An antioxidant and high-stability PSE pork myofibrillar protein complex and a preparation method thereof

Abstract

The application discloses an antioxidative and high-stability PSE pork myofibrillar protein complex and a preparation method thereof, and belongs to the technical field of protein processing. In order to solve the technical problems that the traditional polyphenol covalent grafting modification efficiency of PSE pork protein is low due to a natural aggregate structure, the modified product has poor thermal stability and is difficult to be applied to a thermal processing food system and the like, the application provides an antioxidative and high-stability PSE pork myofibrillar protein complex and a preparation method thereof. The method is characterized in that, through the synergistic effect of octenyl succinic acid modification and polyphenol targeted crosslinking, high-density negative charges, space steric hindrance and a covalent network are introduced, the self-assembly and thermal aggregation behaviors of the protein in a low-salt system are inhibited, the solubility, thermal stability and antioxidative activity of the protein are significantly enhanced, and technical support is provided for high-value utilization of PSE pork.

Description

Technical Field

[0001] This invention belongs to the field of protein processing technology, specifically relating to an antioxidant and highly stable PSE pork myofibrillar protein complex and its preparation method. Background Technology

[0002] Pork is an important source of high-quality protein for humans and has extremely high nutritional value. However, during meat processing, factors such as abnormal post-slaughter glycolysis rates and a sharp drop in pH often result in PSE (pale, soft, exudative) pork, characterized by its pale color, soft texture, and poor water retention. The core reason for this quality deterioration lies in the varying degrees of denaturation of myofibrillar proteins, leading to a significant decrease in protein solubility and severe degradation of processing properties such as emulsification and gelation. Consequently, PSE pork is unsuitable for use in traditional meat processing systems, resulting in a huge waste of high-quality protein resources. Therefore, improving the solubility and processing properties of PSE pork protein has become one of the key issues urgently needing to be addressed in the field of meat science.

[0003] Epigallocatechin gallate (EGCG) is the most abundant catechin monomer in green tea, possessing a variety of remarkable physiological and health-promoting functions, including antioxidant, anti-inflammatory, and anti-tumor effects. In recent years, the introduction of natural polyphenols such as EGCG into protein molecules through covalent cross-linking has been proven to be an effective way to endow protein complexes with potential health benefits. Simultaneously, the introduction of polyphenols can improve the processing properties of proteins, such as solubility, emulsification, and thermal stability, to some extent, by altering their structure and charge distribution. Therefore, utilizing polyphenol modification to improve the processing adaptability of inferior meat proteins has dual significance: the high-value utilization of protein resources and the development of functional ingredients.

[0004] However, in its natural state, myofibrillar proteins exist primarily as large molecular aggregates in aqueous solutions, with their internal hydrophobic groups and active functional groups embedded deep within the protein backbone. This leads to significant limitations in traditional single-step covalent grafting methods. On the one hand, a single grafting reaction can only utilize a limited number of reaction sites on the surface of the protein aggregates, resulting in low grafting efficiency. On the other hand, this method cannot effectively dissociate the protein aggregate backbone; the modified protein complex remains in an aggregated state, making it highly susceptible to irreversible secondary aggregation and precipitation under heating conditions. This severely restricts its application in heat-processed food systems.

[0005] Therefore, developing a modification method that can simultaneously achieve efficient dissociation of myofibrillar protein aggregates and covalent grafting of polyphenols is of great scientific research value and industrial application prospects for fundamentally solving the processing bottlenecks such as poor solubility and low thermal stability of PSE pork protein, and realizing the transformation of inferior meat protein into high-value-added functional food ingredients and nutrient delivery carriers. Summary of the Invention

[0006] To address the technical challenges of low efficiency and poor thermal stability of traditional polyphenol covalent grafting modification of PSE pork protein due to its natural aggregated structure, which hinders its application in heat-processed food systems, this invention provides an antioxidant and highly stable PSE pork myofibrillar protein complex and its preparation method. The method involves extracting PSE pork myofibrillar protein and dispersing it in an aqueous phase. The system is adjusted to alkalinity (pH=9.0), and acylation is initiated by adding 2-octenyl succinic anhydride. After the reaction is complete, the mixture is mixed with an epigallocatechin gallate solution, and the mixture is continuously stirred under alkaline aerobic conditions. Finally, the reaction is terminated by adjusting the system to neutrality (pH=7.0) to obtain the myofibrillar protein complex. This invention utilizes the high-density negative charge and steric hindrance introduced by octenyl succinylation modification to break the protein's self-assembled aggregated state, exposing internal binding sites, and then covalently cross-linking it with the ortho-quinone intermediate generated by the oxidation of epigallocatechin gallate. The prepared complex constructs a stable covalent network to inhibit the self-assembly and thermal aggregation of proteins in a low-salt system, and has both high solubility and strong antioxidant activity. It improves the defects of poor solubility and limited processing of inferior PSE pork protein, and provides an effective way for the in-depth development and high-value utilization of PSE pork in the field of functional food base.

[0007] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a method for preparing an antioxidant and highly stable PSE pork myofibrillar protein complex, the method comprising the following steps: S1. Extracting myofibrillar protein from PSE pork; S2. Disperse the myofibrillar protein obtained in S1 evenly in water to obtain a myofibrillar protein suspension. S3. Adjust the pH of the myofibrillar protein suspension obtained in S2 to 9.0, add 2-octenyl succinic anhydride dropwise, and stir in a water bath at 37°C to obtain mixture A. S4. Mix the mixed solution A obtained in S3 with the aqueous solution of epigallocatechin gallate, and stir to obtain mixed solution B. S5. Adjust the pH of the mixed solution B obtained in S4 to 7.0 and store it at 4°C to obtain the complex solution.

[0008] In one embodiment of the present invention, the PSE pork mentioned in S1 is obtained by heating freshly slaughtered pork in a water bath at 37°C for 7 hours.

[0009] In one embodiment of the present invention, the mass concentration of the myofibrillar protein suspension in S2 is 20 mg / mL.

[0010] In one embodiment of the present invention, the mass of the 2-octenyl succinic anhydride in S3 is 8% of the myofibril protein mass.

[0011] In one embodiment of the present invention, the stirring rate in S3 is 800 r / min, and the time is 2 h.

[0012] In one embodiment of the present invention, the concentration of the epigallocatechin gallate aqueous solution in S4 is 0.6 mmol / L.

[0013] In one embodiment of the present invention, the volume ratio of the mixed solution A and the aqueous solution of epigallocatechin gallate in S4 is 1:1.

[0014] In one embodiment of the present invention, the stirring rate in S4 is 500 r / min, and the time is 2 h.

[0015] The second objective of this invention is to provide a PSE pork myofibrillar protein complex prepared by the above-described preparation method.

[0016] A third objective of this invention is to provide the application of the above-mentioned PSE pork myofibrillar protein complex in the preparation of functional foods, low-sodium protein beverages, or bioactive substance delivery carriers.

[0017] The beneficial effects of this invention are: This invention innovatively employs a "depolymerization-crosslinking" synergistic strategy, utilizing octenyl succinic acid modification to introduce high-density negative charges and steric hindrance, effectively dissociating the dense aggregated state of natural myofibrillar proteins, promoting highly extended peptide chains and exposing reaction sites, thus achieving efficient targeted covalent grafting of epigallocatechin gallate. This study successfully solved the problems of poor solubility and processing limitations of inferior PSE pork myofibrillar proteins by constructing a stable covalent crosslinking network. This network effectively inhibits conformational collapse and thermally induced aggregation of proteins under low-salt conditions, maintaining the system solubility above 90%, while significantly improving thermal stability and antioxidant activity. This method uses safe and compliant food modifiers and natural active ingredients (2-octenyl succinic anhydride and epigallocatechin gallate), with mild and controllable preparation conditions, requiring no large or complex equipment. This technology not only opens up new pathways for the high-value processing of PSE pork but also provides practical technical support for the development of functional low-salt protein beverages and nutrient delivery carriers. Attached Figure Description

[0018] Figure 1 The graph shows the results of determining the free thiol content and the amount of epigallocatechin gallate bound in the PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3. Different lowercase letters in the graph indicate significant differences in the free thiol content (P < 0.05), and different uppercase letters in the graph indicate significant differences in the amount of epigallocatechin gallate bound (P < 0.05). Figure 2 The graph shows the results of solubility and turbidity determination of the PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3; different lowercase letters in the graph indicate significant differences in solubility (P < 0.05), and different uppercase letters in the graph indicate significant differences in turbidity (P < 0.05). Figure 3 The fluorescence spectrum results of the PSE pork myofibrillar protein complex solution prepared in Example 1 and Comparative Examples 1-3 are shown in the figure. Figure 4 The graph shows the potential measurement results of the PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3; different lowercase letters in the graph indicate significant differences in potential (P < 0.05). Figure 5 The figure shows the particle size distribution of the PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3; different lowercase letters in the figure indicate significant differences in particle size (P < 0.05). Figure 6 The images show the microscopic morphology of the PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3. Figure 7 The graph shows the observation results of the heating characteristics of the PSE pork myofibrillar protein complex solution prepared in Example 1 and Comparative Examples 1-3. Figure 8 The graph shows the thermal stability test results of the PSE pork myofibrillar protein complexes prepared in Example 1 and Comparative Examples 1-3. Figure 9 ABTS of the PSE pork myofibrillar protein complex prepared in Example 1 and Comparative Examples 1-3 + The graph shows the results of the determination of free radical scavenging rate and DPPH free radical scavenging rate; where A represents ABTS. + The graph shows the results of the free radical scavenging rate determination. B represents the results of the DPPH free radical scavenging rate determination. Different lowercase letters in the graph indicate significant differences (P < 0.05). Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0021] The PSE pork used in this invention is obtained by heating freshly slaughtered pork in a water bath at 37°C for 7 hours.

[0022] Example 1: This embodiment provides a method for preparing an antioxidant and highly stable PSE pork myofibrillar protein complex. The method is as follows: S1. Extracting myofibrillar protein from PSE pork; S2. The myofibrillar protein obtained in S1 is uniformly dispersed in water to obtain a myofibrillar protein suspension with a mass concentration of 20 mg / mL. S3. Adjust the pH of the myofibrillar protein suspension obtained in S3 to 9.0, add 2-octenylsuccinic anhydride dropwise, and stir at 800 r / min for 2 h in a water bath at 37°C to obtain mixture A; the mass of the 2-octenylsuccinic anhydride is 8% of the myofibrillar protein mass. S4. Mix the mixed solution A obtained in S4 with an equal volume of epigallocatechin gallate aqueous solution, and stir at a stirring rate of 500 r / min for 2 h to obtain mixed solution B; the concentration of the epigallocatechin gallate aqueous solution is 0.6 mmol / L. S5. Adjust the pH of the mixed solution B obtained in S4 to 7.0 and store it at 4°C to obtain the PSE pork myofibrillar protein complex solution.

[0023] Comparative Example 1: This comparative example does not contain 2-octenyl succinic anhydride or epigallocatechin gallate. The specific preparation method is as follows: S1. Extracting myofibrillar protein from PSE pork; S2. The myofibrillar protein obtained in S1 is uniformly dispersed in water to obtain a myofibrillar protein suspension with a mass concentration of 10 mg / mL. S3. The myofibrillar protein suspension with a mass concentration of 10 mg / mL obtained in S2 is stirred at a stirring rate of 400 r / min for 2 h to obtain an aqueous solution containing myofibrillar protein. S4. The aqueous solution containing myofibrillar protein obtained in S3 is stored at a temperature of 4°C to obtain PSE pork myofibrillar protein complex solution.

[0024] Comparative Example 2: This comparative example does not contain 2-octenylsuccinic anhydride; the specific preparation method is as follows: S1. Extracting myofibrillar protein from PSE pork; S2. The myofibrillar protein obtained in S1 is uniformly dispersed in water to obtain a myofibrillar protein suspension (mixture A) with a mass concentration of 20 mg / mL. S3. Mix the mixture A obtained in S2 with an equal volume of epigallocatechin gallate aqueous solution, adjust the pH of the mixture to 9.0, and stir at a stirring rate of 500 r / min for 2 h to obtain mixture B; the concentration of the epigallocatechin gallate aqueous solution is 0.6 mmol / L. S4. Adjust the pH of the mixed solution B obtained in S3 to 7.0 and store it at 4℃ to obtain the PSE pork myofibrillar protein complex solution.

[0025] Comparative Example 3: This comparative example does not contain 2-octenylsuccinic anhydride; the specific preparation method is as follows: S1. Extracting myofibrillar protein from PSE pork; S2. The myofibrillar protein obtained in S1 is uniformly dispersed in water to obtain a myofibrillar protein suspension with a mass concentration of 10 mg / mL. S3. Adjust the pH of the myofibrillar protein suspension obtained in S2 to 9.0, add 2-octenylsuccinic anhydride dropwise, and stir at 800 r / min for 2 h in a water bath at 37°C to obtain a mixed solution; the mass of the 2-octenylsuccinic anhydride is 8% of the myofibrillar protein mass. S4. Adjust the pH of the mixed solution obtained in S3 to 7.0 and store it at 4°C to obtain the PSE pork myofibrillar protein complex solution.

[0026] The PSE pork myofibrillar protein complexes prepared in Example 1 and Comparative Examples 1-3 were analyzed through the following experiments: Experiment 1: Determination of Free Thiol Group Content and Bound Amount of Epigallocatechin Gallate (1) Determination of free thiol content The PSE pork myofibrillar protein complex solutions prepared in Examples 1 and 1-3 were diluted with deionized water to 1 mg / mL, mixed with Ellman's reagent at a volume ratio of 1:4, and incubated in the dark for 30 min. Afterwards, they were centrifuged at 10000 g and 4°C for 15 min, and the absorbance at 412 nm was recorded. Free thiol groups were calculated using the following formula: Thiol content (μmol / g) = 73.53 × 10 × sample absorbance value.

[0027] (2) Determination of the amount of epigallocatechin gallate bound to gallic acid Take 1.0 mL of the PSE pork myofibrillar protein complex solution prepared in Example 1 and Comparative Examples 1-3, respectively, and mix with 5.0 mL of Folin-Ciocalteu reagent. React at room temperature in the dark for 5 min. Then add 4 mL of Na₂CO₃ solution, vortex to mix, and incubate at room temperature in the dark for 120 min. After the reaction, centrifuge the mixture at 6000 g for 15 min at 4°C, collect the supernatant, and measure the absorbance at 700 nm using a UV-Vis spectrophotometer. Calculate the amount of epigallocatechin gallate bound to the sample based on the free epigallocatechin gallate standard curve.

[0028] This experiment was performed in 3 independent replicates (n=3), and the data are expressed as mean ± standard deviation.

[0029] like Figure 1 As shown, the free thiol content in Comparative Example 1 was 111.52 ± 2.58 μmol / g, while the free thiol content in Comparative Example 3 significantly increased to 143.38 ± 1.95 μmol / g (P < 0.05). This indicates that octenyl succinic anhydride altered the dense assembly structure of myofibrillar protein, causing a large number of previously deeply buried, highly active thiol groups to be exposed to the molecular surface. In Example 1, after grafting epigallocatechin gallate, the polyphenol binding amount reached 0.82 ± 0.05 mg / g, significantly better than Comparative Example 2 (P < 0.05), while a large amount of free thiol groups were consumed. The above data indicate that the method provided in Example 1 of this invention dissociates the dense internal structure of PSE pork myofibrillar protein, causing the ortho-quinone intermediate generated by the oxidation of epigallocatechin gallate to preferentially undergo a Michael addition reaction with free thiol groups, achieving efficient targeted cross-linking of polyphenols.

[0030] Experiment 2: Determination of Solubility and Turbidity (1) Determination of solubility The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were diluted with deionized water to 2 mg / mL. They were centrifuged at 5000 g and 4°C for 10 min using a refrigerated centrifuge. The protein concentration before and after centrifugation was determined using the biuret method. The final solubility result was calculated using the following formula: Solubility (%) = Protein concentration in supernatant / Protein concentration in original solution × 100.

[0031] (2) Turbidity determination The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were diluted with deionized water to 1 mg / mL. The absorbance of the samples at 600 nm was measured using a visible spectrophotometer and used as the turbidity of the samples.

[0032] This experiment was performed in 3 independent replicates (n=3), and the data are expressed as mean ± standard deviation.

[0033] like Figure 2As shown, in Comparative Example 1, myofibrillar proteins readily aggregate under neutral, low-salt conditions, exhibiting extremely low solubility (2.53±1.00%) and a high turbidity of 1.00±0.01. While Comparative Example 2 improved protein dispersibility to some extent, it failed to fundamentally address the tendency to aggregate. In contrast, the solubility of the system in Comparative Example 3 jumped to 90.64±2.52%, and the turbidity decreased to 0.026±0.005. This was attributed to the electrostatic repulsion and steric hindrance introduced by octenyl succinic anhydride, which disrupted the protein's self-assembly structure. In Example 1, after efficient grafting with epigallocatechin gallate, the solubility remained stably high at 89.94±1.64%, and the turbidity remained at an extremely low level of 0.03±0.01, demonstrating that no hydrophobic aggregation or precipitation occurred. The above results show that, while achieving efficient grafting of polyphenols, the present invention locks the newly constructed protein-polyphenol covalent network in a highly extended and completely dissolved dispersion state, thus endowing the complex with excellent physical stability.

[0034] Experiment 3: Measurement of Fluorescence Spectroscopy The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were diluted with deionized water to a concentration of 1 mg / mL. Emission spectra were measured using a fluorescence spectrophotometer in the 300 nm to 400 nm wavelength range at a scan rate of 600 nm / min. The excitation wavelength was set to 283 nm, the excitation and emission slit widths were 5.0 nm, and the data interval was 1 nm.

[0035] like Figure 3 As shown, the fluorescence intensity of Comparative Example 1 was at an extremely low level, indicating that its tryptophan residues were deeply buried inside the hydrophobic core, leading to fluorescence quenching. The fluorescence intensity of Comparative Example 3 showed a dramatic increase, possibly because electrostatic repulsion caused the protein backbone to stretch deeply, exposing a large number of tryptophan residues to the polar environment. In Example 1, after grafting epigallocatechin gallate, the fluorescence intensity significantly decreased compared to Comparative Example 3, confirming that the polyphenol oxidation intermediate had been successfully covalently bound to the peptide chain, resulting in static fluorescence quenching.

[0036] Experiment 4: Measurement of Potential The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were diluted with deionized water to 0.1 mg / mL, and the zeta potential was measured using a particle size potentiometer.

[0037] This experiment was performed in 3 independent replicates (n=3), and the data are expressed as mean ± standard deviation.

[0038] like Figure 4As shown, the Zeta potential value of Comparative Example 2 was -21.37 ± 0.91 mV, with a significant increase in absolute value. This is because the alkali treatment and polyphenol grafting altered the polar microenvironment of the protein surface, increasing its negative charge. The negative potential value of Comparative Example 3 further increased to -25.77 ± 0.64 mV. This is because the octenyl succinylation reaction consumes a large number of positively charged ε-amino groups on the protein surface and introduces easily dissociated carboxyl groups, thereby altering the charge distribution on the protein surface and increasing the absolute value of the potential. The potential value of Example 1 remained stable and slightly increased to -25.93 ± 1.40 mV. We speculate that the electrophilic intermediate generated by the oxidation of epigallocatechin gallate crosslinks with the exposed free thiol groups, without consuming the negatively charged carboxyl centers introduced by octenyl succinylation. The electrostatic repulsion between protein molecules with the same negative charge and the large-scale covalent network significantly increases, inhibiting the contractile tendency induced by polyphenol crosslinking and the self-assembly of myofibril proteins. Therefore, Example 1 has a strong inhibitory effect on the spatial collapse of the complex, and improves the solubility and stability of myofibrillar protein in aqueous solution.

[0039] Experiment 5: Particle Size Determination The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were used to directly measure the particle size of the complex using a laser particle size analyzer.

[0040] This experiment was performed in 3 independent replicates (n=3), and the data are expressed as mean ± standard deviation.

[0041] like Figure 5 As shown, the average particle size of Comparative Example 1 was 28.51 ± 0.80 nm, reflecting the spontaneous aggregation state of natural myofibrillar proteins in a low-salt system. The particle size of Comparative Example 3 decreased significantly, plummeting to 5.56 ± 1.22 nm (P < 0.05), indicating that the high-density negative charge and steric hindrance introduced by octenyl succinylation completely disrupted the non-covalent forces maintaining the assembly of the natural protein, causing it to dissociate into highly dispersed, extended, flexible polypeptide chains. The particle size of Example 1 was significantly larger than that of Comparative Example 3. This substantial increase in particle size is due to network expansion driven by covalent cross-linking, where the bound orthoquinone intermediates weave the free single chains into a large-scale polymeric covalent network.

[0042] Experiment 6: Observation of Microscopic Morphology Take 4 mL of the PSE pork myofibrillar protein complex solution prepared in Example 1 and Comparative Examples 1-3, respectively, mix with a staining solution containing 1.0 g / L Nile blue, shake well, and let stand for 30 min. Drop 5 μL of the stained sample onto a glass slide, cover with a glass slide, and seal. Observe the microstructure and distribution of the aqueous solution containing myofibrillar protein using a confocal laser scanning microscope, and take typical fluorescence images under a 40x objective lens.

[0043] like Figure 6 As shown, the image corresponding to Comparative Example 1 reveals a large, highly ordered, dense fibrous structure, a typical morphology of strong self-assembly of natural myofibrillar proteins. In the images corresponding to Comparative Examples 2 and 3, the coarse fibrous structures are significantly disrupted and deaggregated into diffuse, fragmented states. In the image corresponding to Example 1, the myofibrillar proteins lose their highly ordered rigid fiber bundles, exhibiting larger flocculent aggregates and displaying a highly hydrated and loosely porous structure in three-dimensional space. Despite a significant increase in particle size, excellent macroscopic dispersion stability is maintained.

[0044] Experiment 7: Observation of Heating Characteristics The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were respectively placed into 20 mL sample vials, allowed to stand for 3 min (25℃) to observe the appearance of the samples, and then placed in a water bath for heating at 70℃ for 30 min. After the heating was completed, the samples were transferred to ice water for rapid cooling, and the appearance of the samples was observed again.

[0045] like Figure 7 As shown, after heating at 70°C for 30 minutes, the protein complex solutions prepared in Comparative Examples 1, 2, 3, and 1 exhibited varying degrees of thermally induced aggregation and phase separation. The myofibrillar protein prepared in Comparative Example 1 produced a large number of white aggregates upon heating and almost completely precipitated and migrated to the bottom of the bottle, indicating that the aqueous solution of natural myofibrillar protein has extremely poor thermal stability. The sample prepared in Comparative Example 2 still showed significant phase separation and bottom precipitation after heating; the sample prepared in Comparative Example 3 showed a small amount of flocculation at the bottom; while the sample prepared in Example 1 showed almost no visible turbidity, phase separation, or precipitation migration, and the solution remained clear and transparent before and after heating, effectively resisting high-temperature-driven protein molecule recombination and significantly enhancing the thermal stability of the protein. This indicates that the complex prepared in Example 1 of this invention possesses excellent anti-thermal aggregation properties, maintaining a clear and homogeneous liquid phase without turbidity or flocculation after conventional heat sterilization at 70°C. This fully guarantees the excellent sensory quality of the final product and provides broad application prospects for the industrial production of novel transparent protein beverages, low-salt liquid foods, and liquid nutrient carriers.

[0046] Experiment 8: Test of Thermal Stability The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were freeze-dried. 5.0 mg of each freeze-dried protein sample was placed in a sealed aluminum dish, and the samples were heated from 20°C to 90°C at a constant rate of 10°C / min. The conformational stability of the different samples was measured using differential scanning calorimetry.

[0047] like Figure 8 As shown, Comparative Example 1 exhibits the lowest thermal denaturation temperature (51.65℃), indicating that natural myofibril proteins primarily rely on non-covalent interactions to maintain their structure and are highly susceptible to conformational collapse upon heating. Comparative Examples 2 and 3, after single-modification, show increased thermal denaturation temperatures, suggesting that local grafting or steric hindrance enhances the thermal resistance of the protein backbone. Example 1 reaches the highest level at 55.76℃, likely because the protein-polyphenol-protein covalent network constructed between highly extended protein single chains effectively restricts the intense thermal motion of the polypeptide backbone at high temperatures in a dehydrated environment. This microscopic thermodynamic evidence elucidates, at the molecular level, the potential mechanism by which the aqueous solution of the complex prepared in Example 1 effectively inhibits thermally induced aggregation and maintains a homogeneous and clear state at 70℃.

[0048] Experiment Nine: ABTS + Determination of free radical scavenging rate and DPPH free radical scavenging rate (1) ABTS + Determination of free radical scavenging rate The PSE pork myofibrillar protein complex solutions prepared in Examples 1 and Comparative Examples 1-3 were diluted with deionized water to 0.1 mg / mL. 1 mL of the protein solution was mixed with 3 mL of ABTS working solution, and the mixture was reacted for 30 minutes in the dark. The absorbance at 734 nm was recorded. For the control group, 1 mL of deionized water was used instead of the protein solution and mixed with 3 mL of ABTS working solution. The formula for calculating the ABTS+ free radical scavenging rate is as follows: ABTS+ free radical scavenging rate (%) = (absorbance of control group - absorbance of sample group) / absorbance of control group.

[0049] This experiment was performed in 3 independent replicates (n=3), and the data are expressed as mean ± standard deviation.

[0050] (2) Determination of DPPH free radical scavenging rate The PSE pork myofibrillar protein complex solutions prepared in Example 1 and Comparative Examples 1-3 were diluted with deionized water to 1 mg / mL. 2.5 mL of the sample solution was mixed with 2.5 mL of DPPH solution, reacted under light-protected conditions for 30 min, centrifuged at 2000 r / min for 10 min, and the absorbance of the supernatant was measured at 514 nm. Deionized water was used instead of the sample solution for the control group.

[0051] DPPH radical scavenging rate (%) = (Absorbance of control group - Absorbance of sample group) / Absorbance of control group This experiment was performed in 3 independent replicates (n=3), and the data are expressed as mean ± standard deviation.

[0052] like Figure 9 As shown, ABTS in Comparative Example 1 + The DPPH radical scavenging rates were extremely low, at only 2.15±1.11% and 8.37±0.38%, respectively. This is attributed to the lack of highly active hydrogen-donating groups in PSE myofibril protein, and its self-assembly behavior in aqueous solution, which encapsulates a limited number of antioxidant amino acid residues within a hydrophobic core, restricting the contact between these groups and free radicals. Comparative Example 2, due to the covalent grafting of epigallocatechin gallate, directly introduced a large number of phenolic hydroxyl groups with strong hydrogen-donating capabilities, resulting in ABTS... + The DPPH scavenging rate was significantly increased to 15.73±0.73% and 37.84±1.48%, respectively; Comparative Example 3, by dissociating the protein's self-assembled structure, exposed the internally hidden potential antioxidant residues, resulting in a slight improvement in its antioxidant activity. ABTS from Example 1 + The DPPH radical scavenging rates surged to 24.53±0.42% and 41.43±0.68%, respectively, significantly higher than those of comparative examples 1-3 (P<0.05), exhibiting the highest levels. This indicates that octenyl succinic anhydride inhibits protein self-assembly and that the targeted cross-linking of epigallocatechin gallate plays a positive synergistic role.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an antioxidant and highly stable PSE pork myofibrillar protein complex, characterized in that, Includes the following steps: S1. Extracting myofibrillar protein from PSE pork; S2. Disperse the myofibrillar protein obtained in S1 evenly in water to obtain a myofibrillar protein suspension. S3. Adjust the pH of the myofibrillar protein suspension obtained in S2 to 9.0, add 2-octenyl succinic anhydride dropwise, and stir in a water bath at 37°C to obtain mixture A. S4. Mix the mixed solution A obtained in S3 with the aqueous solution of epigallocatechin gallate, and stir to obtain mixed solution B. S5. Adjust the pH of the mixed solution B obtained in S4 to 7.0 and store it at 4°C to obtain the complex solution.

2. The preparation method according to claim 1, characterized in that, The PSE pork described in S1 is made by heating freshly slaughtered pork in a water bath at 37°C for 7 hours.

3. The preparation method according to claim 1, characterized in that, The mass concentration of the myofibrillar protein suspension described in S2 is 20 mg / mL.

4. The preparation method according to claim 1, characterized in that, The mass of the 2-octenyl succinic anhydride mentioned in S3 is 8% of the myofibril protein mass.

5. The preparation method according to claim 1, characterized in that, The stirring rate described in S3 is 800 r / min, and the stirring time is 2 h.

6. The preparation method according to claim 1, characterized in that, The concentration of the epigallocatechin gallate aqueous solution described in S4 is 0.6 mmol / L.

7. The preparation method according to claim 1, characterized in that, The volume ratio of the mixed solution A and the epigallocatechin gallate aqueous solution in S4 is 1:

1.

8. The preparation method according to claim 1, characterized in that, The stirring rate described in S4 is 500 r / min, and the stirring time is 2 h.

9. The PSE pork myofibril protein complex prepared by any of the preparation methods described in claims 1-8.

10. The use of the PSE pork myofibrillar protein complex of claim 9 in the preparation of functional foods, low-sodium protein beverages, or bioactive substance delivery carriers.

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