87%. After microenvironment reconstruction, the turbidities of the protein nanoaggregates prepared in Example 1 and Example 2 are 3.87±0.41% and 2.04±0.38% respectively, both significantly lower than that of Comparative Example 3 (P<0.05). In an alkaline environment, the charge distributed on the protein surface increases, and the electrostatic repulsion increases, thus effectively inhibiting the aggregation between proteins and reducing the light scattering effect. Natural egg white protein forms a gel after heating at 100°C for 30 min, which is due to the fact that high temperature causes the protein to unfold and exposes hydrophobic groups, and the protein molecules randomly crosslink through hydrophobic interactions and disulfide bonds to form a dense gel network and completely lose fluidity. The turbidities of Example 1 and Example 2 after secondary heating in a neutral environment are 3.32±0.51% and 1.98±0.26% respectively, and remain transparent, indicating that the protein is still in a dispersed state during the reheating process, without significant aggregation or denaturation, and has good thermal stability.

[0109] 3) Detect the solubility of protein aggregates in the egg white protein solution;

[0110] The determination of solubility is of great significance for evaluating the dispersion state and stability of proteins under different conditions. By comparing the changes in the solubility of protein nanoaggregates under different treatment conditions, it helps to clarify the protein aggregation mechanism and its thermal stability regulation strategy.

[0111] As Fig.18 shown, the solubilities of Example 1 and Example 2 are close to 100%, and there is no significant difference among groups. Under higher pH conditions, protein molecules carry more net charges, the electrostatic repulsion is enhanced, the formation of aggregates is inhibited, and thus a higher solubility and clear appearance are maintained. After reheating, Example 1 and Example 2 still maintain a high solubility (98.5±0.24%, 99.67±0.38%).

[0112] The above description is only used to help understand the method of the present invention and its core essence, but the protection scope of the present invention is not limited thereto. For those skilled in the art in the art, equivalent replacement or change according to the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for regulating the thermal stability of egg white protein based on microenvironment reconstruction, characterized in that: The following steps are involved: S1, pre-treating egg white protein to remove water-insoluble protein to obtain water-soluble egg white protein; S2, diluting the water-soluble egg white protein to a protein concentration of 0.5%-3% (w / v), adjusting the pH to 10.1-10.5, and heating at 90-100° C. for 20-40 min; S3, cooling the heated solution and collecting the protein by centrifugation or concentrating by ultrafiltration to obtain a neutral protein concentrate; S4. The neutral protein concentrate is freeze-dried or spray-dried to obtain heat-stable nano-scale recombinant egg white protein with a full bactericidal spectrum for the food industry.

2. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 1, characterized in that: The pretreatment in S1 is specifically as follows: mixing and diluting egg white protein with pure water in a volume ratio of 1:0-2, adjusting the pH to neutral, mixing the egg white liquid and centrifuging to remove water-insoluble protein to obtain water-soluble egg white protein.

3. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 2, characterized in that: In the S1, egg white protein and pure water are mixed and diluted in a volume ratio of 1:

1.

4. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 1, characterized in that: In the S2, the water-soluble egg white protein is diluted to a protein concentration of 1%-3%.

5. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 1, characterized in that: The centrifugal collection of protein in S3 is specifically as follows: cooling the heated solution and adjusting the pH value to 4-6, centrifugally collecting the protein and adding pure water to re-dissolve it, and adjusting the pH to neutral again to obtain a neutral protein concentrate.

6. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 1, characterized in that: The ultrafiltration concentration in S3 specifically comprises: cooling the heated solution and adjusting the pH to neutral, and performing ultrafiltration concentration using a 5-30 kDa filter membrane to obtain a neutral protein concentrate.

7. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 1, characterized in that: The nano-scale recombinant egg white protein is a nano-scale aggregate, and the average fluid dynamics diameter of the egg white protein is 20-100 nm.

8. The method for regulating the thermal stability of egg white protein based on microenvironment reconstruction according to claim 1, characterized in that: The heat-stable nano-scale recombinant egg white protein with a full sterilization spectrum of the food industry is specifically: the nano-scale recombinant egg white protein is re-dissolved and then subjected to a heat treatment at 70-135° C. at a protein concentration of 6-10%, and still has transparency and its rheological properties conform to typical shear-thinning behavior.

9. Nano-scale recombinant egg white protein prepared according to any one of claims 1 to 8.

10. Use of the nano-scale recombinant egg white protein as claimed in claim 9 in high-protein transparent beverages.

Images