Application and method for intervening cod protein modification by polyphenols

Abstract

The invention relates to the technical field of protein modification intervention, and discloses application of polyphenol substances in intervention of cod protein modification, and cod protein is cod protein induced and modified by alpha, beta-unsaturated aldehyde in a multi-interface system. According to the method, a cod protein (CP) solution and n-hexadecane are used for constructing a multi-interface model system, a clickable probe yne-ACR is added to simulate the modification effect of alpha, beta-unsaturated aldehyde substances on cod protein, meanwhile, six polyphenols are added to interfere the modification process, and it is verified that the polyphenols interfere the alpha, beta-unsaturated aldehyde substances in the multi-interface system. The unique effect of beta, beta-unsaturated aldehyde substance modified protein provides a potential strategy for regulation and control of protein quality in a multi-interface food system.

Description

Technical Field

[0001] The present invention relates to the technical field of protein modification intervention, and particularly to the use of polyphenols to intervene in the modification of cod protein. Background Art

[0002] Atlantic cod (Gadus morhua) is one of the most important marine fish species globally, with both high production and consumption. Cod is renowned for its high nutritional value, being rich in protein and having a relatively high content of polyunsaturated fatty acids (PUFAs) (Jensen, Larsen, Rustad, & Eilertsen, 2013). In addition to basic processed products such as frozen fish fillets or fish blocks, cod can be further processed into products such as cod cakes, cod puddings, and cod sausages to meet consumer demands (Hou, Xia, Ma, Xue, & Jiang, 2023). These cod products are complex food matrices composed of water, protein, lipids, and other components, and are heterogeneous systems. Therefore, the stability of the interfaces in these matrices, especially the oil-water interface, is crucial for the overall quality of the products.

[0003] Cod protein (CP) is well-known for its strong interfacial activity (Ma et al., 2020; Ma et al., 2021). It stabilizes the food multi-interface system by diffusing, adsorbing, and rearranging at the interface, thereby enhancing the system stability. It should be noted that lipid peroxidation (LPO) in the food multi-interface system can also be regarded as an interfacial phenomenon (Aslam & 2023; Hennebelle et al., 2024). LPO usually originates at the interface and propagates into the bulk phase. During the LPO process of fish oil or other edible fats, a large number of α,β-unsaturated aldehydes are generated due to the stepwise cleavage of polyunsaturated fatty acids (such as acrolein and 4-hydroxy-2-nonenal) (Kanner, 2007). Compared with other aldehydes, these α,β-unsaturated aldehydes are more electrophilic and thus have a stronger ability to modify proteins. They modify proteins by reacting with nucleophilic residues (such as lysine, cysteine, and histidine) in the primary structure of proteins, and even induce cross-linking or aggregation within or between peptide chains, thereby changing the structure and function of proteins (Jiang et al., 2022). Given the coexistence of protein interfacial arrangement and LPO as two interfacial phenomena, the interface may provide a unique environment for α,β-unsaturated aldehydes to modify proteins, and thus may affect their interfacial activity.

[0004] During the processing and storage of aquatic products and their emulsion-based products, LPO and the resulting protein modifications are key factors leading to quality deterioration (Bayram & Decker, 2023). Therefore, natural antioxidants, especially polyphenols, are widely used in foods to inhibit the LPO process, improve product stability (Maqsood, Benjakul, Abushelaibi, & Alam, 2014), and prevent the generation of off-flavors (Falowo, Fayemi, & Muchenje, 2014). Polyphenols can capture free radicals generated during the LPO process (Zamora & Hidalgo, 2016), and can also act as effective scavengers of α,β-unsaturated aldehydes (Jiang et al., 2024; Lund, 2021; Zhu, Sun, Jiang, Chen, & Wang, 2011). Recently, Zhu and his colleagues reported the intervention effect of polyphenols on the modification of proteins by aldehyde LPO products (Lian et al., 2023; Yao et al., 2024; Zhang et al., 2024). They used Western blot analysis to find that in fish oil oxidation emulsions or aqueous systems, phloretin and epigallocatechin could effectively interrupt the modification of whey protein by acrolein or malondialdehyde, indicating that polyphenols may act as inhibitors of LPO-related protein modifications.

[0005] Cod protein and its related products can be regarded as multi-interface heterogeneous systems. Although previous studies have reported the modification of proteins by α,β-unsaturated aldehydes and the intervention of polyphenols in these modifications, most studies have focused on homogeneous systems (such as a single aqueous phase), ignoring the influence of the interface on protein modification and the effect of polyphenols in regulating these modifications. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art, and to propose the use of polyphenolic substances to intervene in the modification of cod protein.

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

[0008] The use of polyphenolic substances for intervening in the modification of cod protein, wherein the cod protein is the cod protein induced by α,β-unsaturated aldehydes in a multi-interface system.

[0009] Preferably, the polyphenolic substances include 3,4-dihydroxybenzoic acid (protocatechuic acid, PA), 2,5-dihydroxybenzoic acid (GeA), gallic acid (GA), 3,5-dihydroxybenzoic acid (RA), epicatechin (EC), epigallocatechin gallate (EGCG).

[0010] Preferably, the multi-interface system is an interface system including one or more of gas-liquid, liquid-liquid, and solid-liquid interfaces.

[0011] A method for intervening in the modification of cod protein, which uses polyphenols as an intervention agent to intervene in the modification of cod protein induced by α,β-unsaturated aldehydes.

[0012] Preferably, the polyphenols are used as scavengers of a,β-unsaturated aldehydes or antioxidants of proteins to intervene in the modification of cod protein induced by α,β-unsaturated aldehydes in the multi-interface system.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The present invention constructs a multi-interface model system with cod protein (CP) solution and hexadecane, adds a clickable probe yne-ACR to simulate the modification of cod protein by α,β-unsaturated aldehydes, and simultaneously adds six polyphenols to intervene in the modification process, verifying the unique role of polyphenols in the modification of proteins by α,β-unsaturated aldehydes in the multi-interface system, providing a potential strategy for regulating the quality of proteins in multi-interface food systems, and having a broad potential market and application demand. Brief Description of the Drawings

[0015] Figure 1 (A) is a schematic diagram of the experimental procedure of in-gel fluorescence imaging for the modification of CP induced by yne-ACR; (B) is the structural diagram of six polyphenols; (C) is the electrophoresis pattern of polyphenols intervening in the modification of CP by yne-ACR;

[0016] Figure 2 It is a data graph of the influence of different polyphenols (500 μM) and yne-ACR (500 μM) on the interfacial tension (γop) of CP (2 mg / mL) at the hexadecane-water interface;

[0017] Figure 3 It is a graph of the change in the diffusion rate of CP in the interfacial simulation system intervened by yne-ACR and polyphenols;

[0018] Among them, (A) is a theoretical model, where kd refers to the diffusion rate; B and C are the influence schematic diagrams of different polyphenols (500 μM) and yne-ACR (500 μM) on the π-t 1 / 2 curve of CP (2 mg / mL) at the hexadecane-water interface, and the slope of the initial linear region is defined as the diffusion rate (kd) of CP.

[0019] Figure 4 It is a schematic diagram of the influence of different polyphenols (500 μM) and yne-ACR (500 μM) on the time-dependent dilatational elastic modulus (Ed) of CP (2 mg / mL) at the hexadecane-water interface;

[0020] Figure 5 Schematic diagram of the influence of different polyphenols (500 μM) and yne-ACR (500 μM) on the swelling rheological properties of CP (2 mg / mL). The slope of the E-π curve (K) reflects the equilibrium state of the adsorbed CP at the interface;

[0021] Figure 6 Schematic diagram of the stability change of different interfacial simulation systems;

[0022] Among them, (A) is a schematic diagram of the appearance of the CP-Hex interfacial simulation system after centrifugation using a LUMiSizer; (B) is a schematic diagram of the relationship between the instability index and time; (C) is a zeta potential diagram; (D) is a schematic diagram of the microscopic morphology of the Hex-CP interfacial simulation system;

[0023] Figure 7 In, (A) is an in-gel fluorescence imaging map of unadsorbed protein and interfacial protein; (B) is a map of the degree of protein carbonylation of unadsorbed protein and interfacial protein; (C) is a map of the quinoprotein content of unadsorbed protein and interfacial protein. Detailed implementation manners

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0025] As Figures 1-7 shown, to verify the use of polyphenolic substances to intervene in the modification of cod protein, in the present invention, a clickable probe yne-ACR was introduced to simulate the modification of cod protein by α,β-unsaturated aldehyde substances. A cod protein solution interfacial model system containing hexadecane and 6 typical polyphenols was prepared, and interfacial hydrodynamic techniques were used to monitor the changes in the interfacial behavior of cod protein with or without polyphenols during the modification process of yne-ACR. Then, the interfacial protein and unadsorbed protein were separated from the model system, and the modification differences of yne-ACR on these two parts of proteins were analyzed by in-gel fluorescence imaging to evaluate the intervention effect of these polyphenols on protein modification. These findings can provide valuable insights for understanding the role of the interface in LPO-related protein modification and formulating regulatory strategies. The specific process is as follows:

[0026] I. Materials and methods:

[0027] 1. Materials:

[0028] Atlantic cod was purchased from Qingdao Yihexing Trading Co., Ltd. (Qingdao, China). n-Hexadecane (Hex, analytical grade, 98%) was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). 3,4-Dihydroxybenzoic acid (protocatechuic acid, PA, 99%), 2,5-dihydroxybenzoic acid (GeA, 99%), gallic acid (GA, 99%), 3,5-dihydroxybenzoic acid (RA, 99%), epicatechin (EC, 98%), and epigallocatechin gallate (EGCG, 98%) were provided by Energy Chemical (Anhui, China). The clickable probe (yne-ACR) was synthesized in our laboratory. All other chemical reagents and solvents used in this invention were of analytical grade.

[0029] 2. Cod protein extraction:

[0030] The frozen cod fillets were mixed with deionized water at a ratio of 1:10 (mass ratio) at 4 °C using a homogenizer (T18 digital set type, IKA company, Germany). The pH of the cod mixture was adjusted to 10.5 using 1 M NaOH solution and stirred at 4 °C for 2 hours, during which the pH was redetected and adjusted to 10.5 every 30 minutes. The adjusted cod mixture was centrifuged at 15,000 g for 30 minutes at 4 °C. The obtained supernatant was adjusted to pH 4.5 using 1 M HCl solution and centrifuged again at 10,000 g for 15 minutes at 4 °C. Subsequently, the precipitate was resuspended in deionized water (1:8, mass ratio) at 4 °C and the pH was adjusted to 7.0. The suspension was stirred at 4 °C for 1 hour and then centrifuged at 15,000 g for 30 minutes at the same temperature. The obtained supernatant was freeze-dried and stored at -20 °C. The protein content of the finally obtained cod protein (CP) powder was determined to be 85% (mass ratio) by the Kjeldahl method.

[0031] 3. Dynamic interfacial property measurement:

[0032] The dynamic interfacial tension of the n - hexadecane - protein system was recorded using a dynamic contact angle measuring instrument (LSA 100, LAUDA, Germany). According to the Food Additive Use Standard (China), the maximum allowable concentration of tea polyphenols as an antioxidant in aquatic products is 0.3 g / kg. Therefore, a polyphenol concentration of 500 μM was used in this experiment. At the beginning of the experiment, yne - ACR (500 μM) was added to n - hexadecane, and different kinds of polyphenols (500 μM) were added to the cod protein (CP) solution (2 mg / mL). Subsequently, the interfacial tension of the n - hexadecane - protein system was recorded to explore the effects of yne - ACR and polyphenols on the interfacial behavior of cod protein. Then, 15 μL of the cod protein solution droplet was suspended at the syringe needle tip and placed in a transparent glass container filled with n - hexadecane for 10,800 seconds to ensure complete protein adsorption. Generally, the interfacial pressure is used to evaluate the protein adsorption ability; to minimize the influence of the oil phase, the normalized π * value was used for evaluation.

[0033] π = γ ow - γ op (1)

[0034]

[0035] where γ ow represents the interfacial tension of the n - hexadecane - water system, and γ op represents the interfacial tension of the n - hexadecane - protein system.

[0036] According to the research of Rillaerts and Joos (Rillaerts & Joos, 1982) and Ward and Tordai (1946) (Ward & Tordai, 1946), the diffusion rate (k d , mN·m -1 ·s -1 / 2 ) can be calculated by the following formula:

[0037]

[0038] where C represents the initial molar concentration of protein in the bulk phase, R is the ideal gas constant, T represents the absolute temperature, D is the diffusion coefficient, C0 refers to the initial mass concentration of protein in the bulk phase, and K is the Boltzmann constant. The relationships between C and C0, and between R and K are as follows:

[0039] C0 = C × N A (5)

[0040]

[0041] where N A is the Avogadro constant.

[0042] When protein migration is diffusion-controlled and there are no energy barriers during the diffusion process, Equations (3) and (4) apply. If adsorption is diffusion-controlled, the π-t 1 / 2 curve will be linear, and the slope represents the diffusion rate constant (k d ).

[0043] After protein diffusion, the adsorption rate is affected by protein penetration and rearrangement. Using the first-order linear fitting method, the penetration rate (k p , s -1 ) and the rearrangement rate (k r , s -1 ) can be determined according to the method described by Graham and Phillips (Graham&Phillips, 1979) (Equation 7). The slopes of the first and second linear regions of Equation (7) correspond to k p and k r , respectively.

[0044]

[0045] where π 10800 , π t , and π0 represent the interfacial pressure at any time and the initial time at 10,800 s, respectively. k i represents the first-order rate constant.

[0046] 4. Measurement of swelling rheological properties:

[0047] The swelling rheological properties of hexadecane-CP were investigated using dynamic ccodontact angle measurement (LSA 100, LAUDA Germany) in the swelling mode. First, yne-ACR (500 μM) was added to hexadecane, and different polyphenols (500 μM) were added to the CP solution (2 mg / mL). Next, a drop of CP solution (15 μL) was suspended in a quartz cuvette filled with hexadecane. The measurement was carried out using the pendant drop method, and the sinusoidal oscillation cycle consisted of 7 effective cycles and 1 blank cycle to record the swelling modulus (E), elastic modulus (Ed), and loss modulus (Ev). The oscillation frequency was set to 0.1 Hz, and the amplitude was 20% (ΔA / A).

[0048] E d = E|cosσ| (8)

[0049] E v = E|sinσ| (9)

[0050] where σ is the phase angle between stress and strain.

[0051] 5. Preparation of the hexadecane-CP interface simulation system:

[0052] First, 2 mM of different types of polyphenols were added to the CP solution (8 mg / mL) and mixed well. Then, 2 mM yne-ACR was added to hexadecane and mixed. Next, the CP solution was mixed with hexadecane at a ratio of 4:1 (v / v) and homogenized using a high-speed homogenizer at 12,000 rpm for 2 minutes. Homogenization was repeated every 2 hours for a total duration of 6 hours to obtain the hexadecane-CP interface simulation system. The blank control group was the simulation system without polyphenols and yne-ACR.

[0053] 6. Zeta potential of the interface simulation system:

[0054] The Zeta potential (mV) of the interface simulation system was measured using a Zeta potential meter (Nano Brook Omni, Brookhaven, NY, USA). Different interface simulation system samples were diluted 1,000 times with deionized water and tested at 25 °C.

[0055] 7. Microscopic morphology of the interface simulation system:

[0056] The microscopic structure of different interface simulation system samples was observed using an optical microscope (ECLIPSE Si, Nikon, Tokyo, Japan). A drop of freshly prepared emulsion was placed on a glass slide, covered with a coverslip, and the microscopic morphology was examined at 25 °C.

[0057] 8. Stability measurement of the interface simulation system:

[0058] The stability of the interface simulation system was evaluated using a LUMiSizer 651 (LUM GmbH, Berlin, Germany) under the following conditions: wavelength of 870 nm, centrifugal force of 1,000 g, test duration of 75 minutes, and temperature of 20 °C.

[0059] 9. Separation and extraction of unadsorbed protein and interfacial protein from the interface simulation system:

[0060] The freshly prepared hexadecane-CP interface simulation system was centrifuged at 13,000 g and 4 °C for 30 minutes. The precipitate representing the unadsorbed protein was collected. The emulsion layer was washed 3 times with deionized water to remove any attached unadsorbed protein. Then, 2% Tween was added, and the mixture was stirred at 300 rpm at 37 °C for 3 h to displace the interfacial protein in the emulsion. The resulting mixture was centrifuged at 13,000 g and 4 °C for 60 minutes, and the precipitate representing the interfacial protein was collected. The protein concentrations of the unadsorbed protein and interfacial protein were measured using the BCA assay.

[0061] 10. Modification of CP by yne-ACR and click labeling:

[0062] The CP solution (4 mg / mL) and different polyphenols (500 μM) were incubated with different concentrations of yne-ACR probe at 37 °C for 12 h. To observe the modification level on CP by in-gel fluorescence, 10 μL of the modified CP solution was vortexed with 18 μL of phosphate buffer (PBS, 0.2 M, pH 7.4), 4 μL of FAM-azide solution (2.5 mM), 2.5 μL of THPTA solution (10 mM), 2.5 μL of CuSO4 solution (20 mM), and 2.5 μL of NaVc solution (300 mM). The resulting mixture was incubated at 37 °C for 2 h. The labeled samples were separated by 12% SDS-PAGE gel and imaged by ChemiDoc XRS+. Subsequently, the gel was stained with Coomassie Brilliant Blue.

[0063] 11. Quinoprotein content of unadsorbed protein and interfacial protein:

[0064] To quantify the quinoprotein levels in unadsorbed protein and interfacial protein, the NBT (nitroblue tetrazolium chloride) / glycine assay (Zhang et al., 2017) was performed. Briefly, the separated unadsorbed protein and interfacial protein were centrifuged at 15,800 g and 4 °C for 30 min. The resulting pellet was dissolved with 2 M glycine buffer, and the protein concentration of each sample was determined by BCA protein assay kit. To detect quinoprotein, 200 μL aliquots of the protein solution were mixed with 4 μL of NBT (12 mM) and incubated in the dark at 37 °C for 1 h. After the reaction, the absorbance at 530 nm was measured using a microplate reader (INFINITE E PLEX, Austria). The quinoprotein level was OD 530 / mg protein.

[0065] 12. Protein carbonyl content:

[0066] First, the concentration of unadsorbed protein and interface protein was determined using a BCA assay kit. Next, 50 μL of protein solution was mixed with 50 μL of 10 mM 2,4-dinitrophenylhydrazine (DNPH dissolved in 2 M HCl) and incubated in the dark for 60 minutes, vortexing every 10 minutes. After the reaction was completed, 100 μL of 10% trichloroacetic acid (TCA) was added, and the mixture was centrifuged at 13,000 rpm and 4°C for 10 minutes to obtain a protein precipitate. The precipitate was then washed with an ethanol-ethyl acetate mixture (1:1) and centrifuged at 13,000 rpm and 4°C for 5 minutes. This washing step was repeated until the supernatant became colorless. Finally, the protein precipitate was dissolved in 150 μL of 6 M guanidine hydrochloride solution, and 100 μL was transferred to a microplate to measure the absorbance at a wavelength of 366 nm (A366). The carbonyl content in proteins was calculated using the following formula (Nuerjiang et al., 2023; Oliver, Ahn, Moerman, Goldstein, & Stadtman, 1987).

[0067]

[0068] where 0.3125 is the height of the reaction mixture in the microplate, 22 is the extinction coefficient of DNPH, and 150 is the volume of the protein solution.

[0069] 2. Results and Discussion

[0070] 1. Interventional effects of polyphenols on yne-ACR modified CP in homogeneous aqueous systems:

[0071] The probe yne-ACR is designed to be equipped with an electrophilic warhead to mimic the modification of proteins by α,β-unsaturated aldehydes generated during LPO. The clickable alkyne handle in the structure allows for fluorescence imaging of the modified proteins via the classic CUAAC click reaction. Figure 1 A). We first studied six common polyphenols (PA, GeA, GA, RA, EC and EGCG, such as Figure 1 B) on the effect of yne-ACR induced CP modification in homogeneous aqueous solution. As shown in the fluorescent gel ( Figure 1C), after incubation with 500 μM yne-ACR at room temperature for 12 h, significant modification of CP occurred. Among the polyphenols tested, PA, GeA, and RA had no significant effect on the level of protein modification. However, co-incubation with EC further enhanced the modification effect of yne-ACR. Both GA and EGCG effectively reduced the fluorescence intensity of modified CP, indicating that these polyphenols significantly inhibited the modification of proteins by yne-ACR. The pyrogallol units present in GA and EGCG may be the key structural fragments for inhibiting protein modification. The above results suggest that the effectiveness of polyphenols as intervention agents for α,β-unsaturated aldehyde-induced CP modification may be related to their structural characteristics.

[0072] 2. Dynamic interfacial properties of CP under yne-ACR modification and intervention of polyphenols in the interfacial simulation system:

[0073] To investigate the effects of the modification of α,β-unsaturated aldehydes and the intervention of six common polyphenols on the modification on the interfacial properties of cod protein (CP), the dynamic adsorption behavior at the hexadecane-water interface was measured in this study. As Figure 2 [[ID=⑧]]shown, with the increase of adsorption time, the interfacial tension of all samples decreased steadily and tended to be stable after 10800 s, indicating that the adsorption saturation state was reached. When only yne-ACR was added to hexadecane, the interfacial tension of cod protein decreased significantly, indicating that α,β-unsaturated aldehydes enhanced the interfacial activity of cod protein. After adding different types of polyphenols to the cod protein solution, GeA, RA, EC, and EGCG all decreased the interfacial tension, while PA and GA had little effect on the interfacial activity. When yne-ACR and polyphenols with different structures were added simultaneously, in the presence of PA, GeA, and GA, the effect of yne-ACR on the interfacial activity of cod protein dominated. In contrast, RA, EC, and EGCG synergistically acted with yne-ACR to enhance the interfacial activity of cod protein.

[0074] To minimize the interference of the oil phase polarity on the interfacial pressure (π), the normalized interfacial pressure (π, i.e., π / γ ow ) was used in this study to analyze the adsorption capacity of cod protein. The π-t curve can be divided into three stages: induction period, rapid increase period, and slow increase period. In the case of adding only polyphenols, PA and GA had little effect on the adsorption capacity of cod protein, while the addition of GeA, RA, EC, and EGCG enhanced its adsorption capacity. When yne-ACR and polyphenols were present in the system simultaneously, the equilibrium π* value (at 10800 s) was higher. These results indicate that α,β-unsaturated aldehydes modified cod protein at the interface and affected its interfacial activity, while polyphenols interfered with the modification of α,β-unsaturated aldehydes and showed a structure-activity relationship.

[0075] To further explore the effects of α,β-unsaturated aldehydes with different structures and polyphenols on the dynamic changes of cod protein (CP) at the interface, the kinetic parameters of CP adsorption at the interface were analyzed ( Figure 3 ). In the initial process, CP must diffuse from the bulk phase to the interface. When the interfacial pressure is low, the adsorption kinetics of CP at the interface is diffusion-controlled, where K d represents the diffusion rate of CP. However, when yne-ACR is added to hexadecane, PA and GA have little effect on the diffusion rate of CP, while GeA increases it, and RA, EC, and EGCG decrease it. These results indicate that in a multi-interface system, the intervention of polyphenols on the interfacial diffusion activity of proteins in α,β-unsaturated aldehydes depends on the structure of the polyphenols. As the adsorption time increases, the π-t 1 / 2 curve significantly deviates from linearity, and diffusion no longer controls the adsorption kinetics. At this time, the ability of the protein to unfold (K p ) and rearrange (K r ) at the interface determines the adsorption rate. These findings suggest that the intervention of α,β-unsaturated aldehydes and polyphenols changes the structure of CP molecules and promotes their unfolding at the interface. However, the unfolded protein molecules cannot maintain a stable arrangement at the interface.

[0076] 3. Swelling rheological properties of CP under the intervention of yne-ACR and polyphenols

[0077] Figure 4 shows the dynamic changes in the elastic modulus (E d ) of the adsorption layer during protein adsorption. The E d of all samples gradually increases with the increase of adsorption time, which is attributed to the adsorption of proteins at the interface. In the CP+yne-ACR interface system, the addition of yne-ACR produces a stronger viscoelastic interfacial structure. However, in the CP-polyphenol system, the addition of polyphenols results in a decrease in E d , indicating that polyphenols disrupt the interactions between proteins and weaken the network structure of the surface film. It is worth noting that when both yne-ACR and polyphenols are present in the interface system, the addition of PA, GeA, GA, and RA has little effect on the E d value, indicating that the effect of yne-ACR on the interfacial viscoelasticity of proteins dominates. In contrast, the addition of EC weakens the viscoelasticity of the system, while EGCG combines with yne-ACR to synergistically enhance the interfacial viscoelasticity of proteins.

[0078] Figure 5Shows the variation of the surface expansion complex modulus (E) with the interfacial pressure (π). The E-π curve and its slope reflect the equilibrium state of the adsorbed substances at the interface. For CP, the slope of the E-π curve is 7.157, indicating strong interactions between the CP molecules adsorbed at the interface. For the CP+yne-ACR and CP+polyphenol systems, the curve slopes are higher than that of CP alone, and the slope of the CP+yne-ACR system is steeper than that of the CP+polyphenol system. In the presence of EGCG, the curve slope in the CP+polyphenol+yne-ACR system is significantly higher than that of CP. These results suggest that lipid oxidation and polyphenol intervention in the multi-interfacial system strengthen the protein-protein interactions at the interface.

[0079] 4. Stability of the interfacial simulation system constructed by CP modified with yne-ACR and polyphenol intervention:

[0080] Based on the above results, it was found that both α,β-unsaturated aldehyde and polyphenol intervention affect the interfacial properties of proteins. Increasing evidence indicates that the adsorption, rearrangement, and penetration of proteins at the interface form a dense and highly viscoelastic network structure, serving as a defense against external barriers, which is crucial for the stability of the emulsion system. Generally, the higher the interfacial activity of proteins, the better the stability of the formed emulsion. Therefore, to further investigate the effects of α,β-unsaturated aldehyde and polyphenol intervention on the stability of the multi-interfacial system, centrifugal stability tests were first conducted on different samples. Figure 6 A shows the macroscopic images of different samples after the centrifugal stability test. Figure 6 B shows the variation of the centrifugal instability index of different samples with time. These data indicate that α,β-unsaturated aldehyde and polyphenol intervention have the least effect on the centrifugal stability of CP in the interfacial simulation system. The results of the dynamic interfacial tension study (3.2) showed that the addition of yne-ACR significantly enhanced the interfacial activity of CP. However, from the perspective of macroscopic centrifugal stability, no effect of yne-ACR on the stability of the interfacial simulation system was observed. This indicates that evaluating the stability of the system in a complex food multi-phase interfacial system from a single perspective cannot fully reflect the true behavior of the system. Next, the Zeta potential of different multi-interfacial systems was analyzed. As Figure 6 shown in C, yne-ACR has no significant effect on the Zeta potential of the CP interfacial system. Similarly, the addition of RA, EC, and EGCG has no significant effect on the Zeta potential of the system. However, the addition of PA, GA, and GeA reduces the absolute value of the Zeta potential. Finally, the microstructures of different interfacial systems were observed. As Figure 6 shown in D, yne-ACR increases the particle size of the CP interfacial simulation system. Except for EC, the addition of other polyphenols increases the particle size of the system to varying degrees, and there is also a tendency for the interfacial area to increase.

[0081] 5. Influence of the interface on the modification of CP by yne-ACR and the intervention of polyphenols:

[0082] In the multi-interface system established with Hex and CP solutions, the interfacial proteins and unadsorbed proteins were extracted, and the modified proteins were subjected to fluorescence imaging. As Figure 7 shown in A, in the multi-interface system, the modification of interfacial proteins by yne-ACR was significantly stronger than that of unadsorbed proteins. This indicates that compared with homogeneous aqueous solutions, the formation of the interface is conducive to protein modification, and the interface provides a unique environment for the modification reaction. In addition, the addition of PA, GeA, GA, RA, and EGCG alleviated the modification of unadsorbed proteins in the aqueous phase, while only GA, RA, and EGCG could alleviate the modification of interfacial proteins. However, the addition of EC significantly increased the modification levels of unadsorbed and interfacial proteins in the emulsion system. Therefore, the presence of an interface in the CP emulsion leads to the modification of proteins by α,β-unsaturated aldehydes, which is different from that in single-phase aqueous solutions, and the interface promotes the modification reaction. Among the tested polyphenols, EC promoted the modification of unadsorbed and interfacial proteins by α,β-unsaturated aldehydes, while EGCG inhibited the modification of both protein types. The structure of polyphenols may determine their preferential intervention in the modification of proteins induced by α,β-unsaturated aldehydes.

[0083] Since α,β-unsaturated aldehydes can modify nucleophilic residues through Michael addition and induce protein carbonylation, the levels of protein carbonylation in unadsorbed and interfacial proteins in the CP multi-interface simulation system were also measured ( Figure 7 B). It was found that 2 mM yne-ACR did not significantly increase the carbonylation of unadsorbed proteins in the inner phase within 6 h, but significantly promoted the carbonylation of interfacial proteins. In the CP system incubated with polyphenolic compounds, the carbonylation levels of interfacial proteins in the PA, GeA, and RA groups were still significantly higher than those of unadsorbed proteins. However, in the GA incubation group, the carbonylation levels of interfacial and unadsorbed proteins were similar, and the carbonylated interfacial proteins were significantly lower than those of unadsorbed proteins in the EGCG incubation group. Therefore, among these six polyphenols, GA and EGCG can intervene in the modification of CP induced by α,β-unsaturated aldehydes by inhibiting the carbonylation of interfacial proteins, while the intervention of other polyphenols may involve different modification pathways (such as the formation of Schiff bases).

[0084] The oxidation of polyphenols can produce quinones, which then modify nucleophilic amino acid residues such as lysine and cysteine, and finally form quinone proteins. Since quinones and α,β-unsaturated aldehydes have similar modification sites, it is hypothesized that the formation of quinone proteins may be one of the ways for polyphenols to intervene in the modification of α,β-unsaturated aldehydes on CP. Therefore, NBT staining was used to measure the levels of quinone proteins in each CP system ( Figure 7C). In the CP system containing GeA, PA, GA, EC, and EGCG, the level of quinoprotein in the interfacial protein was significantly higher than that in the unadsorbed protein, indicating that the presence of the interface in the system also promoted the formation of quinoprotein. However, a significant increase in the quinoprotein level was not detected in the RA addition group, which might be due to the difficulty of the resorcinol fragment to form stable oxidation products compared with other diphenols. It can be inferred that the quinoprotein formed during the oxidation of GA, EGCG, and EC might contribute significantly to the intervention of α,β-unsaturated aldehyde-modified CP. In addition, it has been reported that the resorcinol structure can effectively promote the scavenging ability of polyphenols for α,β-unsaturated aldehyde. Therefore, RA might inhibit CP modification by effectively scavenging free α,β-unsaturated aldehyde.

[0085] In summary, the above implementation process investigated the influence of the interface on α,β-unsaturated aldehyde-modified CP and the roles of six typical polyphenols in regulating these modifications. Different from the single-phase system, the present invention verified that the interface provided a unique platform for α,β-unsaturated aldehyde-modified proteins and polyphenol intervention in the modification. These findings provided valuable insights into the influence of α,β-unsaturated aldehyde on protein properties from the perspective of the interface.

[0086] As mentioned above, the above are only the preferred specific implementation manners of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. Use of polyphenols for intervening in the modification of cod protein, characterized in that: The cod protein is a cod protein modified by α,β-unsaturated aldehydes in a multi-interface system.

2. Use of polyphenols for intervening in the modification of cod protein according to claim 1, characterized in that: The polyphenolic substances include 3,4-dihydroxybenzoic acid (protocatechuic acid, PA), 2,5-dihydroxybenzoic acid (GeA), gallic acid (GA), 3,5-dihydroxybenzoic acid (RA), epicatechin (EC), and epigallocatechin gallate (EGCG).

3. Use of polyphenols for intervening in cod protein modification according to claim 1, characterized in that: The multi-interface system is an interface system containing one or more of gas-liquid, liquid-liquid, and solid-liquid interfaces.

4. Method for intervening in the modification of cod protein, characterized in that: The method uses the polyphenolic substances described in any one of claims 1-3, and uses the polyphenolic substances as intervention agents to intervene in the modification of cod protein induced by α,β-unsaturated aldehydes.

5. The method for intervening in the modification of cod protein according to claim 4, wherein: The polyphenolic substances, as eliminators of α,β-unsaturated aldehydes or antioxidants of proteins, intervene in the modification of cod protein induced by α,β-unsaturated aldehydes in a multi-interface system.

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