Method for regulating and controlling stability of compound protein aquatic product based on structural dependence of high-load polyphenol

Abstract

The invention discloses a method for regulating and controlling the stability of a compound protein aquatic product based on high-load polyphenol structure dependence, which comprises the following specific operation steps: extracting myofibrillar protein, adding vegetable protein into a myofibrillar protein solution to construct a compound protein matrix, and introducing polyphenol modified compound protein with a specific structure. Then, a Fenton system is used for simulating a food processing oxidation environment; and after the oxidation is terminated, heating in a water bath at 90 DEG C for 30 minutes to obtain the polyphenol-modified compound protein gel system. The compound protein aquatic product prepared by the method shows improved gel property and stability. The gel strength of the prepared polyphenol-modified compound protein aquatic product can be improved by 50% or above after the polyphenol-modified compound protein aquatic product is oxidized in a simulated processing environment, the product is stable in texture and high in nutrition retention rate, chemical additives do not need to be added, and a new strategy is provided for developing high-nutritional-value clean tag protein products.

Description

Technical Field

[0001] This invention discloses a method for regulating the gelation properties and stability of composite protein aquatic products under oxidative stress based on structured polyphenol modification, belonging to the field of aquatic food processing technology. Background Technology

[0002] Surimi products, such as fish balls, fish cakes, and fish sausages, are popular with consumers due to their high protein, low fat, and unique flavor, and are an important part of the global seafood processing industry. The textural properties of these products, such as elasticity, firmness, and water retention, directly determine their sensory quality and commercial value. The formation of these textural properties relies primarily on the three-dimensional network gel structure formed by myofibrillar protein (MP), the main component of fish meat, during the heating process.

[0003] However, not all fish myofibrillar proteins possess good gelling properties. Especially for some freshwater fish, low-value marine fish, or raw fish that have undergone multiple freeze-thaw cycles, the myofibrillar proteins themselves have poor stability. During processing and heating, they are prone to protein denaturation and aggregation, resulting in a coarse gel network structure with low strength and poor water retention. This directly manifests as a loose texture, easy water separation (poor water separation properties), and poor slicing properties in surimi products, severely restricting the product quality and market development of related surimi products.

[0004] Currently, methods for improving the gel properties of myofibrillar proteins mainly include physical modification, chemical modification, enzymatic modification, and modification with natural additives. Among these, utilizing natural active substances, especially plant polyphenols (such as tea polyphenols, tannic acid, gallic acid, etc.) to interact with proteins and improve their functional properties has become a current research hotspot. The phenolic hydroxyl groups in polyphenol molecules can bind to amino acid residues of proteins (such as lysine and arginine) through non-covalent interactions (such as hydrogen bonds and hydrophobic interactions) and covalent interactions (the formation of quinones after oxidation and their reaction with nucleophilic groups), thereby cross-linking proteins and filling the gel network, theoretically improving gel performance and oxidative stability.

[0005] While polyphenol modification of myofibrillar proteins shows promise, current techniques face a significant bottleneck: natural myofibrillar proteins, due to their specific spatial structure and limited reactive sites, have a low polyphenol loading capacity. Studies have shown that at low polyphenol concentrations, the binding of myofibrillar proteins to polyphenols tends to saturate. This low-level polyphenol modification, while potentially improving certain gel properties (such as antioxidant capacity), cannot fundamentally and significantly strengthen the three-dimensional gel network structure of myofibrillar proteins. Insufficient polyphenol coverage at effective cross-linking points results in limited improvement in the strength and density of the gel network, leading to a less than expected improvement in gel performance.

[0006] Therefore, there is an urgent need to develop a method that can effectively overcome the saturation limit of polyphenol loading on myofibrillar proteins, thereby achieving a significant and synergistic improvement in the gel performance and stability of myofibrillar proteins at high polyphenol content. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a method for regulating the stability of composite protein aquatic products based on the structure-dependent regulation of high-load polyphenols. This method utilizes plant proteins and myofibrillar proteins to form a composite protein system. After polyphenol modification of the composite protein, an oxidative induction reaction is carried out using a hydroxyl radical (OH·) reaction system. Finally, the formed system is subjected to gelation treatment, enabling the composite protein gel system to exhibit higher gel strength and stability even with high polyphenol addition.

[0008] To achieve the above objectives, the technical solution adopted by the present invention includes the following steps: S1. Preparation of the complex protein system: Myofibrillar protein MP was prepared into an MP solution, and plant protein and calcium chloride were added to it and homogenized to mix it thoroughly. S2, Polyphenol modification: Add polyphenols to the composite protein system obtained in step S1, and stir magnetically for 2 hours at 4°C to ensure thorough mixing; S3, Oxidation-induced reaction: The composite system obtained in step S2 is mixed with the hydroxyl radical (OH·) reaction system and reacted at 4°C for 7-9 hours. After the reaction is completed, butylated hydroxytoluene is added to stop the reaction. The hydroxyl radical (OH·) reaction system is composed of 100 μM FeCl3, 100 μM ascorbic acid and 1 mM H2O2. S4. Centrifugation: Add 0.02 M phosphate buffer (pH 6.0) to the mixture obtained in step S3, wash with this solution 2-5 times and then centrifuge to completely remove the reagents in the hydroxyl radical reaction system; S5. Gelation treatment: The precipitate obtained in step S4 is heated in a water bath to induce its thermal gelation, and then immediately placed in an ice bath to cool for 10 min and stored at 4 °C. In one embodiment of the present invention, the method for extracting myofibrillar proteins used in step S1 includes the following steps: (1) Yellowfin tuna raw material processing: Take out the frozen yellowfin tuna and thaw it overnight in a 4 ℃ refrigerator; manually remove the head and tail and remove the fascia to obtain the white meat part of the back and belly of the fish, and store it in a -30 ℃ refrigerator. (2) Extraction of MP: Frozen yellowfin tuna white meat was taken out of the freezer at -30 ℃ and thawed overnight in a refrigerator at 4 ℃; then, the yellowfin tuna white meat was placed in a meat grinder and ground until fine (30 s / time, 1-3 min). 100 g of the ground fish meat was weighed and placed in a centrifuge bottle, and 4 times the volume of ice-cold buffer (0.1 M NaH2PO4 / Na2HPO4 with a volume ratio of 39:61, 0.1 M NaCl, 0.002 M MgCl2, 1 mM EDTA, pH 7.0) was added, and homogenized for 1-3 min at 11000-13000 rpm / min; the resulting homogenate was filtered through two layers of 80 gauze to remove connective tissue, and centrifuged at 4 ℃ and 3500 ×g for 10 min, discarding the supernatant and keeping the precipitate. The above operation was repeated 3 times. Then, add 4 volumes of 0.1 M NaCl solution to the precipitate, homogenize for 1 min at 11000-13000 rpm / min, and centrifuge at 4 ℃ and 3500 ×g for 10 min. Discard the supernatant and repeat the above operation 3 times. The precipitate collected is the protein protein (MP) of yellowfin tuna. Finally, the protein concentration is detected by the Bradford assay kit, and the prepared MP is stored at 4 ℃ for subsequent analysis.

[0009] In one embodiment of the present invention, in step S1, the MP solution is prepared by dissolving MP in a 0.3M sodium chloride solution, and the concentration of the MP solution is 20~40 mg / mL.

[0010] In one embodiment of the present invention, in step S1, the plant protein includes one of soy protein isolate (SPI), pea protein isolate, and mung bean protein. The amount of plant protein added is 30-70% of the mass of MP. After adding calcium chloride, the concentration of calcium chloride is 50-100 mM.

[0011] In one embodiment of the present invention, the polyphenols in step S2 are selected from at least one of gallic acid (GA), taurine (TF) and epigallocatechin gallate (EGCG), preferably taurine (TF).

[0012] In one embodiment of the present invention, after adding polyphenols in step S2, the concentration of polyphenols in the composite system is 40~100 μmol / g, preferably 80~100 μmol / g.

[0013] In one embodiment of the present invention, in step S3, the volume ratio of the composite system to the hydroxyl radical (OH·) reaction system is 40~60mL:100μL, and the mass ratio of the added butyl hydroxytoluene to the volume of the reaction system is 0.015~0.025%.

[0014] In one embodiment of the present invention, in step S5, the water bath heating temperature is 70~100℃ and the heating time is 30~50min.

[0015] In one embodiment of the present invention, the fish selected in step (1) is not limited to yellowfin tuna.

[0016] In a preferred embodiment of the present invention, in step (2), the white flesh of yellowfin tuna is placed in a meat grinder and ground until it becomes fine (30 s / time, 2 min). 100 g of the ground fish meat is weighed and placed in a centrifuge bottle, 4 times the volume of ice-cold buffer is added, and homogenized for 2 min at 12000 rpm / min; 4 times the volume of 0.1 M NaCl solution is added to the precipitate, and homogenized for 1 min at 12000 rpm / min. The present invention also provides a composite protein gel prepared according to the above method.

[0017] The present invention also provides an application of the above-mentioned composite protein gel in the food industry.

[0018] Beneficial effects: (1) This invention utilizes plant protein and myofibril protein to form a composite system, then modifies polyphenols in this system, and uses a hydroxyl radical system to induce oxidation, thereby constructing an "interfacial molecular bridge", which simultaneously achieves efficient oxidation inhibition and significant improvement in gel quality of the composite protein gel system.

[0019] (2) When the present invention selects taurine as a polyphenol to react with the composite protein system, it is found that taurine as a polyphenol can not only significantly enhance the gel performance of the composite protein gel and maintain its stability, but also maintain the color stability of the composite system during the oxidation process, which greatly improves the color stability of the myofibrillar protein composite gel.

[0020] (3) This invention adds calcium chloride to the complex protein system. The addition of calcium chloride helps neutralize the charge on the surface of the complex protein, reduces the electrostatic repulsion between MP and SPI, and promotes the orderly arrangement of protein molecules. This weakened electrostatic shielding effect helps to lock water molecules in the grid and pores of the gel network structure. In addition, calcium ions that are easily and strongly hydrated may induce the formation of a well-structured three-dimensional network, which is beneficial for the physical capture of water molecules.

[0021] (4) This invention proposes a method for regulating the stability of composite protein aquatic products based on the structure-dependent regulation of high-load polyphenols. This method avoids the gel degradation problem commonly seen in single-protein systems with high-dose polyphenols by "bridging polyphenols at the binary protein interface". (5) The emulsified fish products prepared by this invention have simple production steps, low cost, and contain no additives or preservatives, enabling large-scale factory production. This provides an effective technical solution for the high-value utilization of low-value yellowfin tuna and the development of high-quality, high-stability novel composite protein aquatic products, enhancing the product's market competitiveness and economic benefits, and possessing broad market application prospects. Attached Figure Description

[0022] Figure 1 A comparison of the gel strength of the composite protein gels in Comparative Example 1 and Comparative Example 3. Figure 2 Analysis of crosslinking of composite protein gels in Examples 1-3 and Comparative Examples 1-2; Figure 3 The rheological properties of the composite protein gels in Examples 1-3 and Comparative Examples 1-2; Figure 4 Lissajous figures of the composite protein gels of Examples 1-3 and Comparative Examples 1-2; Figure 5 The images show the apparent color of the composite protein gels from Examples 1-3 and Comparative Examples 1-2. Figure 6 The gel strength of the composite protein gels of Examples 1-3 and Comparative Examples 1-2; Figure 7 The gel strength of the composite protein gels in comparative examples 4-6; Figure 8 Moisture distribution of the composite protein gels of Examples 1-3 and Comparative Examples 1-2 Figure 9 The water-holding capacity of the composite protein gels in Examples 1-3 and Comparative Examples 1-2; Figure 10 The microstructures of the composite protein gels in Examples 1-3 and Comparative Examples 1-2 are shown. Figure 11The images show the infrared microscopy of the composite protein gels of Examples 1-3 and Comparative Examples 1-2. Figure 12 This is an energy dispersive spectroscopy (EDS) analysis diagram of the composite protein gel; Figure 13 To utilize the hydroxyl radical (OH) of the present invention The cell viability results after the reaction system was subjected to the MTT assay. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] The test methods involved in the embodiments and comparative examples of this invention are as follows: SDS-PAGE gel electrophoresis: 0.1 g of gel sample was completely dissolved in 2 mL of solution (containing 8 M urea, 2% SDS, 2% β-mercaptoethanol, and 20 mM Tris-HCl). The sample was centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was collected, and the protein concentration was adjusted to 1 mg / mL using sampling buffer. 10 μL of the sample was subjected to constant-voltage SDS-PAGE electrophoresis (prefabricated gel: 4% stacking layer, 12% separating layer). The gel was then stained in Thomas Brilliant Blue solution for 2 h, followed by three repeated washings with destaining solution and deionized water. Gel images were obtained using a DNR bioimaging system (Bio-Rad, Hercules, California, USA).

[0025] Rheological property determination: The rheological properties of the gel samples were evaluated using a rheometer equipped with a 40 mm parallel plate (TA Instruments, USA). First, the linear viscoelastic region (LVR) of the samples was determined by amplitude scanning. Subsequently, strain scanning was performed at a rate of 10 rad / s, with the scan range set from 0.01% to 1000%, and Lissajous figures were plotted using Metalab software. Frequency scanning was performed at 1% strain, with the frequency range set from 0.1 to 100 Hz. Creep and recovery tests were performed by applying a constant stress of 10 Pa for 200 s, followed by stress release and a 400 s recovery test.

[0026] Gel strength analysis: All composite protein gel samples were uniformly shaped into cylinders 2 cm high and 2 cm in diameter. The surfaces were smoothed, and a P / 0.5s probe was used, perpendicularly pressed onto the top of the oleogel product. The speed before, during, and after the test was 1 mm / s. Each sample underwent two consecutive compressions with a compression height of 10 mm, while the trigger force was set to 5 g. The gel strength was calculated as the product of the breaking force (g) and the breaking distance (mm).

[0027] Determination of gel moisture distribution: After weighing, the protein gel samples were wrapped in plastic wrap and measured in an NMR tube. The relaxation time (T2) distribution curve of each sample was determined using a low-field NMR analyzer with Q-CPMG pulse sequence, and the data were analyzed using dedicated software.

[0028] Microstructure determination: The composite protein gel sample was frozen in liquid nitrogen for 1 minute and then subjected to gold sputtering. The microstructure of the sample was observed using a Hitachi SU8000 cryo-electron microscope manufactured in Tokyo, Japan, at 10 kV and 2,000x magnification.

[0029] Micro-infrared measurements: Frozen protein gel sections were placed on a glass slide and then on the microscope stage. A PerkinElmer Spectrum Spotlight 400 micro Fourier transform infrared spectrometer (μ-FTIR; PerkinElmer, USA) was used, with measurements taken at 4000 to 400 cm⁻¹. -1 Within the specified wavenumber range, spectral measurements were performed in transmission mode, with an average of 25 scans per spectrum. The imaging area was 900 × 900 micrometers. Single-wavelength spectra were then plotted based on the characteristic absorption peaks of the protein gel.

[0030] Example 1 A method for regulating the stability of complex protein aquatic products based on the structure-dependent regulation of high-load polyphenols includes the following steps: S1. Yellowfin tuna raw material processing: Remove the frozen yellowfin tuna and thaw it overnight in a 4°C freezer; manually remove the head and tail and remove the fascia to obtain the white meat from the back and belly of the fish, and store it in a -30°C freezer.

[0031] Extraction of S2 and MP: Remove the frozen yellowfin tuna white meat from the freezer at -30°C and thaw it overnight in a refrigerator at 4°C. Place the yellowfin tuna white meat in a meat grinder and grind until smooth (30 s / time, 2 min). Weigh 100 g of the ground fish meat into a centrifuge bottle, add 4 times the volume of ice-cold buffer (0.1 M NaH2PO4 / Na2HPO4, 0.1 M NaCl, 0.002 M MgCl2, 1 mM EDTA, pH 7.0), and homogenize for 2 min (12000 rpm / min). The obtained homogenate was filtered through two layers of 80 gauze to remove connective tissue. It was centrifuged at 4 °C and 3500 ×g for 10 min, the supernatant was discarded and the precipitate was retained. The above operation was repeated 3 times. Four times the volume of 0.1 M NaCl solution was added to the precipitate and homogenized for 1 min (12000 rpm / min). Then it was centrifuged at 4 °C and 3500 ×g for 10 min, the supernatant was discarded and the above operation was repeated 3 times. The precipitate collected was the MP of yellowfin tuna. Preparation of the S3, MP-SPI complex protein system: The MP extracted in step S2 was prepared into an MP solution with a final concentration of 25 mg / mL; SPI was added to the MP solution at an amount of 50% of the MP mass, and 50 mM CaCl2 was added. The solution was thoroughly mixed using a homogenizer (T25, IKA, Germany) to obtain a complex protein system. S4. Construction of polyphenol-modified MP-SPI composite system: Gallic acid (GA) was added to the complex protein system in step S3 at a final concentration of 80 μmol / g, and the mixture was magnetically stirred at 4 °C for 2 h to ensure thorough mixing. S5, Oxidation-induced reaction: 50 mL of the composite system obtained in step S4 was mixed with 100 μL of hydroxyl radicals (OH). The reaction system (100 μM FeCl3, 100 μM ascorbic acid, 1 mM H2O2) was mixed and reacted at 4 ℃ for 8 h. Then, 0.02% (w / v) butylated hydroxytoluene was added to the system to stop the reaction. After the reaction was completed, the mixture was washed twice with 20 mL of 0.02 M phosphate buffer (pH 6.0). The precipitate obtained after centrifugation was the complex protein system.

[0032] S6. Preparation of composite protein gel: The composite protein system obtained in step S5 was continuously heated in a 90 °C water bath for 30 min to induce its thermal gelation. Immediately after the gelation reaction, the thermo-induced emulsion gel was placed in an ice bath to cool for 10 min and stored at 4 °C.

[0033] Example 2 The difference between Example 2 and Example 1 is that gallic acid (GA) is replaced with taurine (TF). All other steps remain the same as in Example 1, resulting in a composite protein gel.

[0034] Example 3 The difference between Example 3 and Example 1 is that gallic acid (GA) is replaced with epigallocatechin gallate (EGCG). All other steps remain the same as in Example 1, resulting in a composite protein gel.

[0035] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that steps S4 and S5 are omitted, and the composite protein system obtained in step S3 is directly heated in a 90 °C water bath for 30 min to induce its thermal gelation.

[0036] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that step S4 is omitted, and the composite protein system obtained in S3 is subjected to an oxidation-induced reaction.

[0037] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the addition of CaCl2 in step S3 is omitted. After adding SPI to the MP solution at an amount of 50% of the MP mass, the solution is thoroughly mixed using a homogenizer (T25, IKA, Germany). Steps S4 and S5 are omitted. The other steps are the same as in Example 1.

[0038] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that steps S3 and S4 are omitted, and 50 mL of 25 mg / mL MP solution is directly mixed with 100 μL of hydroxyl radicals (OH). The reaction system (100 μM FeCl3, 100 μM ascorbic acid, 1 mM H2O2) was mixed to carry out an oxidation-induced reaction.

[0039] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that step S3 is omitted, and GA is added to the 25 mg / mL MP solution to make the final concentrations of GA in the system 40, 60 and 80 μmol / g, respectively. The other steps are the same as in Example 1.

[0040] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that step S3 is omitted, and TF is added to the 25 mg / mL MP solution to make the final concentrations of TF in the system 40, 60 and 80 μmol / g, respectively. The other steps are the same as in Example 1.

[0041] The experiment investigated the effect of calcium chloride addition on the complex protein system and gel properties. In Comparative Example 3, without the addition of calcium chloride, electrostatic repulsion occurred between MP and SPI, affecting the orderly arrangement of protein molecules, thereby affecting the binding of the complex protein to polyphenols, and consequently affecting the gel properties of the complex protein gel. Figure 1 The gel strength results for the composite gels of Comparative Example 1 and Comparative Example 3 are presented. Figure 1 As can be seen, the gel strength of the composite protein gel in Comparative Example 3 without calcium chloride was significantly lower than that in Comparative Example 1 with calcium chloride. This indicates that Ca... 2+ It neutralizes the negative charge on the surface of binary proteins, thereby weakening the electrostatic repulsion between protein molecules and promoting the regular aggregation of protein molecules. Meanwhile, Ca... 2+ Forming ion bridges between adjacent protein molecules helps to strengthen the gel network structure.

[0042] Figure 2 The effects of different phenolic substances on the MHC bands of composite protein gel electrophoresis were shown. In the absence of β-ME, the MHC band intensity of the composite protein gel treated with •OH-induced oxidation was significantly reduced, and new bands were generated at the top of the gel due to the polymerization of protein aggregates. However, in Example 2, the optical density value of the weakened MHC band gradually increased, indicating that the addition of TF in the oxidative environment can neutralize •OH and reduce MP aggregation caused by oxidation. Effective binding between SPI and TF also alleviates the effect of TF on MP oxidative stress induction. Notably, although the polymer at the top of the stacked gels did not increase significantly in Examples 1 and 3, the MHC bands were significantly weakened. This is mainly because the GA and EGCG with resorcinol structures, pre-oxidized to ortho-diquinones, bind to the amino and thiol groups of the protein, occupying the Coomassie Brilliant Blue binding sites on the protein chain, thus preventing effective binding of Coomassie Brilliant Blue. Figure 2 As can be seen in B, the protein bands recovered in the presence of β-ME, indicating that under •OH-induced oxidation conditions, disulfide bonds are the main factor leading to the cross-linking and aggregation of binary proteins in gels. Figure 3The effect of adding structured polyphenols on the support properties and stability of binary protein gels was evaluated by frequency scanning. The G' and G" values ​​of all samples showed a frequency-dependent increase. Compared with the composite protein gel of Comparative Example 1, the protein gels induced by Comparative Example 2 and Examples 1-3 had higher modulus values. Oxidation altered the amino acid side chains in the protein, enhancing non-covalent and covalent interactions, including hydrogen bonds, hydrophobic interactions, and electrostatic forces, as well as disulfide bonds, inducing cross-linking and aggregation of binary proteins and enhancing the rigidity and elasticity of the gel network. Furthermore, oxidation promoted head-to-tail interactions of myosin and, together with soy protein, promoted the construction of a dense gel network. The addition of phenolic substances further improved the viscoelasticity of the protein gel. This indicates that the addition of phenolic substances protected the structural integrity of the protein by reducing oxidative degradation and retaining more effective cross-linking sites. The high-strength hydrogen bond interactions formed promoted cross-linking of the gel network, enhancing the mechanical strength and elastic response of the gel. The ability of the binary protein gel to recover its original state after stress release was further evaluated using creep recovery mode. Generally, protein gels with higher gel strength deform less under the same stress. Figure 3 As shown, the protein gel of Comparative Example 1 exhibits greater strain than the oxidation-induced protein gels of Comparative Example 2 and Examples 1-3, indicating weaker gel strength, consistent with the results regarding protein gel strength. Oxidation modification promotes cross-linking within the binary protein gel, enhancing its structural stability. The addition of phenolic substances, particularly in Examples 2 and 3, significantly reduces the creep strain of the binary protein gel, mitigating stress-induced structural damage during creep and minimizing the negative impact of external stress on the gel structure.

[0043] Based on Lissajous figure analysis, the relationship between instantaneous cyclic internal stress and applied strain in binary protein gel samples was visualized by the addition of phenolic substances. Within the linear viscoelastic region, the stress curve exhibits a standard sine waveform, matching the frequency of the applied stress-strain curve, forming a standard elliptical Lissajous figure. Upon entering the nonlinear viscoelastic region, the G' and G" values ​​of all samples plummeted, causing distortion of the shear stress waveform and deviation from sinusoidal characteristics. As shown in the figure, at a strain of 1%, the elastic Lissajous curves of all samples were compact ellipses, indicating a perfect elastic response to stress and strain. However, after OH oxidation induction, when the strain reached 5%, the shear stress waveform of the composite protein gel in Comparative Example 2 began to change. When the strain reached 50%, the shear stress waveform expanded and deviated from the central axis. As the strain continued to increase, the shape of the Lissajous curve changed to a quadrilateral, indicating an increase in viscous behavior and a shift from elastic to viscous-dominated behavior. The addition of phenolic compounds can effectively delay this change in behavior and maintain the elastic behavior of the composite protein gel at higher strains, which is directly related to the rigid protein conformation induced by polyphenol binding in the simulation. Furthermore, the influence of different phenolic compounds on the viscoelasticity of the protein gel is consistent with the trends obtained from other rheological tests.

[0044] The composite protein gels of Comparative Examples 1-2 and Example 2 showed no significant difference in color. However, the composite protein gels of Examples 1 and 3 were brown and pink, respectively. The hydroxyl radicals generated in the Fenton oxidation system attack the phenolic hydroxyl groups of GA and EGCG, causing them to lose hydrogen atoms and form phenoxy radicals. These phenoxy radicals are further oxidized to form ortho- or para-quinones. GA, with its three adjacent phenolic hydroxyl groups, can form stable ortho-quinone structures, which are typically brownish-green. EGCG contains multiple phenolic hydroxyl groups and an ester bond. Its oxidation process generates complex quinone polymers (benzoquinone, hydroquinone, etc.), which are pink due to their extended conjugated structures. TF, with its dihydroflavonoid skeleton, lacks a double-bonded conjugated structure. This lack of strong conjugation with electrons makes it less likely to form colored quinone compounds during oxidation, thus maintaining the inherent color of the protein gel. Color stability is a key factor affecting consumer acceptance of food products. Undesirable colors in gel-based foods may be perceived as unnatural changes, thus reducing consumer appeal. Choosing phenolic compounds like TF, which can maintain antioxidant efficacy while barely altering the sample's color, offers a clear advantage for developing visually appealing and stable functional foods.

[0045] like Figure 6As shown, the gel strength of the composite protein gel significantly improved after oxidation induction, which may be due to the enhanced non-covalent and covalent interactions between proteins resulting from oxidation modification, thus improving the cross-linking of the gel network structure. Protein gels prepared from composite proteins treated with phenolic substances exhibited even higher gel strength. This is because the addition of polyphenols anchors key amino acid residues of the composite protein through non-covalent interactions, forming stable bi-protein interface bridges, inhibiting oxidative aggregation of proteins, and promoting ordered cross-linking. Generally, the larger the molecular weight of the phenolic compound, or the more binding sites (phenolic rings) it has, the stronger its binding to the protein. Among the three phenolic substances, the addition of polyphenols in Example 1 contributed the least to the gel strength of the composite protein gel. This may be because GA has a small molecular weight and contains only three phenolic hydroxyl groups, making it easily degraded under oxidative conditions, leading to a lower effective concentration and weakening the interaction with the protein, similar to the findings of Cao et al. The addition of polyphenols in Examples 2 and 3 showed comparable gel strength. The addition of TF exposed enhanced hydrophobic interactions caused by hydrophobic groups, promoting initial hydrophobic aggregation between protein molecules and enhancing the structure of the gel network. The EGCG molecule contains multiple phenolic hydroxyl groups and galloyl groups, which gives it a stronger ability to form hydrogen bonds and covalent crosslinks, helping to form a uniform and stable gel network framework.

[0046] Figure 7 The effect of polyphenol concentration on the gel strength of a single protein protein (MP) system was investigated. It can be seen that the increased gel strength in Comparative Examples 5 and 6 compared to Comparative Example 4 indicates that the addition of polyphenols significantly improved the gel performance of MP. However, when the polyphenol concentration reached 80 μmol / g protein, the gel strength of the single MP system began to decrease significantly, indicating that this concentration is a critical threshold for the gel strength of a single protein system. It is evident that in Comparative Example 5, when the GA concentration was 80 μmol / g protein, the gel strength of Comparative Examples 5 and 4 showed no significant difference. Figure 6 It can be observed that, with the same polyphenol concentration, Example 1 exhibits significantly higher gel strength than Comparative Example 2. This clearly demonstrates that the binary protein system, under hydroxyl radical oxidation conditions, can improve the poor gel performance of single polyphenols at this critical concentration.

[0047] Figure 8Four peaks were identified in the protein gel samples, labeled T21, T22, T23, and T24. Water in all protein gel samples was primarily in the form of fixed water, playing a dominant role in the overall water composition variation. In Comparative Example 2 and Examples 1-3, the relaxation times of bound and fixed water in the composite protein gels were prolonged, while the relaxation time of free water was shortened. The addition of phenolic substances can shift water in the binary protein gel towards shorter relaxation times, particularly Example 3, which significantly shortened the relaxation times of each peak. Furthermore, structural changes after protein oxidation disrupt the original bound water layer, leading to a shift of fixed water content to free water. The addition of phenolic substances effectively mitigated the increase in free water content, especially Example 2, which minimized the increase. It is also noteworthy that phenolic substances promote covalent cross-linking of the protein gel, which further increases the conversion of fixed water to bound water, enhancing the stability and binding capacity of water molecules retained within the gel network structure. T2-weighted imaging represents changes in water fixation. Unoxidized protein gel samples show more red and yellow pixels, indicating a higher water proton density, consistent with the experimental results above. After oxidation induction, the red signal value of the protein gel is significantly lower than the blue signal value. The addition of phenolic substances enhances the red signal value; the ordered gel network of the protein gel effectively retains more water within the homogeneous gel structure. Changes in the fluidity and relative content of water in the gel system ultimately affect the quality characteristics of the product.

[0048] from Figure 9 As can be seen, the water-holding capacity of the binary protein gel significantly decreased after hydroxyl oxidation induction. This is mainly due to the excessive aggregation of proteins caused by oxidation, which increased the particle size and thus formed more internal voids and gaps within the gel matrix. Examples 1-3 improved the stability of Comparative Example 2, and the strong interaction between the protein and the complex further enhanced the protein gel network, which is beneficial for the physical capture of water molecules. The polyphenols of the three structures inhibited the excessive oxidation of proteins to varying degrees. The study found that moderately oxidized proteins can induce oxidized histidine and peptide fragments to generate additional charges, thereby improving the water-holding capacity of the protein gel. The dense gel network structure is also considered to be a key factor contributing to high water stability.

[0049] Figure 10The microstructure of the composite protein gel is shown. The composite protein gel of Comparative Example 1 exhibits a uniform network morphology. With the proceeding of the oxidation reaction, although the enhanced cross-linking between protein molecules reduces the porosity of the binary protein gel, the composite protein gel of Comparative Example 2 shows partial network rupture. This may be due to uneven protein oxidation leading to localized over-oxidation and protein structural damage, resulting in a loose gel structure and rough surface. Examples 1-3 effectively inhibited excessive protein oxidation, and this protective effect reduced protein degradation. Furthermore, polyphenols bridged the binary proteins through hydrogen bonds and hydrophobic interactions, effectively suppressing conformational fluctuations in proteins. Molecular-scale stability helps maintain the density and uniformity of the gel network. Although Example 1 maintained the uniformity of the composite protein gel network structure, it was insufficient to form high-density cross-linking, and the porosity was greater than that of the composite protein gels prepared in Examples 1 and 3. TF and EGCG, due to their phenolic hydroxyl structure and molecular size advantages, promoted the cross-linking of the gel network structure, contributing to the formation of an organized gel network structure.

[0050] Figure 11 A and B show the average absorbance and amide band (1700–1500 cm⁻¹) of the composite protein gel frozen sections under a microscope. 1) Imaging (10 μm × 10 μm). Red-yellow indicates a high signal density value for the characteristic group at this location, while blue-violet indicates a low signal density value for the characteristic group at this location. Comparative Example 2 and Examples 1-3 showed a relatively enhanced signal value compared to Comparative Example 1, which is related to the changes in protein structure caused by oxidation and the increase in cross-linking. The composite protein gel with added phenolic substances showed an enhanced amide band signal on the gel backbone, and the pores of the gel network backbone decreased, becoming more regular and uniform. This may be because the addition of phenolic substances promoted the conformational folding transition of the protein, adjusted the morphology of the protein network, and promoted the intermolecular interactions between the phenolic substances, the composite protein, and the other two components, thereby optimizing the structural integrity and stability of the gel and making the gel texture more stable. At the same time, the phenolic substances introduced increased hydrogen bonds, improved the hydrogen bonding force in the system, and strengthened the binding of water in the network. The difference in the intensity of the amide band signal brought about by the phenolic substances is mainly attributed to the structural differences of the three phenolic substances themselves. Polyphenolic hydroxyl groups can bind to the amino, thiol, or carbonyl groups of proteins through hydrogen bonding, hydrophobic interactions, or covalent cross-linking (such as quinone-amino reactions), promoting intermolecular cross-linking of proteins and forming a denser gel network. The polyphenol in Example 1 contains only 3 hydroxyl groups and has low structural rigidity, resulting in fewer interaction sites with proteins and lower cross-linking efficiency. In contrast, the polyphenol in Example 3 contains 8 phenolic hydroxyl groups and ester bonds, while the polyphenol in Example 2 has a conjugated benzene ring structure and 5 hydroxyl groups. The greater number of polyphenolic hydroxyl groups and the more complex stereochemical structure provide more possibilities for cross-linking with proteins.

[0051] To further illustrate the hydroxyl radical (OH) of the present invention The reaction system does not produce harmful elements or pose a threat to food safety. Energy dispersive spectroscopy (EDS) analysis was performed on the gel prepared in Comparative Example 2. Figure 12 The results show that, via hydroxyl radicals (OH) After oxidation induction in the reaction system, the resulting gel system does not contain residual iron ions. The main reason for this is that, on the one hand, hydroxyl radicals (OH-) The reaction system only provides an inducing environment for the complex protein system; substances such as ferric chloride do not participate in the actual reaction. On the other hand, after the oxidative induction reaction, hydroxyl radicals (OH-) are generated. The reagents involved in the reaction system will be removed, and the hydrogen peroxide will also decompose during the water bath heating process to prepare the gel. In the end, there will be no ferric chloride and hydrogen peroxide residue in the gel system.

[0052] To further demonstrate the safety of the composite gel obtained in this invention, hydroxyl radicals (OH) are utilized. The reaction system (Fenton system) underwent an MTT assay. The specific procedure of the MTT assay is as follows: Caco-2 cells were cultured in complete EMEM medium containing 10% fetal bovine serum at 37°C with a 5% CO2 flow rate. After seeding cells into 96-well plates, the corresponding Fenton concentration used in the experiment, along with a washed Fenton concentration solution, was added. Cell viability was assessed using the MTT assay after 24 hours of culture. Data are presented as a percentage of cell viability, with the control group as a baseline.

[0053] The corresponding Fenton system concentration solution: Take 50 mL of MP sample with a concentration of 1 mg / mL, and add an equal amount (100 μL) of hydroxyl radicals (OH) to it. The reaction system (100 μM FeCl3, 100 μM ascorbic acid, 1 mM H2O2) was reacted at 4 ℃ for 8 h. Then, 0.02% (w / v) butylhydroxytoluene was added to the system to stop the reaction. After mixing, 200 μL was added to a 96-well plate. The washed Fenton system concentration solution: Take 50 mL of MP sample with a concentration of 1 mg / mL, and add an equal amount (100 μL) of hydroxyl radicals (OH) to it. The reaction system (100 μM FeCl3, 100 μM ascorbic acid, 1 mM H2O2) was reacted at 4 ℃ for 8 h. Then, 0.02% (w / v) butylated hydroxytoluene was added to the system to stop the reaction. After the reaction was completed, 20 mL of 0.02 M phosphate buffer (pH 6.0) was added to the system to wash the precipitate twice and centrifuge it. 200 μL of the obtained precipitate was mixed and added to a 96-well plate.

[0054] Figure 13 The results of the MTT experiment are presented. As can be seen from the figure, the hydroxyl radical (OH) of this invention... After the reaction system and the washed system were subjected to the MTT assay, the cell viability was greater than 80%. Under normal circumstances, a cell viability greater than 70% is considered to indicate that there is no potential toxicity. Therefore, the Fenton system introduced in the experiment is safe and has no potential toxicity.

[0055] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for regulating the stability of a complex proteinaceous aquatic product based on high loadings of polyphenols depending on the structure, characterized by, The method comprises the following steps: (1) Preparation of a composite protein system: muscle fiber protein MP is prepared into an MP solution, plant protein and calcium chloride are added to the MP solution, and homogenization is performed to fully mix the MP solution; (2) Polyphenol modification: polyphenol is added to the composite protein system obtained in step (1), and magnetic stirring is performed at 4°C for 2h to fully mix the polyphenol; (3) Oxidation induction reaction: the composite system obtained in step (2) is mixed with a hydroxyl radical reaction system, and the mixture is reacted at 4°C for 7-9h; after the reaction is completed, butylated hydroxytoluene is added to stop the reaction, and the hydroxyl radical reaction system is composed of 100 μM FeCl3, 100 μM ascorbic acid and 1 mM H2O2; (4) Centrifugal treatment: 0.02 M phosphate buffer is added to the mixture obtained in step (3), and the mixture is washed with the phosphate buffer for 2-5 times and then centrifuged to completely remove the reagents in the hydroxyl radical reaction system; (5) Gelation treatment: the precipitate obtained by centrifugation in step (4) is heated under water bath conditions to induce thermal gelation, and then immediately placed in an ice bath for cooling for 10 min, and stored at 4 ℃.

2. The method of claim 1, wherein, In step (1), the concentration of the MP solution is 20-40 mg / mL.

3. The method of claim 1, wherein, In step (1), the plant protein includes one of soybean protein isolate SPI, pea protein isolate and mung bean protein, and the addition amount of the plant protein is 30-70% of the mass of the MP; after the calcium chloride is added, the concentration of the calcium chloride is 50-100 mM.

4. The method of claim 1, wherein, In step (1), the polyphenol is taxifolin.

5. The method of claim 1, wherein, After the polyphenol is added in step (2), the concentration of the polyphenol in the composite system is 40-100 μmol / g.

6. The method of claim 1, wherein, In step (3), the volume ratio of the composite system to the hydroxyl radical reaction system is 40-60 mL:100 μL, and the mass / volume ratio of butylated hydroxytoluene added after the reaction to the reaction system is 0.015-0.025%.

7. The method of claim 1, wherein, In step (5), the temperature of water bath heating is 70-100°C, and the heating time is 30-50 min.

8. A composite protein gel prepared by the method according to any one of claims 1-7.

9. Application of the composite protein gel according to claim 8 in the field of food.

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