Method for improving lipid loss resistance of emulsified fish product based on polyphenol modified compound protein

Abstract

The invention discloses a method for improving lipid loss resistance of an emulsified fish product based on polyphenol modified compound protein, and belongs to the technical field of food processing. The method for improving the lipid loss resistance of the emulsified fish product specifically comprises the following steps: forming a compound protein system by using MP of fish meat and plant source protein, modifying the compound protein system by adopting polyphenol, adding fish oil into the polyphenol modified MP-SPI compound system, homogenizing and emulsifying, heating and inducing to gelatinize, and cooling, so as to obtain the emulsified fish product. The emulsified fish product obtained by the method shows enhanced interfacial strength and oxidation resistance, significantly inhibits lipid droplet coalescence and oxidation loss in the hot working process, reduces the lipid loss rate by 40% or more after heat treatment, has stable texture and high nutrition retention rate, does not need chemical additives, and is suitable for industrial production. The method is suitable for producing functional emulsified fish products rich in highly unsaturated fatty acids, and has a wide practical application prospect.

Description

Technical Field

[0001] This invention relates to a method for improving the lipid loss resistance of emulsified fish products based on polyphenol-modified complex proteins, belonging to the field of food processing technology. Background Technology

[0002] Emulsified fish products, with their fresh and bouncy texture and extremely high nutritional value, have become an important part of healthy diets worldwide. Myofibrillar protein (MP), as the dominant protein in aquatic muscle, is prone to interfacial membrane strength decay and gel network loosening during heat processing due to its inherent environmental sensitivity. This leads to lipid droplet aggregation and oxidation, resulting in problems such as oil seepage and texture deterioration in the product.

[0003] Existing research focuses on introducing exogenous components such as plant proteins, polysaccharides, or cross-linking enzymes in a green and safe manner, providing a basic solution for improving the functional properties of polysaccharides (MPs). However, it remains insufficient in the face of the dual challenges of high unsaturated fatty acid content in high-value-added products and high-temperature processing (which disrupts cross-linking).

[0004] Plant polyphenols, due to their diverse molecular structures and excellent antioxidant activity, are considered ideal natural additives for inhibiting protein oxidative modification. They primarily scavenge free radicals and chelate transition metal ions through phenolic hydroxyl donors, effectively blocking oxidative chain reactions and thus mitigating the functional degradation, nutritional decline, and food quality deterioration caused by protein oxidation. However, the molecular rigidity and degree of polymerization of polyphenols significantly affect their interaction patterns with proteins, exerting a complex influence on the gel properties of food systems. On the one hand, they can bind to protein molecules through non-covalent and covalent interactions, enhancing the stability of the gel network structure and improving the texture and functional properties of protein gels. On the other hand, high-dose addition may lead to excessive cross-linking or non-selective binding, disrupting matrix integrity and impairing gel performance, thereby affecting the lipid loss resistance of emulsified fish products. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for enhancing the lipid loss resistance of emulsified fish products based on polyphenol-modified composite proteins. This method modifies the interface of the composite protein with polyphenols, enhancing the mechanical strength of the interfacial membrane, the amount of interfacial protein adsorbed, and the density of the gel network, thereby blocking the vicious cycle of lipid droplet aggregation and oxidation and solving the problem of lipid loss caused by thermal processing in traditional processes. By constructing a binary protein system, SPI competes with MP for polyphenol binding sites, thereby improving the gelation properties of MP and mitigating the adverse effects of high-dose polyphenols on MP. It fully leverages the molecular advantages of phenolic substances to achieve a synergistic effect of structural optimization and lipid oxidation inhibition, which aligns with the trend of healthy consumption and has broad market application prospects.

[0006] To achieve the above objectives, the following technical solution is provided: The first objective of this invention is to provide a method for improving the lipid loss resistance of emulsified fish products based on polyphenol-modified complex proteins, the method comprising the following steps: (1) Raw material processing Remove the head and tail of the fish, and remove the fascia to obtain the white meat from the back and belly of the fish. (2) Extraction of MP Place the white meat from the back and belly of the fish obtained in step (1) into a meat grinder and grind it until it becomes fine; then take the ground fish meat into a centrifuge bottle, add phosphate buffer, homogenize, filter, centrifuge and collect the precipitate; add NaCl solution to the precipitate, homogenize, centrifuge, discard the supernatant, and collect the precipitate as MP; (3) Preparation of polyphenol-modified MP-SPI composite system The MP solution prepared by step (2) is mixed with plant-derived protein and CaCl2 to obtain the MP-plant-derived complex protein system; then polyphenols are added to the MP-plant-derived complex protein system and stirred thoroughly to obtain the polyphenol-modified MP-plant-derived complex system. (4) Preparation of emulsion gel Fish oil is added to a polyphenol-modified MP-SPI composite system, homogenized and emulsified, then heated to induce gelation, and cooled to obtain emulsified fish products.

[0007] In one embodiment, the fish mentioned in step (1) includes one or more of yellowfin tuna, cod, and ribbonfish.

[0008] In one embodiment, the meat grinder in step (2) is used to grind meat for 30 seconds per cycle, for 1-3 minutes.

[0009] In one embodiment, the phosphate buffer in step (2) has a pH of 7.0 and a concentration of 0.1~0.5 mol / L.

[0010] In one embodiment, the amount of phosphate buffer added in step (2) is 4 to 6 times the volume of the fish meat.

[0011] In one embodiment, the homogenization conditions in step (2) are: 11000-13000 rpm for 1-3 min.

[0012] In one embodiment, the filtration in step (2) is performed using gauze filtration.

[0013] In one embodiment, the centrifugation parameters in step (2) are 3000~4000×g and the time is 10~15min.

[0014] In one embodiment, the concentration of the NaCl solution in step (2) is 0.1~0.5 mol / L.

[0015] In one embodiment, the amount of NaCl solution added in step (2) is 4 to 6 times the volume of the precipitate.

[0016] In one embodiment, the plant-derived protein in step (3) includes any one or more of soy protein isolate, quinoa protein, mung bean protein, and pea protein isolate.

[0017] In one embodiment, the polyphenol in step (3) includes one or more of GA, TF and EGCG; preferably any one of GA and TF.

[0018] In one embodiment, the concentration of MP in the polyphenol-modified MP-plant-derived complex system in step (3) is 20-40 mg / mL.

[0019] In one embodiment, the concentration of plant-derived protein in the polyphenol-modified MP-plant-derived complex system in step (3) is 10-20 mg / mL.

[0020] In one embodiment, the concentration of CaCl2 in the polyphenol-modified MP-plant-derived complex system in step (3) is 10-100 mM.

[0021] In one embodiment, the concentration of polyphenols in the polyphenol-modified MP-plant-derived complex system in step (3) is 70-90 mol / g protein.

[0022] In one embodiment, the fish oil in step (4) includes one or more of tuna oil, soybean oil, and perilla oil.

[0023] In one embodiment, the volume fraction of fish oil in the emulsion system of step (4) is 30%-50%; preferably 40%.

[0024] In one embodiment, the protein concentration in the polyphenol-modified MP-SPI complex system in step (4) is 20-40 mg / mL.

[0025] In one embodiment, the homogenization emulsification conditions in step (4) are: 10,000 to 15,000 rpm, time of 1 to 3 min, and temperature of 25 to 30°C.

[0026] In one embodiment, the temperature induced by heating in step (4) is 90~100℃ and the time is 20~40min.

[0027] A second objective of this invention is to provide a fish product obtained by the method described above.

[0028] A third objective of this invention is to provide an application of the method described above in the field of food deep processing technology.

[0029] Beneficial effects: (1) The present invention provides a method for improving the anti-lipid loss performance of emulsified fish products based on polyphenol-modified composite protein. The method modifies the interface of the composite protein with polyphenol, which enhances the mechanical strength of the interface membrane, the amount of interface protein adsorption and the density of the gel network, blocks the vicious cycle of lipid droplet aggregation and oxidation, and solves the problem of lipid loss caused by heat processing in traditional processes. (2) This invention modifies complex proteins with polyphenols, and reduces the adverse effects of high doses of polyphenols on MP by competing for polyphenol binding sites among complex proteins. It fully utilizes the molecular advantages of phenolic substances and achieves synergistic dual functions of structural optimization and lipid oxidation inhibition, which is in line with the trend of healthy consumption. (3) The emulsified fish products obtained by the method of the present invention are low in cost, contain no additives or preservatives, and can be produced on a large scale in factories. This provides a practical solution for developing high-end functional seafood products, enhances the market competitiveness and economic benefits of the products, and has broad market application prospects.

[0030] Attached image caption Figure 1 SAXS spectra of the composite protein emulsion gels of Examples 1-3 and Comparative Examples 1-2; Figure 2 Small-angle diffraction patterns of the composite protein emulsion gels of Examples 1-3 and Comparative Examples 1-2; Figure 3 The graph shows the POV value data of the composite protein emulsion gels of Examples 1-3 and Comparative Examples 1-2 during storage; Figure 4 The graph shows the TBARS values ​​of the composite protein emulsion gels of Examples 1-3 and Comparative Examples 1-2 during storage. Figure 5 The graph shows the oil-holding capacity data of the composite protein emulsion gels of Examples 1-3 and Comparative Examples 1-2 during storage; Figure 6 The graph shows the adsorption data of the interface proteins in Examples 1-3 and Comparative Examples 1-2. Figure 7 Transmission electron microscopy images of the interface proteins in Examples 1-3 and Comparative Examples 1-2; Figure 8 These are atomic force micrographs of the interface proteins in Examples 1-3 and Comparative Examples 1-2; Figure 9 The XRD patterns of the interface proteins in Examples 1-3 and Comparative Examples 1-2 are shown. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0032] The testing method involved in this invention 1. Cold field electron microscopy The distribution of lipid droplets in the gel network of the emulsion gel was observed using cryo-scanning electron microscopy. The sample was placed in a copper sample bath and pre-cooled by immersion in liquid nitrogen, then transferred to a vacuum preparation chamber and sublimated at -90 °C for 30 min, followed by gold sputtering. The microstructure of the sample was analyzed using a cryo-electron microscope (model SU8000, manufactured by Hitachi, Tokyo, Japan) at an operating voltage of 10 kV and a magnification of 5,000x.

[0033] 2. Lipid peroxide determination Fresh emulsion gels were placed in sealed screw-cap tubes and stored in an oven at 55 °C for 7 days to accelerate oxidation. Lipid peroxide, as a marker of primary lipid oxidation products, was measured according to a previous method. Briefly, 0.1 g of emulsion gel was mixed with 1.5 mL of isooctane / isopropanol (3:1, v / v), vortexed for 30 s, and centrifuged at 10,000 xg at 4 °C for 2 min. 50 μL of the supernatant and 1.45 mL of methanol / butanol solution (2:1, v / v) were collected, followed by the addition of 10 µL of ammonium ions and 10 µL of Fe. 2+ Ions were collected, and the mixture was vortexed and reacted in the dark for 20 min. The absorbance was measured at 510 nm using a microplate reader. The concentration of lipid hydrogen peroxide was calculated from a hydrogen peroxide standard curve.

[0034] 3. Determination of thiobarbituric acid value 0.1 g of the emulsion was mixed thoroughly with 2 mL of chloroform / ethanol (1:1, v / v). Then, 2.5 mL of a mixed reagent (15% (w / v) trichloroacetic acid, 0.375% thiobarbituric acid, and 0.25 M HCl) was added to the mixture. After thorough mixing, the mixture was boiled in a water bath for 10 min, then rapidly cooled to room temperature. Finally, the resulting mixture was centrifuged (4,000 g, 10 min), and the supernatant was collected. The absorbance of the supernatant was measured at 532 nm. The TBARS concentration was determined using a standard curve prepared from 1,1,3,3-tetramethoxypropane.

[0035] 4. Determination of oil holding capacity Weigh (m1) an appropriate amount of emulsion gel and centrifuge at 15,000 rpm for 20 min. Then, invert the sample for 20 min, remove excess oil with oil-absorbing paper (m2), and calculate the oil holding capacity according to the reported formula.

[0036] 5. SAXS measurement A wavelength of 1.54 Å and a sample-to-detector distance of 2.4 m were used. A circular sample holder with a diameter of approximately 1.5 mm was used and sealed with a Kappton window. The sample was held in a vacuum during the measurement. Data reduction was performed using XSACT software (Xenics), and the scattering intensity was normalized to the transmitted intensity of the direct beam to obtain data at absolute scale. This calibration was checked by comparing the scattering with that measured on glassy carbon.

[0037] 6. Determination of interfacial adsorption protein content The emulsion gel was centrifuged at 10,000 xg for 30 min, the aqueous phase was collected, and the water was dried at 100 °C. The concentration of unadsorbed protein was determined using the Bradford Protein Kit and calculated according to the following equation: Interfacial adsorption protein content (%) = CINI CSER CINI×100 CINI and CSER refer to the protein concentrations in the initial phase and aqueous phase of the emulsion, respectively.

[0038] 7. Transmission electron microscopy The microstructure of all samples was observed using a transmission electron microscope (JSM-6390LV, Japan). First, each sample was diluted with deionized water to 1 mg / mL, then coated onto a carbon-stabilized copper mesh, stained with 0.5% 12-wolyrine phosphate, and dried at room temperature. Finally, the dried copper mesh was placed under an accelerating voltage of 80 kV, and each sample was observed and photographed using a JSM-6390LV transmission electron microscope.

[0039] 8. Extraction of surfactant proteins Take 20g of emulsion gel and centrifuge at 10,000×g at 4 °C for 30 min to remove unabsorbed particles. After centrifugation, collect the emulsion layer. Then mix it with methanol at a ratio of 1:2 and incubate at room temperature for 10 min. Centrifuge the mixture at 5000×g for 10 min and remove the methanol phase at the top. Repeat this operation three times. Then wash three times with n-hexane and three times with ethanol. The collected precipitate is washed three times with phosphate buffer and then lyophilized to obtain the surface-active protein extracted from the composite protein emulsion gel.

[0040] Example 1 A method for preparing high-elasticity tuna surimi products by one-step ultra-high pressure auxiliary heating includes the following steps: (1) Processing of yellowfin tuna raw materials Remove the frozen yellowfin tuna and thaw it overnight in a 4°C refrigerator; then manually remove the head and tail and remove the fascia to obtain the white meat from the back and belly of the fish; (2) Extraction of MP Place the white meat from the back and belly of the fish obtained in step (1) into a meat grinder and grind it until it becomes fine (30s / time, 2 min); weigh 100 g of the ground fish meat and place it in a centrifuge bottle, add 4 times the volume of 4℃ buffer solution (0.1 M NaH2PO4 / Na2HPO4, 0.1 M NaCl, 0.002 M MgCl2, 1 mM EDTA, pH 7.0), homogenize it at 12000 rpm for 2 min to obtain a homogenate; then filter the homogenate through two layers of 80-mesh gauze to remove connective tissue, centrifuge the filtrate at 4℃ and 3500 ×g for 10 min, discard the supernatant and keep the precipitate, and repeat the above operation 3 times; Add 4 volumes of 0.1 M NaCl solution to the obtained precipitate, homogenize at 12000 rpm for 1 min, then centrifuge at 4 ℃ and 3500 ×g for 10 min, discard the supernatant, repeat the above operation 3 times, and collect the precipitate, which is the myofibrillar protein MP of yellowfin tuna; the protein concentration is detected by Bradford kit, and the prepared MP is stored at 4 ℃ for later use. (3) Preparation of MP-SPI complex protein system The MP obtained in step (2) was prepared into a solution with a final concentration of 25 mg / mL using a 0.3 mol / L sodium chloride solution; then, soybean protein isolate (SPI) was added to it with a content of 50% MP (w / w), and CaCl2 was added with a concentration of 50 mmol / L in the system; the solution was thoroughly mixed using a homogenizer to obtain the MP-SPI complex protein system. (4) Construction of polyphenol-modified MP-SPI composite system Gallic acid (GA) was added to the MP-SPI complex protein system at a concentration of 80 μmol / g and magnetically stirred at 4 °C for 2 h to ensure thorough mixing, thereby obtaining the polyphenol-modified MP-SPI complex system. (5) Preparation of emulsion gel The protein concentration of the polyphenol-modified MP-SPI complex was determined using the Bradford assay kit, and then diluted with 0.3 mol / L sodium chloride aqueous solution to adjust the protein concentration of the polyphenol-modified MP-SPI complex to 30 mg / mL. Tuna oil was added to the adjusted polyphenol-modified MP-SPI composite system to ensure that the oil phase volume fraction reached 40%. The emulsion was obtained by emulsifying it at 25 °C for 2 min using a digital homogenizer at 10,000 rpm. Then, 20 g of the emulsion was slowly transferred to a glass bottle and heated continuously in a 90 °C water bath for 30 min to induce its thermal gelation. After that, it was placed in an ice bath to cool for 10 min to obtain the emulsion gel, which is the high-elastic tuna surimi product.

[0041] Example 2 The only difference from Example 1 is that GA in step (4) is changed to TF, while other parameters and conditions are the same as in Example 1.

[0042] Example 3 The only difference from Example 1 is that the GA in step (4) is changed to epigallocatechin gallate (EGCG), and the other parameters and conditions are the same as in Example 1.

[0043] Comparative Example 1 The only difference from Example 1 is that the MP obtained in step (2) is prepared into a suspension with a concentration of 30 mg / mL; then tuna oil is added to ensure that the oil phase volume fraction reaches 40%, and emulsification is performed at 25°C for 2 min using a digital homogenizer at a speed of 10000 rpm; other parameters and conditions are the same as in Example 1.

[0044] Comparative Example 2 The only difference from Example 1 is that the concentration of the MP-SPI complex protein system obtained in step (3) was adjusted to 30 mg / mL, and then tuna oil was added to ensure that the oil phase volume fraction reached 40%. The mixture was emulsified at 25 °C for 2 min using a digital homogenizer at a speed of 10,000 rpm. All other parameters and conditions were the same as in Example 1.

[0045] Results Analysis 1. The performance of the composite protein emulsion gel products prepared in the examples and comparative examples was tested. Oil droplets in emulsion gels can be stabilized by interfacial protein films and the gel matrix, which directly reflects the network structure of the gel. Figure 1As can be seen, the three-dimensional structure of the emulsion gel constructed in Comparative Example 1 is not obvious, and the lipid droplets aggregate to a large size, indicating that single MP is prone to aggregation, which is not conducive to maintaining the gel network structure. Proteins and the oil phase separate, exhibiting an "oil leakage" phenomenon. In contrast, the emulsion gel prepared with composite proteins shows a clear gel skeletal structure and smaller lipid particles. When GA and TF from Examples 1 and 2 are introduced into the system, the emulsion gel exhibits a denser and more uniform protein matrix. However, for the EGCG-treated sample in Example 3, lipid droplets aggregate, forming discontinuous patches, indicating that the addition of EGCG disrupts the structural and functional properties of MP, leading to poor interfacial protein interactions. This may be because when EGCG binds to proteins, some essential amino acids are blocked, affecting their availability. EGCG modification-directed non-covalent and covalent interactions and steric hindrance effects affect the rate of protein cross-linking reactions, thus affecting the interactions between protein molecules and the formation of the gel network. The emulsion gel prepared with TF has a more uniform microstructure and fewer voids than that prepared with GA. This is due to the significant increase in phenolic hydroxyl content leading to an increase in hydrogen bond content, which further promotes the bridging of polyphenols and binary proteins. The size of oil droplets embedded in the network is significantly reduced, and adjacent dispersed oil droplets gradually approach each other, indicating that the introduction of TF can impart a finer quality to the gel structure, making the oil droplets less prone to clumping. At the same time, the interaction between the interfacial protein and the proteins in the gel matrix is ​​enhanced, forming a tight and uniform gel network, and the oil droplets adhere perfectly to the gel matrix.

[0046] 2. SAXS spectra were determined for the emulsion gels prepared in the examples and comparative examples. The results are as follows Figure 2 As shown, the SAXS curves of all emulsion gels exhibit a typical trend of decreasing scattering intensity with increasing scattering vector Q, reflecting the hierarchical structural heterogeneity from the nanoscale to the mesoscale. All emulsion gel systems show little difference in intensity in the low Q region, but exhibit significant differences in the mid-to-high Q region. At Q ≈ 0.4 Å... - ¹A distinct diffraction peak was observed, which is attributed to the short-range ordered structure in the aqueous protein matrix. A higher peak value indicates a higher degree of order in the sheets, while a wider peak width indicates lower uniformity between the sheets. The peak intensity is positively correlated with the electron density difference Δρ between the crystalline and amorphous layers.

[0047] As shown in the figure, both polyphenol modification and non-modification of the complex protein enhanced the peak intensity of single MP, indicating that the formation of the complex system effectively altered the amorphous layer rather than the crystalline layer, leading to increased lamellar order and electron density difference. SPI synergistically strengthens the gel network framework with MP, improving local density and order. Taxiardin modification exhibits the highest peak intensity, attributed to its protein conformation and hydrophobic-hydrophilic balance, which allows it to bridge MP and SPI via hydrophobic interactions and hydrogen bonds, inhibiting the disordering of protein thermal aggregation and promoting the formation of short-range ordered domains. In contrast, gallic acid, containing only three phenolic hydroxyl groups and with a smaller molecule, induces weaker order than taxiardin but stronger order than the unmodified MS complex. EGCG, due to its high degree of polyphenol polymerization and large steric hindrance, easily undergoes excessive cross-linking with proteins, restricting protein chain movement, disrupting the synergistic effect of the complex protein, and interfering with the formation of ordered structural regions, resulting in a lower peak intensity than unmodified MS. The differences in nanoscale order revealed by SAXS provide a structural basis for the macroscopic properties of the emulsion gel. Enhanced short-range ordering may improve gel strength and oil droplet embedding efficiency by forming a denser protein network, while the disordering caused by EGCG leads to network fragility.

[0048] 3. The oxidative stability of emulsion gels prepared with different protein matrices was evaluated by monitoring the dynamic accumulation of primary and secondary lipid oxidation products in the emulsion gel samples throughout the entire storage period from 0 to 7 days.

[0049] The results are as follows Figure 3 and 4As shown, the POV and TBARS values ​​of all emulsion gels increased in a time-dependent manner, indicating that lipid oxidation of the samples continued during storage. Compared with the emulsion gel prepared in Comparative Example 1, the composite protein emulsion gel in Comparative Example 2 had lower POV and TBARS values. This may be because the composite system formed by MP and SPI improved the interfacial encapsulation ability, effectively reducing lipid precipitation during storage. Maintaining the intact three-dimensional network structure of the emulsion gel during storage reduced the contact between lipids and external oxygen, effectively reducing the formation of oxidation products. The presence of polyphenols, regardless of molecular structure, significantly improved the oxidative stability of the emulsion gel. Its antioxidant capacity is generally positively correlated with the phenolic structure and the number of hydroxyl groups, consuming free radicals through the H atoms on the phenolic hydroxyl groups and preventing the cycle of oxidation reactions. This was most significant in the emulsion gel prepared from the polyphenol-modified composite protein in Example 2. The 2,3-double bond and 4-oxo group in the C ring of taurine contribute to its strong antioxidant activity. Although the octahydroxy structure of the triphenol ring of EGCG in Example 3 can significantly inhibit the formation of oxidation products. However, due to the poor stability of EGCG-modified composite protein emulsion gels, destructive factors such as high temperatures and chemical interactions during storage accelerate the collapse of their gel structure, causing oil droplets to precipitate and coalesce from the system, thus accelerating the oxidation reaction. This is why its antioxidant effect is inferior to TF but superior to GA.

[0050] 4. The oil-holding capacity of the emulsion gel samples prepared in the examples and comparative examples was measured. from Figure 5As can be seen, the oil-holding capacity of the emulsion gel in Example 2 is better than that of Example 1 (gallic acid modification), which is better than that of Comparative Example 2, which is better than that of Example 3 (EGCG modification), which is better than that of Comparative Example 1. The differences stem from the differentiated regulation of the gel network structure and interfacial stability by different protein matrices. Heat treatment easily causes disordered aggregation of Mp, forming a loosely structured gel network that cannot effectively retain oil droplets. Simultaneously, the poor continuity of the MP interfacial film leads to easy oil droplet precipitation during storage, resulting in a decrease in oil-holding capacity. The composite protein system, through hydrophobic complementarity and enhanced interaction forces, forms a more uniform and dense interfacial film and a three-dimensional network structure, significantly reducing oil droplet precipitation. The effects of the three polyphenol modifications on the oil-holding capacity of the composite protein show a pattern of "significant enhancement by taxonomist, moderate enhancement by gallic acid, and inhibition by EGCG," mainly due to the differences in polyphenol structure and their interaction with the composite protein in regulating interfacial film formation. The non-covalent and covalent interactions between tuftinoside and the complex protein promote the cross-linking of the dense gel network structure, enhance the interfacial encapsulation capability of the complex system, and significantly improve the oil droplet retention efficiency. GA has a small molecular weight and a fast diffusion rate, allowing it to penetrate into the gaps in the gel network, fill structural defects in the complex protein, and enhance network continuity; however, its relatively small number of phenolic hydroxyl groups limits the strength of its cross-linking with the protein. Notably, oil holding capacity is positively correlated with oxidative stability. Inhibiting lipid oxidation can maintain the stability of the gel network structure, thereby improving oil holding capacity; while excessive cross-linking of EGCG damages the network structure, accelerates the oxidation of precipitated oil, and forms a vicious cycle of "interfacial oxidation - structural damage - decreased oil holding capacity".

[0051] 5. Determination of oil-interfacial protein content in the emulsion gel samples prepared in the examples and comparative examples. Figure 6The changes in the interfacial protein content of MP, composite protein, and composite proteins modified with different polyphenol structures encapsulating tuna oil are shown. Compared with Comparative Example 1, the interfacial protein adsorption rate of Comparative Example 2 is significantly increased. The smaller droplet size increases the specific surface area, promoting the increase in protein adsorption. The polyphenol modification in Examples 1 and 3 further increased the interfacial protein adsorption rate of the composite protein. Among them, the polyphenol-modified composite protein in Example 2, when used as an aqueous emulsifier, has the highest interfacial adsorption capacity. This indicates that the TF modification makes the composite protein prone to multilayer adsorption, thereby forming a thicker interfacial film. In addition, the increase in interfacial protein adsorption may also be related to the diffusion rate of surfactant to the interface. MP-SPI-TF promotes the formation of interfacial film and prevents the aggregation of emulsion gel by rapidly adsorbing at the water-in-oil interface. The polyphenol modification in Example 3 further increased the interfacial protein adsorption rate of the composite protein. Among them, the addition of the polyphenol-modified composite protein in Example 2 reduced the interfacial protein adsorption rate of the composite protein, which may be due to the steric hindrance caused by its larger molecular weight, leading to an increase in the rearrangement of protein molecules adsorbed on the interface. Meanwhile, the presence of excess unadsorbed proteins in the aqueous phase led to depletion flocculation, reducing the adsorption content of interfacial proteins. These findings indicate that protein solutions modified with suitable polyphenol structures possess better oil-water interfacial adsorption capacity and superior emulsifying properties.

[0052] 6. Transmission electron microscopy was used to observe the interfacial proteins extracted from the emulsion gel samples prepared in the examples and comparative examples. TEM was used to observe the nanoscale microstructure and aggregation state of interfacial proteins extracted from emulsion gels prepared from different protein systems. The microstructure and aggregation state of the interfacial proteins were observed using TEM. All samples exhibited spherical particles, attributed to structural remodeling of the proteins during emulsification and heat-induced gelation. Myofibril aggregates dissociated into subprotein species, such as coarse / fine filaments, oligomers, or monomers, to degrade the myofibril aggregates.

[0053] The results are as follows Figure 7 As shown, in Comparative Example 1, the single MP protein exhibits the largest size when used as the interfacial protein. When adsorbed at the oil-water interface, it readily aggregates to form a coarse interfacial film. Heat treatment further induces this aggregation, disrupting the actin-myosin filament structure. In contrast, the interfacial proteins in the composite protein emulsion gel show a significantly reduced aggregation state. The hydrophobic complementarity of SPI inhibits the disordered aggregation of MP, reducing the size of the aggregates. In Example 3, the large molecular weight and highly rigid planar structure of EGCG lead to steric hindrance when it binds to the protein, while excessive cross-linking forces the protein molecules to form coarse aggregates. In contrast, the modification with GA and TF in Examples 1 and 2 enhances the interaction with the composite protein, promotes the dispersion of the interfacial proteins, and contributes to the formation of a tighter interfacial film.

[0054] Further analysis using AFM (Aspect-Factor Mechanism) was conducted to compare the surface uniformity and roughness of interfacial proteins. Figure 8 As can be seen, the interfacial protein surface of the single MP emulsion gel in Comparative Example 1 showed large surface undulations and poor uniformity. This is attributed to the irreversible aggregation of myofibrillar proteins under interfacial adsorption and heat treatment, forming large-sized aggregates. The composite protein system in Comparative Example 2 significantly reduced the surface roughness of the interfacial proteins and improved surface uniformity. Further investigation into the effect of polyphenol modification in Examples 1-3 revealed that polyphenols of appropriate molecular weight can penetrate into the interstitial network of the composite protein and specifically regulate the surface structure through structural differences. The phloroglucinol group of GA formed non-covalent and covalent interactions with the hydroxyl and amino groups of the protein, compensating for the structural defects of the composite protein and reducing surface voids and undulations. The strong interaction between the saturated pyran ring of dihydroflavonol in TF and the composite protein stabilized the interfacial film structure, making the surface more dense and uniform. This dense and uniform surface structure significantly improved the continuity and stability of the interfacial film. In general, the AFM analysis results are consistent with the morphological changes of the interfacial proteins observed by TEM, effectively reflecting the degree of MP aggregation and the regulation of interfacial structure by polyphenol-protein interactions.

[0055] The crystallization state and structural characteristics of interfacial protein molecules were assessed using XRD patterns. Figure 9 As can be seen, Examples 1-3 and Comparative Examples 1-2 all exhibit a characteristic broad peak around 20° (2θ), and the characteristic sharp peak of polyphenols is absent, indicating that the interfacial proteins exist in an amorphous state. This also verifies that phenolic substances and proteins are tightly bound through strong interactions, inhibiting the crystallization of phenolic substances. Compared with MP, the intensity of the characteristic peak around 20° in the other protein samples is significantly reduced, indicating a decrease in protein particle crystallization. Furthermore, this broad peak usually corresponds to the ordered secondary structure of the protein. This change stems from the construction of the complex protein system and the modification of polyphenols, which promotes the complete unfolding of the MP conformation, disrupting the original level of MP forming actin-myosin filament structures, resulting in a reduction in the content of α-helical structures and a decrease in the degree of ordered aggregation. This makes it easier for interfacial proteins to unfold during interfacial adsorption, enhancing the continuity of the interfacial film, which is consistent with the improved oil holding capacity. In particular, we found that the sharp small peaks around 30° and 45° in the XRD patterns of all samples may be due to the weak diffraction peaks caused by trace amounts of salt particles remaining during the extraction process.

[0056] 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 enhancing the lipid loss resistance of emulsified fish products based on polyphenol-modified complex proteins, characterized in that, The method includes the following steps: (1) Raw material processing Remove the head and tail of the fish, and remove the fascia to obtain the white meat from the back and belly of the fish. (2) Extraction of MP Place the white meat obtained in step (1) into a meat grinder and grind it until it becomes fine; then place it in a centrifuge bottle, add phosphate buffer, homogenize, filter, centrifuge and collect the precipitate; add NaCl solution to the precipitate, homogenize, centrifuge, discard the supernatant, and collect the precipitate as MP; (3) Preparation of polyphenol-modified MP-SPI composite system The MP solution prepared by step (2) is mixed with plant-derived protein and CaCl2 to obtain the MP-plant-derived complex protein system; then polyphenols are added to the MP-plant-derived complex protein system and stirred thoroughly to obtain the polyphenol-modified MP-plant-derived complex system. (4) Preparation of emulsion gel Fish oil is added to a polyphenol-modified MP-SPI composite system, homogenized and emulsified, then heated to induce gelation, and cooled to obtain emulsified fish products.

2. The method according to claim 1, characterized in that, The plant-derived protein in step (3) includes any one or more of soy protein isolate, quinoa protein, mung bean protein, and pea protein isolate.

3. The method according to claim 1, characterized in that, The polyphenols in step (3) include one or more of GA, TF and EGCG.

4. The method according to claim 1, characterized in that, In step (3), the concentration of MP in the polyphenol-modified MP-plant-derived complex system is 20-40 mg / mL.

5. The method according to claim 1, characterized in that, In step (3), the concentration of polyphenols in the polyphenol-modified MP-plant-derived complex system is 70-90 mol / g protein.

6. The method according to claim 1, characterized in that, The volume fraction of fish oil in the emulsion system described in step (4) is 30%-50%.

7. The method according to claim 1, characterized in that, In step (4), the protein concentration in the polyphenol-modified MP-SPI complex system is 20-40 mg / mL.

8. The method according to claim 1, characterized in that, The heating induction temperature in step (4) is 90~100℃, and the time is 20~40min.

9. The emulsified fish product obtained by the method according to any one of claims 1 to 8.

10. The application of the method according to any one of claims 1 to 8 in the field of food deep processing technology.

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