Preparation method of lysozyme-nanoselenium complex and application thereof in ROS scavenging

Abstract

The application discloses a preparation method of a lysozyme-nano selenium compound and application of the lysozyme-nano selenium compound in ROS removal. To mix sodium selenite solution and lysozyme uniformly, a lysozyme-sodium selenite solution is obtained, then vitamin C is added, and the mixture is uniformly mixed under the condition of light shielding and at room temperature, the pH value is adjusted to 6.5-7.5, a red solution is obtained, and dialysis is performed, so that the lysozyme-nano selenium compound is obtained. The lysozyme-nano selenium compound has the advantages of simple process, mild preparation condition, higher stability, biological safety, efficient ROS removal in egg white, and no influence on the regulation ability of the functional property, edible quality and other characteristics of the egg white, so that the deterioration problem of the egg white in the storage process is solved. The lysozyme-nano selenium compound has good application prospect in ROS removal, and can be applied to the fields of biomedical science and material science.

Description

Technical Field

[0001] This invention relates to the field of ROS scavenging technology, and more specifically, to a method for preparing a lysozyme-nanoselenium complex and its application in ROS scavenging. By constructing a lysozyme-nanoselenium complex system to enhance its ROS (reactive oxygen species) scavenging ability, its application potential in the anti-oxidative stress response of egg white is explored. Background Technology

[0002] Reactive oxygen species (ROS) refer to a class of highly reactive oxygen-containing molecules or free radicals, including superoxide anions, hydroxyl radicals, hydrogen peroxide, and singlet oxygen. Appropriate amounts of ROS participate in physiological processes such as cell signaling and immune defense; however, excessive ROS accumulation leads to oxidative stress, causing DNA damage, protein denaturation, and lipid peroxidation, which in turn induces various diseases. In the food industry, particularly in egg white systems, egg white is rich in high-quality protein and is commonly processed and stored in liquid form. However, it is highly susceptible to ROS attack. The oxidative effect of ROS accelerates protein structural denaturation, reduces functional properties, and promotes lipid peroxidation, resulting in deterioration of egg product quality, nutrient loss, and a significantly shortened shelf life. Furthermore, ROS removal is also in high demand in fields such as biomedicine and materials science.

[0003] In the field of antioxidants, lysozyme (LYZ) mainly relies on its "self-sacrifice" mechanism to scavenge reactive oxygen species (ROS). For example, Chen J et al. disclosed that LYZ protects egg white protein (EWP) from oxidation by scavenging ROS through "self-sacrifice," providing a scientific basis for the regulation of EWP storage stability (Chen J, Wang Z, Xiao N, Guo S, Ai M. Lysozyme "self-sacrificing"quenching reactive oxygen species blocks egg white protein oxidation and dilution degradation. Food Chem. 2025 Nov 30;493(Pt 3):145919. doi: 10.1016 / j.foodchem.2025.145919. Epub 2025 Aug 14. PMID: 40840385.). However, this "self-sacrifice" approach means that the structure of lysozyme itself is easily destroyed by reactive oxygen species, thus being permanently consumed in a single action, resulting in limited stability and durability. Currently, research and applications of lysozyme in ROS removal mainly focus on its individual effects, but its removal capacity has certain limitations. To overcome this deficiency, there is an urgent need to construct a composite system that is simple to process, highly stable, and has high ROS removal efficiency. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and provide a method for preparing a lysozyme-nanoselenium complex. This invention utilizes lysozyme and nanoselenium as core functional components, and under mild reaction conditions, employs a biomolecule-mediated in-situ reduction-complexation one-step method to construct a stable lysozyme-nanoselenium complex system, successfully preparing a highly efficient, low-toxicity, and stable ROS scavenging and antioxidant complex for resisting oxidative stress.

[0005] A second objective of this invention is to provide a lysozyme-nano selenium complex prepared by the aforementioned preparation method.

[0006] A third objective of the present invention is to provide the lysozyme-nano selenium complex.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: A method for preparing a lysozyme-nano selenium complex includes the following steps: S1. Mix sodium selenite solution with lysozyme to obtain lysozyme-sodium selenite solution; S2. Add vitamin C to the lysozyme-sodium selenite solution from step S1, mix well under light-protected and room temperature conditions, adjust the pH to 6.5-7.5 to obtain a red solution, dialyze to obtain the lysozyme-nano selenium complex.

[0008] This invention addresses the shortcomings of lysozyme in ROS scavenging by introducing nano-selenium as a synergistic antioxidant component. Selenium, an essential micronutrient, plays a core role in various biological processes. Nano-selenium, with its high antioxidant activity and low toxicity, can directly neutralize hydroxyl radicals and superoxide anions through electron transfer. It can also act as a selenium source to activate endogenous antioxidant systems such as glutathione peroxidase, providing multi-pathway and regenerative antioxidant protection. This allows it to complement and synergize with lysozyme, reducing the self-sacrificing burden on lysozyme. In the lysozyme-nano-selenium complex prepared by this invention, lysozyme effectively inhibits the aggregation of nano-selenium, significantly increasing the dispersion stability of nano-selenium. After lysozyme and nano-selenium are combined, the stability of nano-selenium is significantly improved, resulting in a final complex with a sheet-like morphology and a larger particle size. Among them, the nano-selenium peroxidase exhibits catalytic activity, enabling the continuous decomposition of reactive oxygen species precursors such as hydrogen peroxide; while lysozyme not only quenches free radicals through electron transfer, but more importantly, it stabilizes the dispersion of nano-selenium and prevents its aggregation and deactivation, thereby significantly improving the overall ROS removal efficiency of the system. 1Free radicals such as O2 are rapidly eliminated upon generation, forming a highly efficient and synergistic antioxidant system. This invention utilizes a biomolecule-mediated in-situ reduction-complexation one-step method to construct a stable lysozyme-nanoselenium complex system under mild reaction conditions, without the need for a protective atmosphere during preparation. This method achieves stable binding solely through solution mixing and stirring, leveraging the natural protein structure of lysozyme and the surface activity of selenium nanoparticles. This successfully prepares a highly efficient, low-toxicity, and stable ROS scavenging and antioxidant complex for combating oxidative stress.

[0009] Further, the concentration of the sodium selenite solution in step S1 is 10–50 mmol / L (preferably 30 mmol / L).

[0010] Further, the lysozyme concentration in the lysozyme-sodium selenite solution in step S1 is 1–5 mg / mL (preferably 3 mg / mL). That is, the content of lysozyme in the lysozyme-sodium selenite solution is 1–5 mg / mL (preferably 3 mg / mL).

[0011] Further, in step S2, the molar ratio of sodium selenite to vitamin C in the lysozyme-sodium selenite solution is 1:3 to 5 (preferably 1:4).

[0012] Furthermore, the mixing in step S2 is performed at 24°C and stirred at 200–300 rpm for 10–15 min.

[0013] Furthermore, the pH is 7.0.

[0014] Preferably, 0.1 mol / L HCl or 0.1 mol / L NaOH is used as an acid-base regulator to adjust the pH.

[0015] Furthermore, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 0.8 to 1.2 kDa for 70 to 75 hours (preferably using a dialysis bag with a molecular weight cutoff of 1 kDa for 72 hours).

[0016] The present invention also provides a lysozyme-nano selenium complex prepared by any of the above-described preparation methods.

[0017] The lysozyme-nanoselenium complex of the present invention has a simple process and mild preparation conditions. At the same time, the lysozyme-nanoselenium complex has higher stability and biosafety, and can efficiently remove ROS in egg white without affecting the ability to regulate the functional and edible properties of egg white.

[0018] Therefore, the present invention also provides the above-mentioned lysozyme-nano selenium complex as a ROS reactive oxygen species inhibitor / ROS scavenger or in the preparation of ROS reactive oxygen species inhibitors / ROS scavengers.

[0019] This invention also provides the application of the aforementioned lysozyme-nanoselenium complex in the antioxidant activity of egg white. The lysozyme-nanoselenium complex exhibits long-lasting antioxidant capacity in the egg white system, thereby solving the problem of deterioration during egg white storage.

[0020] Specifically, the lysozyme-nano selenium complex achieves antioxidant effects by directly capturing or removing ROS in egg white, inhibiting ROS-mediated tyrosine cross-linking reactions, and / or catalyzing hydrogen peroxide to convert it into water.

[0021] The present invention also provides a ROS reactive oxygen species inhibitor / ROS scavenger containing the above-mentioned lysozyme-nano selenium complex.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a lysozyme-nanoselenium complex. The lysozyme-nanoselenium complex requires no protective atmosphere during preparation; it achieves stable binding solely through solution mixing and stirring, utilizing the natural protein structure of lysozyme and the surface activity of nano-selenium. This simplifies the process, reducing the need for excessive temperature control and multi-step purification, resulting in a more streamlined operation and significant time savings in actual production. The simple operation and low barrier to entry facilitate standardized procedures. Regarding safety, in the lysozyme-nanoselenium complex, lysozyme, a naturally occurring protein in humans and animals, can be degraded into amino acids within the body, while nano-selenium can be converted into selenium metabolites through the body's antioxidant system, preventing accumulation in tissues and enhancing safety. The lysozyme-nanoselenium complex prepared by this invention exhibits higher stability and biosafety, and possesses the ability to efficiently remove ROS from egg white without affecting its functionality or edible quality, thus solving the problem of egg white deterioration during storage. The lysozyme-nanoselenium complex shows promising application prospects in ROS removal and can be used in biomedicine, materials science, and other fields. Attached Figure Description

[0023] Figure 1 The particle size potential of lysozyme-selenium nanocomposites obtained with different lysozyme concentrations is shown in Figure 1. (A) ζ-potential; (B) Particle size. Note: Different letters indicate significant differences between different groups (P < 0.05).

[0024] Figure 2 Microstructure of lysozyme-selenium nanocomplexes obtained with different lysozyme concentrations.

[0025] Figure 3 The results show the stability of the lysozyme-selenium nanocomplex. (A) Ionic stability; (B) pH stability; (C) Storage stability.

[0026] Figure 4The yield of ROS in egg white obtained from lysozyme-nano selenium complexes with different lysozyme concentrations.

[0027] Figure 5 This represents the change in carbonyl content of egg white during storage.

[0028] Figure 6 This represents the change in dityrosine content in egg white during storage.

[0029] Figure 7 The yield change of egg white ROS during storage.

[0030] Figure 8 The results show the foaming properties of egg white under different storage times. (A) Foaming ability; (B) Changes in foaming stability. Note: Different letters indicate significant differences between different groups. P <0.05).

[0031] Figure 9 Results of emulsification performance determination of egg white after emulsification at different storage times. (A) EAI change; (B) ESI change. Note: Different letters indicate significant differences between different groups. P <0.05) Detailed Implementation The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Example 1 Preparation of lysozyme-selenium nanocomplex SeNPs-1 (1) Dissolve sodium selenite in deionized water to obtain a sodium selenite solution with a concentration of 30 mmol / mL; (2) Sodium selenite solution was mixed with lysozyme to obtain lysozyme-sodium selenite solutions with different mass concentrations of lysozyme (5, 3, 1, 0 mg / mL); (3) Add the vitamin C solution to the lysozyme-sodium selenite solution at a molar ratio of sodium selenite to vitamin C (Vc) of 1:4, stir at a constant temperature and in the dark (200-300 rpm for 10-15 min at 24℃), and adjust the pH to 7 after mixing evenly to obtain a red solution. (4) The red solution was dialyzed for 72 h using a dialysis bag with a molecular weight of 1 kDa to obtain lysozyme-nano selenium complex.

[0034] Specifically, in this embodiment, the concentration of lysozyme used is 5 mg / mL.

[0035] The specific preparation method of the lysozyme-selenium nanocomplex SeNPs-1 is as follows: S1: Take a 100 mL volumetric flask and add about 80 mL of deionized water; accurately weigh 0.786 g of sodium selenite, add it to the volumetric flask, and stir magnetically until completely dissolved; S2: Add 0.5g of lysozyme, dissolve it completely, and then bring the volume to 100 mL to obtain a lysozyme-sodium selenite solution; S3: Take a 50 mL beaker, add 20 mL of deionized water, weigh 2.112 g of Vc, dissolve it completely, and then add it to the lysozyme-sodium selenite solution; S4: Transfer the solution to a brown bottle and place it in a 24°C constant temperature incubator; add a magnetic stir bar, adjust the speed to 200 rpm, and stir for 10 min until the system is mixed evenly and turns a stable red color. Use 0.1 mol / L HCl or 0.1 mol / L NaOH as an acid-base adjuster and add it dropwise to adjust the pH to 7. S5: After dialyzing the red solution with a dialysis bag containing a molecular weight of 1 kDa for 72 h, the lysozyme-nano selenium complex, namely SeNPs-1, was obtained.

[0036] Example 2 Preparation of lysozyme-selenium nanocomplex SeNPs-2 (1) Dissolve sodium selenite in deionized water to obtain a sodium selenite solution with a concentration of 30 mmol / mL; (2) Sodium selenite solution was mixed with lysozyme to obtain lysozyme-sodium selenite solutions with different mass concentrations of lysozyme (5, 3, 1, 0 mg / mL); (3) Add vitamin C (Vc) solution to lysozyme-sodium selenite solution at a molar ratio of sodium selenite to Vc of 1:4, stir at a constant temperature and in the dark (200-300 rpm for 10-15 min at 24℃), and adjust the pH to 7 after mixing evenly to obtain a red solution. (4) The red solution was dialyzed for 72 h using a dialysis bag with a molecular weight of 1 kDa to obtain lysozyme-nano selenium complex.

[0037] Specifically, in this embodiment, the concentration of lysozyme used is 3 mg / mL.

[0038] The specific preparation method of the lysozyme-nano selenium complex SeNPs-2 is the same as in Example 1.

[0039] Example 3 Preparation of lysozyme-selenium nanocomplex SeNPs-3 (1) Dissolve sodium selenite in deionized water to obtain a sodium selenite solution with a concentration of 30 mmol / mL; (2) Sodium selenite solution was mixed with lysozyme to obtain lysozyme-sodium selenite solutions with different mass concentrations of lysozyme (5, 3, 1, 0 mg / mL); (3) Add vitamin C (Vc) solution to lysozyme-sodium selenite solution at a molar ratio of sodium selenite to Vc of 1:4, stir at a constant temperature and in the dark (200-300 rpm for 10-15 min at 24℃), and adjust the pH to 7 after mixing evenly to obtain a red solution. (4) The red solution was dialyzed for 72 h using a dialysis bag with a molecular weight of 1 kDa to obtain lysozyme-nano selenium complex.

[0040] In this specific implementation case, the concentration of lysozyme used is 1 mg / mL.

[0041] The specific preparation method of the lysozyme-nano selenium complex SeNPs-3 is the same as in Example 1.

[0042] Preparation of comparative lysozyme-selenium nanocomplexes (SeNPs) (1) Dissolve sodium selenite in deionized water to obtain a sodium selenite solution with a concentration of 30 mmol / mL; (2) Sodium selenite solution was mixed with lysozyme to obtain lysozyme-sodium selenite solutions with different mass concentrations of lysozyme (5, 3, 1, 0 mg / mL); (3) Add vitamin C (Vc) solution to lysozyme-sodium selenite solution at a molar ratio of sodium selenite to Vc of 1:4, stir at a constant temperature and in the dark (200-300 rpm for 10-15 min at 24℃), and adjust the pH to 7 after mixing evenly to obtain a red solution. (4) The red solution was dialyzed for 72 h using a dialysis bag with a molecular weight of 1 kDa to obtain lysozyme-nano selenium complex.

[0043] Specifically, in this comparative example, the concentration of lysozyme used was 0 mg / mL.

[0044] The specific preparation method of the lysozyme-selenium nanocomplex SeNPs is the same as in Example 1.

[0045] Experimental Example 1: Determination of Particle Size Potential of Lysozyme-Nano Selenium Complex 1. Method The average particle size and zeta potential of LYZ and the above-mentioned lysozyme-selenium nanocomplexes SeNPs, SeNPs-1, SeNPs-2, and SeNPs-3 were determined using a nanoparticle size and ZETA potential analyzer. Samples were diluted with distilled water to 1% (particle size, w / v) and 0.1% (zeta potential, w / v), equilibrated at 25°C for 120 s, and measured in triplicate.

[0046] 2. Results Particle size potentials of lysozyme-selenium nanocomplexes obtained with different lysozyme concentrations are as follows: Figure 1 As shown. Figure 1 In component A, LYZ carries a positive charge, while the unmodified blank selenium nanoparticles carry a negative charge. The lysozyme-selenium nanoparticle complexes obtained after lysozyme modification all carry a positive charge, and their zeta potentials lie between those of LYZ and the unmodified selenium nanoparticles, indicating that lysozyme successfully binds to the selenium nanoparticle surface. Zeta potential is one of the important indicators for evaluating the stability of lysozyme-selenium nanoparticle complexes; the higher the potential, the better the stability of the complex. Among them, SeNPs-2 has the highest potential, indicating the best stability. Figure 1 In B, the particle size of LYZ is close to 0, while the unmodified blank selenium nanoparticles have a higher particle size and are prone to aggregation. The particle size of the lysozyme-selenium nanoparticle complex obtained by lysozyme modification is significantly reduced, indicating that lysozyme can effectively inhibit the aggregation of selenium nanoparticles and significantly increase the dispersion stability of selenium nanoparticles.

[0047] Experimental Example 2: Determination of the microstructure of the lysozyme-selenium nanocomplex 1. Method The microstructures of LYZ, Se, and the aforementioned lysozyme-selenium nanoparticle complexes SeNPs-1, SeNPs-2, and SeNPs-3 were observed using scanning electron microscopy (SEM). Thermally induced EWG samples were cut into approximately 1 cm × 1 cm × 1 cm cubes, fixed overnight in 2.5% (v / v) glutaraldehyde solution, and washed with phosphate-buffered saline (PBS). The samples were then dehydrated for 30 min in a series of fractionated ethanol solutions (60%, 70%, 80%, 90%, and 100%) and thoroughly freeze-dried using a vacuum freeze dryer. Finally, the dried samples were sprayed with a gold layer and observed at an accelerating voltage of 20 kV and magnifications ranging from 1 kx to 20 kx.

[0048] 2. Results Scanning electron microscopy (SEM) was used to observe the microstructure of LYZ, Se, and three different SeNPs at different magnifications. The results are as follows: Figure 2 As shown, LYZ particles appear as complete spherical particles with smooth surfaces, uniform distribution, and no aggregation at different magnifications. Se particles are small, uniformly distributed, and aggregated, indicating that individual selenium nanoparticles are prone to aggregation, which is consistent with the results shown in the particle size potential section. The lysozyme-selenium nanoparticle complex appears as a sheet, indicating that the binding of lysozyme and selenium nanoparticles alters the morphology of either particle when they exist alone. This may be due to hydrogen bonding, hydrophobic interactions, and electrostatic adsorption between the protein and nanoparticles, causing conformational rearrangement of the protein molecules and forming a new complex interface.

[0049] Experimental Example 3: Determination of Sample Stability of Lysozyme-Nano Selenium Complex 1. Method Test subjects: LYZ and the above-mentioned lysozyme-nano selenium complex samples SeNPs, SeNPs-1, SeNPs-2, and SeNPs-3; (1) Ionic stability: The ionic stability of each sample was evaluated within a certain range of NaCl concentrations. Each sample was mixed with NaCl solution to obtain solutions with a pH of 7 at concentrations of 0, 25, 50, 100, and 200 mmol / L. The particle size of the samples was determined after storage at room temperature for 2 h.

[0050] (2) pH stability: The pH of each sample was adjusted to 3, 5, 7, 9, and 11 using 1 mol / L HCl and 1 mol / L NaOH. The particle size of the samples was measured after storage at room temperature for 2 h.

[0051] (3) Storage stability: The particle size of the sample was measured after being stored at room temperature for 0, 7, 14, 21 and 28 days.

[0052] 2. Results The colloidal stability of the complex was evaluated by measuring the particle size changes of lysozyme-selenium nanocomplexes obtained from LYZ and different lysozymes under different NaCl concentrations, pH values, and storage times.

[0053] The stability of nanoparticles is closely related to the ion concentration of the system. Increasing the ion concentration will compress the diffuse electric double layer of the particles, change their surface potential, and thus promote particle aggregation and precipitation. Figure 3 A shows the particle size variation of the lysozyme-selenium nanocomposite at different NaCl concentrations. With increasing NaCl concentration, the particle size of all samples increases. This is because salt ions shield the electrostatic repulsion on the particle surface, thus inducing aggregation. The particle size of LYZ remains consistently low. P The concentration >0.05 indicates that LYZ is less affected by salt ions and has good stability. The higher particle size of SeNPs indicates poor stability and greater susceptibility to salt ions. Although the lysozyme-selenium nanoparticle complex modified with lysozyme gradually increases with increasing NaCl concentration, the increase is significantly smaller than that of the unmodified blank selenium nanoparticles. Furthermore, the particle size of SeNPs-2 is the smallest at all salt ion concentrations, indicating that its structure has better salt tolerance.

[0054] pH is one of the key factors affecting the stability of nanoparticles. It can change the surface electrochemical properties of nanoparticles, leading to aggregation or deaggregation between particles. Figure 3In group B, a peak particle size is observed near pH 7 because proteins and nanoparticles have the lowest electrostatic charge and weakest electrostatic repulsion near their isoelectric point. Under strong acid or strong alkaline conditions, the surface charge density of the particles increases, the electrostatic repulsion strengthens, and the degree of aggregation decreases. Similarly, LYZ maintains the smallest particle size at all pH values, while SeNPs exhibit the worst stability.

[0055] Figure 3 C reflects the stability of the sample under different storage times. As time increases, all particle sizes show an increasing trend, with SeNPs-2 exhibiting the best stability and SeNPs exhibiting the worst stability.

[0056] The stability of the complex was observed under different salt ion concentrations, pH values, and storage times. The results showed that the stability of the nano-selenium was significantly improved after being combined with lysozyme.

[0057] Experimental Example 4: Determination of ROS Yield of Lysozyme-Nano Selenium Complex 1. Method ESR spectra were measured using an EMX spectrometer. Freeze-dried egg white (EW) samples were transferred to an ESR capillary tube. The operating parameters were set as follows: modulation frequency 100 kHz, modulation amplitude 4.66 G, frequency 9.84 GHz, microwave power 2 mW, center field 3510 G, scan width 400 G, receiver gain 4104, time constant 327.68 ms, spectral scan time 655.36 s, spectral points 1024, and three scans.

[0058] 2. Results The ROS generation rate of egg white systems containing EW, samples supplemented with 0.3% LYZ, or 0.3% of the above-mentioned lysozyme-nano selenium complex SeNPs-1, SeNPs-2, and SeNPs-3 was determined by ESR. The results are as follows: Figure 4 As shown, the ESR spectrum of the lysozyme-selenium nanocomplex is almost undetectable. 1 The O2 signal is primarily due to the formation of a highly efficient and synergistic antioxidant system by the complex. Specifically, the nano-selenium peroxidase exhibits catalytic activity, continuously decomposing reactive oxygen species (ROS) precursors such as hydrogen peroxide; while lysozyme not only assists in quenching free radicals through electron transfer, but more importantly, it stabilizes the dispersion of nano-selenium, preventing its aggregation and deactivation. This significantly enhances the overall ROS scavenging efficiency of the system. 1 Free radicals such as O2 are rapidly eliminated after they are generated.

[0059] Example 5: Application of lysozyme-nano-selenium complex in regulating oxidative deterioration of egg white during storage S1. Add the prepared SeNPs-2 to egg white (4 mg / kg) and mix well to obtain S-EW; S2. The prepared S-EW and original egg white liquid (EW) were placed in aerobic storage at room temperature for 0, 1, 3, 5 and 7 days, respectively. The samples obtained from different groups were freeze-dried and stored at -20℃ for later use.

[0060] 1. Determination of carbonyl content in egg white (1) Method The carbonyl content of proteins was determined using the 2,4-dinitrophenylhydrazine (DNPH) method. For each of the different sample groups, two equal aliquots of 0.2 mL of 4 mg / mL egg white were taken. One aliquot was added to 0.4 mL of 2 M HCl as a blank control, and the other aliquot was added to 0.4 mL of 10 mM DNPH dissolved in 2 M HCl (sample group). The mixture was reacted in the dark at 37°C for 1 h. Then, 0.5 mL of 20% (w / v) trichloroacetic acid (TCA) was added, and the reaction was allowed to stand for 5 min to terminate the reaction. The mixture was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was removed. The precipitate was washed with 1 mL of ethyl acetate / ethanol (1:1, v / v) and centrifuged at 12000 rpm for 15 min at 4°C. This step was repeated three times to remove excess DNPH. The precipitate obtained after centrifugation was mixed with 1 mL of 6 M guanidine hydrochloride to dissolve the precipitate. Finally, the mixture was centrifuged at 12000 rpm for 15 min. The supernatant was then measured at 370 nm to calculate the carbonyl content. The calculation formula is as follows: Carbonyl content (mmol / mg) = (1) A 370 —Absorbance at 370 nm; D—Dilution factor; C—Protein concentration (mg / mL) 。

[0061] (2) Results Results of protein carbonyl group changes during egg white storage: Figure 5 As shown, the carbonyl groups in egg white increased significantly with increasing storage time. P <0.05), where the carbonyl content in EW is significantly higher than that in S-EW ( P The value <0.05 indicates that SeNPs-2 has a significant inhibitory effect on ROS-mediated protein carbonylation. Protein carbonylation is one of the most prominent manifestations of protein oxidation, with carbonyl groups mainly generated by the oxidative breakage of amino acid side chains that are vulnerable to free radical attack. SeNPs-2 can directly capture or remove ROS in egg white, thereby blocking the oxidative attack of ROS on amino acid side chains and significantly mitigating carbonylation damage.

[0062] 2. Determination of dityrosine content in egg white (1) Method The egg white solution concentration was adjusted to 1 mg / mL using 0.01 mM phosphate buffer, pH 7.4. Fluorescence intensity was recorded using a fluorescence spectrophotometer between excitation wavelengths of 325 nm and emission wavelengths of 420 nm, with a slit width of 3 nm for all wavelengths. The dityrosine content was represented by dividing the measured fluorescence intensity value by the protein concentration.

[0063] (2) Results Dityrosine is formed by the covalent cross-linking of two adjacent tyrosine residues. ROS can oxidize tryptophan and cross-link tyrosine to produce dityrosine. The content of dityrosine is one of the indicators for measuring the degree of protein damage. Figure 6 As shown, the content of dityrosine increased significantly with increasing storage time. P <0.05 indicates that the degree of oxidative cross-linking of proteins is continuously increasing, with the dityrosine content in EW being significantly higher than that in S-EW ( P <0.05), which demonstrates that SeNPs-2 can effectively inhibit ROS-mediated tyrosine cross-linking, thereby slowing down the oxidative polymerization process of egg white proteins.

[0064] 3. Determination of changes in egg white ROS yield (1) Method ESR spectra were measured using an EMX spectrometer. Freeze-dried egg white samples were transferred to an ESR capillary tube. The operating parameters were set as follows: modulation frequency 100 kHz, modulation amplitude 4.66 G, frequency 9.84 GHz, microwave power 2 mW, center field 3510 G, scan width 400 G, receiver gain 4104, time constant 327.68 ms, spectral scan time 655.36 s, spectral points 1024, and three scans.

[0065] (2) Results The yield of ROS in egg white during storage was determined by ESR, and the results are as follows: Figure 7 As shown. It was detected that with prolonged storage time, the egg white content in the S-EW group decreased. 1The O2 signal intensity was lower in the egg white group than in the EW group, indicating that the free radicals in the egg white system were scavenged by the added SeNPs-2, reducing the degree of oxidative attack on egg white proteins by free radicals. During storage, the endogenous oxidation reaction of egg white produces reactive oxygen species, including superoxide anions and hydrogen peroxide. SeNPs-2 can efficiently catalyze the conversion of hydrogen peroxide into water, thereby reducing the ROS concentration. This process fundamentally inhibits the ROS-induced oxidation reaction and slows down a series of oxidative damage processes, including dityrosine cross-linking.

[0066] 4. Determination of the foaming properties of egg whites (1) Method Take 20 mL of 2% (w / v) EWP solution and homogenize it at 10,000 rpm for 1 min. Record the total foam volume V0 and V60 after standing at room temperature for 0 min and 60 min, respectively. Calculate the foaming capacity and foam stability using the following formulas: Foaming capacity (%) (5) Foam stability (%) (6) (2) Results The functional properties of egg white protein are important for its processing applications. The foaming ability of protein largely depends on the adsorption kinetics of protein molecules at the gas-liquid interface. The foaming ability of egg white is as follows... Figure 8 As shown in Figure A, after a week of aerobic storage, the foaming performance of the S-EW group was superior to that of the EW group. ROS attacks the amino acid residues of the side chains of egg white proteins, inducing protein oxidation and thus increasing protein hydrophobicity. However, excessive ROS can lead to over-oxidation of proteins, causing uncontrolled aggregation of protein molecules. SeNPs-2, on the other hand, cleared the ROS generated during storage, inhibiting the oxidative aggregation of egg white proteins, thereby maintaining the structural and functional properties of the proteins. Regarding the stability of egg white foam (… Figure 8 (B) The foam stability of the S-EW group is better than that of the EW group. Foam stability is related to the elasticity of the protein membrane. During storage, due to the continuous action of ROS, proteins undergo oxidative cross-linking and aggregation, which increases the rigidity of the protein membrane and makes it easy to break. The addition of SeNPs-2 effectively removes ROS, inhibits excessive protein oxidation, and helps maintain the flexibility of protein molecules in the protein membrane, thereby delaying foam breakage.

[0067] 5. Determination of the emulsifying properties of egg white (1) Method Mix 10 mL of 2 mg / mL EWP solution with 10 mL of peanut oil and homogenize at 12,000 rpm for 2 min to obtain an emulsion. Take 20 μL of the emulsion and mix thoroughly with 5 mL of 0.1% SDS solution. Use the 0.1% SDS solution as a blank control. Incubate at room temperature for 0 min and 10 min, and measure the absorbance A0 and scan A10 at 500 nm. Calculate the emulsion stability index (EAI) and emulsion activity index (ESI) using the following formulas: EAI (%) (7) ESI (m 2 / g) (8) (2) Results Emulsion stability index (ESI) and emulsion activity index (EAI) are used to assess changes in the emulsifying properties of egg white proteins during storage. EAI characterizes the ability of proteins to be absorbed at the oil-water interface, while ESI determines the ability of proteins to stably emulsify over a specific time period. Figure 9 As can be seen from Figure A, the EAI of the S-EW group was higher than that of the EW group throughout the storage period, indicating that the addition of SeNPs-2 significantly increased the emulsifying activity of the protein. SeNPs-2 can effectively inhibit the accumulation of ROS in the system, thereby reducing oxidative damage to egg white proteins. ROS-induced oxidation usually leads to the conversion of protein sulfhydryl groups into disulfide bonds, thereby forming intramolecular or intermolecular crosslinks. Moderate oxidative crosslinking can enhance the mechanical strength of the protein network structure, but excessive crosslinking can lead to the formation of protein aggregates, destroying the integrity of the emulsion interfacial membrane. The ESI of the EW group showed a trend of first increasing and then decreasing, while the ESI of the S-EW group remained stable in the later stage of storage. Figure 9 B).

Claims

1. A method for preparing a lysozyme-nanoselenium complex, characterized by, Includes the following steps: S1. Mix sodium selenite solution with lysozyme to obtain lysozyme-sodium selenite solution; S2. Add vitamin C to the lysozyme-sodium selenite solution from step S1, mix well under light-protected and room temperature conditions, adjust the pH to 6.5-7.5 to obtain a red solution, dialyze to obtain the lysozyme-nano selenium complex.

2. The preparation method according to claim 1, characterized in that, The concentration of the sodium selenite solution in step S1 is 10–50 mmol / L; preferably 30 mmol / L.

3. According to the preparation method of claim 1, the concentration of lysozyme in the lysozyme-sodium selenite solution in step S1 is 1-5 mg / mL; preferably 3 mg / mL.

4. The method of claim 1, wherein, In step S2, the molar ratio of sodium selenite to vitamin C in the lysozyme-sodium selenite solution is 1:3 to 5.

5. The preparation method according to claim 1, characterized in that, The dialysis described in step S2 is performed for 70 to 75 hours using a dialysis bag with a molecular weight cutoff of 0.8 to 1.2 kDa.

6. The lysozyme-nano selenium complex prepared by any of the preparation methods described in claims 1 to 5.

7. The use of the lysozyme-nano selenium complex of claim 6 as a ROS reactive oxygen species inhibitor / ROS scavenger or in the preparation of a ROS reactive oxygen species inhibitor / ROS scavenger.

8. The application of the lysozyme-nano selenium complex according to claim 6 in the antioxidant activity of egg white.

9. The application according to claim 8, characterized in that, The lysozyme-nano selenium complex achieves antioxidant effects by directly capturing or removing ROS in egg white, inhibiting ROS-mediated tyrosine cross-linking reactions, and / or catalyzing hydrogen peroxide to convert it into water.

10. A ROS (Reactive Oxygen Species) Inhibitor / ROS Scavenger, characterized in that, It contains the lysozyme-nano selenium complex as described in claim 6.

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