Dynamic covalent cross-linked double protein as well as preparation method and application thereof

Abstract

The invention discloses a dynamic covalent cross-linked double protein as well as a preparation method and application thereof, and the dynamic covalent cross-linked double protein is constructed by taking lactoferrin and transferrin as raw materials and reduced glutathione as a tool to enable the lactoferrin and the transferrin to be subjected to disulfide bond exchange reaction. The method comprises the following steps: (1) respectively dissolving lactoferrin and transferrin in a phosphate buffer solution to obtain a lactoferrin solution and a transferrin solution; (2) mixing the solutions, adding reduced glutathione, and oscillating and incubating in a water bath; (3) adding EDTA to terminate the reaction; and (4) dialyzing to remove the free reduced glutathione, thereby obtaining the product. According to the lactoferrin-transferrin compound disclosed by the invention, the bacteriostatic and antioxidant functions of the two proteins are synergistically enhanced, the stability of the lactoferrin-transferrin compound in a weakly acidic environment to a weakly alkaline environment is remarkably improved, and the encapsulation efficiency of the monascus yellow liposome is improved from 55.25% to 76.25%.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a dynamically covalently cross-linked dual protein, its preparation method, and its applications. Background Technology

[0002] Lactoferrin (LF) is a multifunctional iron-binding globulin widely found in the milk of mammals, especially in colostrum, with a molecular weight of approximately 80 kDa. LF consists of a single polypeptide chain containing two iron-binding sites, enabling it to bind to iron with high affinity. This polypeptide chain contains approximately 700 amino acids and two homologous globular domains: an N-ring and a C-ring. Each ring consists of two subdomains: N1, N2 and C1, C2. Lactoferrin is a basic protein with an isoelectric point of approximately 8.7. Due to its rich physiological functions, including antibacterial, antiviral, anti-inflammatory, immunomodulatory, and bone-growth-promoting effects, lactoferrin is widely used in food additives, food preservatives, and functional ingredients, covering multiple industries such as food, health products, cosmetics, and pharmaceuticals.

[0003] Transferrin (TF), also known as transferrin, has a molecular weight of approximately 76-80 kDa and two iron-binding domains (C-lobe and N-lobe), each capable of independently binding one iron ion. TF's antibacterial and antiviral activities are relatively indirect, relying on its iron transport function to indirectly influence the immune system and the survival environment of pathogens. Furthermore, research on transferrin's antioxidant, anti-inflammatory, and immunomodulatory effects is receiving increasing attention, particularly regarding its potential applications in treating anemia, infections, and immune system disorders.

[0004] In recent years, studies have found that the synergistic effect of LF and TF can significantly enhance immunomodulatory, antioxidant, and anti-inflammatory effects. However, conventional compounding methods typically involve simply mixing two proteins, which suffers from drawbacks such as insufficient stability, susceptibility to environmental factors (e.g., pH, temperature, ionic strength), potential decrease in protein activity, and limited bioavailability. Therefore, developing more stable dual-protein complexes to improve the stability and bioactivity of individual proteins is of significant scientific and practical value, and also shows great potential in drug development, immunotherapy, and functional foods. Summary of the Invention

[0005] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a dynamic covalent cross-linked dual protein and its application. The lactoferrin-transferrin complex constructed by this invention through dynamic covalent cross-linking technology achieves synergistic enhancement of the antibacterial and antioxidant functions of the two proteins, significantly improves the stability in weakly acidic to weakly alkaline environments, and increases the encapsulation efficiency of red yeast rice liposomes from 55.25% to 76.25%.

[0006] Technical solution: A dynamic covalently cross-linked dual protein, wherein the dynamic covalently cross-linked dual protein is constructed by using lactoferrin (LF) and transferrin (TF) as raw materials and reduced glutathione (GSH) as a tool to induce disulfide bond exchange reaction between lactoferrin and transferrin (LF-SS-TF).

[0007] This invention also provides a method for preparing dynamically covalently cross-linked biproteins, comprising the following steps: (1) Dissolve lactoferrin and transferrin in phosphate buffer solution to obtain lactoferrin solution and transferrin solution respectively; (2) Mix lactoferrin solution and transferrin solution, add reduced glutathione, and incubate in a water bath at 35-40 ℃ with shaking for 1-4 h; (3) Add EDTA to terminate the reaction; (4) Dialyze at 0-4 ℃ for 24-48 h to remove free reduced glutathione, and obtain LF-SS-TF.

[0008] Furthermore, the phosphate buffer solution in step (1) has a concentration of 5-20 mM and a pH of 6.0-8.0.

[0009] Furthermore, the concentration of the LF solution in step (1) is 1-4 mg / mL; the concentration of the TF solution is 1-4 mg / mL.

[0010] Furthermore, in step (2), the molar ratio of lactoferrin to transferrin is 0.33-4.0.

[0011] Furthermore, the final concentration of GSH in step (2) is 5-30 mM.

[0012] Furthermore, the amount of EDTA added in step (3) is 0.05-0.20% of the total solution volume.

[0013] Furthermore, the dialysis in step (4) uses a dialysis membrane with a molecular cutoff of 10 kDa.

[0014] The present invention also includes the application of a dynamically covalently cross-linked dual protein in loading red yeast rice yellow pigment.

[0015] Furthermore, the application method is as follows: S1. Weigh 30-100 mg of soybean lecithin and cholesterol in a mass ratio of 4:1, add 30 mL of chloroform solution, and dissolve by sonication for 20 min. S2. At atmospheric pressure, rotary evaporate at 50-45℃ and 100-300 r / min for 10-30 min, then at reduced pressure, rotary evaporate at 25-35℃ and 100-300 r / min for 30-100 min until a uniform film is formed on the wall of the tank. Then, nitrogen gas is introduced for protection for 5-20 min. S3. Take 10-60 mL of red yeast rice pigment PBS solution, preheat to 30-50 ℃, place in a rotary evaporation flask, and rotary evaporate in a normal pressure water bath at 50-200 r / min for 10-60 min to ensure complete hydration and obtain liposome suspension. S4. Take 1-10 mL of liposome suspension and mix it with 2-10 mL of dynamic covalently cross-linked double protein solution with a concentration of 0.1-10.0 mg / mL. Stir magnetically in an oil bath at 20-35℃ for 10-60 min to obtain Lf-SS-TF modified red yeast rice liposomes.

[0016] Furthermore, the concentration of the red yeast rice pigment PBS solution is 0.1-1.0 mg / mL.

[0017] Beneficial effects: (1) The LF-SS-TF dual-protein complex constructed by the present invention through dynamic covalent cross-linking technology achieves deep complementarity and synergy between lactoferrin (LF) and transferrin (TF) in both structure and function. Experimental results show that: In the Escherichia coli growth curve experiment, the LF-SS-TF complex had a slightly stronger effect on promoting bacterial growth than the LF and TF single treatment groups, indicating that the complex enhanced its ability to promote growth to a certain extent through the iron ion binding effect. In antioxidant activity tests, the complex exhibited a unique advantage of complementary mechanisms: For ABTS + The free radical scavenging, combined with the electron transfer ability of TF and the iron chelation of LF, maintains the scavenging rate at 14%~18%. For DPPH free radicals, the complex has a better scavenging ability than a single protein (LF-SS-TF>LF>TF), up to 1.5 times that of LF; For hydroxyl radicals (•OH), the complex exhibited the strongest scavenging effect, with a scavenging rate significantly higher than that of the single protein at a concentration of 2.0 mg / mL (LF-SS-TF>TF>LF).

[0018] (2) The dynamic covalent cross-linked dual protein structure of the present invention significantly improves the conformational stability of the protein: Under pH conditions (pH 7-8), the complex still maintains a high content of free thiol groups, indicating that its conformational stability is better than that of a single protein.

[0019] (3) The dynamic covalent dual protein obtained in this invention has multiple functional properties and can be widely used in multiple fields: In the food industry, it can be used as a natural preservative and functional additive to extend the shelf life of food and enhance its nutritional value. In the red yeast rice yellow pigment loading experiment, the encapsulation efficiency of the complex-modified liposomes reached 76.25%, significantly higher than that of the single protein modification group; In the health supplement sector: it can be developed into an immune-modulating and antioxidant supplement, effectively eliminating free radicals in the body and enhancing the body's immunity; Drug delivery system: As a drug carrier, its dynamic covalent structure has responsive release characteristics, which can achieve precise drug release under specific physiological conditions.

[0020] (4) The present invention employs a chemical crosslinking method under mild conditions, which has significant process advantages: Low cost: No expensive exogenous inducers are required; only reduced glutathione is used as the cross-linking tool, thus reducing raw material costs. High efficiency: The reaction can be completed in just 1-4 hours at 35-40℃, which is much shorter than traditional protein modification processes (which usually take 12-24 hours). Easy to purify: Free reduced glutathione can be removed by simple dialysis, without the need for complicated chromatographic purification steps; Scalable: The reaction conditions are mild, making it easy to scale up production and suitable for industrial-scale batch preparation.

[0021] (5) The dynamic covalent cross-linking technology of this invention endows the biprotein complex with unique structural characteristics: Reversibility: Disulfide bonds can break under specific conditions, giving the complex a responsive degradation capability; Biocompatibility: Made from natural proteins, it has good biocompatibility and no risk of immunogenicity. Attached Figure Description

[0022] Figure 1 The free thiol groups of the dynamically covalently cross-linked biproteins prepared in Examples 1-2 and Comparative Examples 1-2; Figure 2 The free amino groups of the dynamically covalently cross-linked biproteins prepared in Examples 1-2 and Comparative Examples 1-2; Figure 3 The zeta potentials of the dynamically covalently cross-linked biproteins prepared in Examples 1-2 and Comparative Examples 1-2; Figure 4The particle size and PDI of the dynamically covalently cross-linked biproteins prepared in Examples 1-2 and Comparative Examples 1-2 are shown. Figure 5 Transmission electron microscope images of LF, TF, NLF (prepared in Comparative Example 1), NTF (prepared in Comparative Example 2), and LF-SS-TF complex (prepared in the optimal example); Figure 6 Growth curves of LF, TF, and LF-SS-TF (prepared in the optimal embodiment) against Escherichia coli; Figure 7 A comparison of the free radical scavenging abilities of LF, TF, and LF-SS-TF (prepared in the optimal embodiment); where A represents the ABTS⁺ free radical scavenging rate; B represents the DPPH free radical scavenging rate; and C represents the ·OH free radical scavenging rate. Figure 8 This is the standard curve for red yeast rice. Detailed Implementation

[0023] This invention proposes a dynamically covalently cross-linked dual protein, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific examples. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0024] Lactoferrin (LF) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of ≥95% and molecular weights of 74 kDa and 78 kDa (provided by the supplier).

[0025] Transferrin (TF) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of 98% (BR) and a molecular weight of 80 kDa (provided by the supplier).

[0026] Example 1 The preparation method of LF-SS-TF is as follows: (1) Dissolve LF and TF in 10mM phosphate buffer solution (pH=8.0) respectively to obtain LF solution with a concentration of 2mg / mL and TF solution with a concentration of 2mg / mL; (2) Mix the LF solution and TF solution at a molar ratio of 0.33, 0.5, 1.0, 1.5 and 2.0 respectively, add GSH (final concentration of 5mM), and incubate in a water bath at 37 ℃ with shaking for 2 h; (3) The reaction was terminated by adding EDTA to a final concentration of 0.05%; (4) Dialyze at 4 ℃ (the molecular cutoff of the dialysis membrane is 10 kDa) for 24 h to remove free GSH, and obtain LF-SS-TF.

[0027] Example 2 The pH-optimized preparation method for LF-SS-TF is as follows: (1) Dissolve LF and TF in 10 mM phosphate buffer solutions (pH=5.0, 6.0, 7.0, 8.0, 9.0, 10.0) respectively to obtain LF solution with a concentration of 2 mg / mL and TF solution with a concentration of 2 mg / mL; (2) Mix the LF solution and TF solution at a volume ratio of 1:1, add GSH (final concentration of 5mM), and incubate in a water bath at 37 ℃ with shaking for 2 h; (3) The reaction was terminated by adding EDTA to a final concentration of 0.05%; (4) Dialyze at 4 ℃ (the molecular cutoff of the dialysis membrane is 10 kDa) for 24 h to remove free GSH, and obtain LF-SS-TF.

[0028] Comparative Example 1 The preparation method of NLF is as follows: (1) Dissolve LF in 10 mM phosphate buffer solution (pH=8.0) to obtain an LF solution with a concentration of 2 mg / mL; (2) Take 2 mL of LF solution, add GSH (final concentration 5 mM), and incubate in a water bath at 37 ℃ with shaking for 2 h; (3) The reaction was terminated by adding EDTA to a final concentration of 0.05%; (4) Dialyze at 4 ℃ (the molecular cutoff of the dialysis membrane is 10 kDa) for 24 h to remove free GSH, and then obtain NLF.

[0029] Comparative Example 2 The preparation method of NTF is as follows: (1) Dissolve TF in 10 mM phosphate buffer solution (pH=8.0) to obtain a TF solution with a concentration of 2 mg / mL; (2) Take 2 mL of TF solution, add GSH (final concentration 5 mM), and incubate in a water bath at 37 ℃ with shaking for 2 h; (3) The reaction was terminated by adding EDTA to a final concentration of 0.05%; (4) Dialyze at 4 ℃ (the molecular cutoff of the dialysis membrane is 10 kDa) for 24 h to remove free GSH and obtain NTF.

[0030] Physical performance testing: (1) Determination of free thiol groups The free thiol content of LF, TF, LF-SS-TF, NLF, and NTF prepared with different molar ratios and pH values ​​was determined using a free thiol assay kit (DTNB method).

[0031] (2) Determination of free amino groups The free amino group content in LF, TF, and LF-SS-TF, NLF, and NTF prepared at different molar ratios and pH values ​​was determined using the OPA method. 1.905 g of borax and 50 mg of SDS were completely dissolved in 25 mL of deionized water. 1 mL of OPA (methanol-soluble, 4%, w / v) and 44 mg of DTT were added, and the volume was adjusted to 50 mL with deionized water to prepare the OPA working solution. For the assay, 20 μL (0.5 mg / mL) of protein solution and 200 μL of OPA working solution were mixed and reacted at 35°C for 2 min. The absorbance at 340 nm was then measured using a microplate reader. The free amino group content was calculated using L-Ser as a standard, expressed as mmol / g NH2 / g protein.

[0032] (3) Measurement of Zeta potential The zeta potentials of LF, TF, LF-SS-TF, NLF, and NTF prepared at different molar ratios and pH values ​​were determined using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The complex solutions were placed in the potential cell, and the scan count was adjusted to 20 times with an equilibration time of 30 s, and the results were performed in triplicate.

[0033] (4) Determination of particle size and PDI The particle sizes of LF, TF, and LF-SS-TF, NLF, and NTF prepared at different molar ratios and pH were determined using a Zetasizer Nano ZS90 (Malvern Instruments, UK). The particle size distribution was expressed using the polydispersity index (PDI). The complex solutions were placed in cuvettes, and the scan count was adjusted to 15, with an equilibration time of 30 s, and the results were performed in triplicate.

[0034] (5) TEM 0.1 mg / mL solutions of LF, TF, LF-SS-TF, NLF, and NTF were added dropwise onto a copper grid. After standing for several minutes, excess liquid was blotted away with filter paper. Then, 1% phosphotungstic acid solution was added for staining, and after standing for 5 minutes, the dye was blotted away with filter paper again. After the sample dried, high-resolution images of the protein morphology, size, and aggregation state were obtained by transmission electron microscopy (TEM).

[0035] Results and Analysis: (1) Analysis of free thiol groups Thiol groups and disulfide bonds are key chemical groups that maintain the spatial stability of protein structures, and changes in their content can serve as important indicators of protein conformational changes. Thiol groups in proteins mainly originate from cysteine ​​(Cys) residues and possess high chemical reactivity. On one hand, thiol groups can form disulfide bonds (–S–S–) through oxidation, thereby stabilizing the higher-order structure of proteins; on the other hand, they can participate in thiol-disulfide bond exchange reactions, initiating protein conformational rearrangements. Furthermore, thiol groups can react with small molecules such as glutathione (GSH) for protein modification or coupling. Figure 1 As shown, LF-SS-TF exhibits higher thiol exposure compared to LF and TF, indicating that its conformation unfolds to some extent under the influence of GSH. However, compared to NLF and NTF, the free thiol content in LF-SS-TF is actually lower, suggesting that some thiol groups participate in disulfide bond formation or thiol-disulfide bond exchange reactions during the recombination process. With the gradual increase of the LF:TF molar ratio, the free thiol content generally shows an upward trend, but when LF:TF = 1.0, the free thiol content is the lowest, and disulfide bond formation is the most abundant, suggesting that its LF-SS-TF recombination rate is the highest. With the gradual increase of pH, the free thiol content shows an initial increase followed by a decrease. However, under pH conditions (pH 5-6), the conformational stability of the protein is significantly affected. Under acidic conditions, the protein may undergo some degree of conformational contraction or denaturation, but the disulfide bonds are relatively stable, and the thiol groups are prone to protonation, leading to a decrease in its reactivity, thus manifesting as a lower free thiol content. In the neutral to slightly alkaline range (pH 7-8), protein structure gradually relaxes, increasing the exposure of buried thiol groups and maximizing the free thiol content. Under alkaline conditions (pH ≥ 9), further protein denaturation may lead to the masking of some disulfide bonds, resulting in a decrease in the free thiol content.

[0036] (2) Analysis of free amino groups The α- and ε-amino content of proteins can be determined using the OPA method, reflecting the degree of protein hydrolysis and information about the lysine groups in the side chains. For example... Figure 2 As shown, the free amino content of the LF-SS-TF complex falls between that of LF and TF, indicating that the structural characteristics of this complex result in an exposure level of free amino groups between the two. It is speculated that during its synthesis, certain disulfide bonds or other chemical bonds may be involved in the synthesis of novel materials, allowing some free amino groups to be exposed and further enhancing its chemical activity. The formation of the LF-SS-TF complex not only alters the structure of a single protein but may also regulate the availability of free amino groups through the formation of disulfide bonds, leading to higher reactivity or binding capacity in certain chemical reactions. In the molar ratio optimization experiment ( Figure 2In Example 1), the content of free amino groups fluctuated with increasing molar ratio, reaching its highest value at a molar ratio of 0.33. For the LF-SS-TF complex, this may mean that at a certain molecular ratio, an optimal balance is reached between disulfide bond formation and free amino group exposure, promoting the formation of more free amino groups. In the pH optimization experiment ( Figure 2 (Example 2) Different pH conditions affect the content of free amino groups, especially at near-neutral and slightly alkaline pH levels, where the content of free amino groups is higher. For the LF-SS-TF complex, the disulfide bonds may be more stable under these pH conditions, thus increasing the availability of free amino groups. This suggests that the LF-SS-TF complex may have higher free amino group activity in a more neutral environment, making it suitable for certain biological or chemical reactions. Both NLF and NTF are proteins obtained by GSH modification of single proteins, such as... Figure 2 (Comparative Examples 1 and 2) show that the free amino content of both NLF and NTF is slightly increased compared to the unmodified protein. This may be because the GSH-modified LF and TF have relatively loose structures, allowing some free amino groups to be exposed. However, their free amino content is significantly different from that of LF-SS-TF, indicating that during the synthesis of LF-SS-TF, some free amino groups are either encapsulated or exposed due to changes in spatial structure.

[0037] (3) Zeta potential analysis The zeta potential (ζ-potential) is an important parameter characterizing the surface charge properties of proteins in solution. It effectively reflects changes in their charged state and interfacial properties, and is therefore often used to analyze the microscopic mechanisms of intermolecular interactions between proteins. Simultaneously, changes in ζ-potential can also serve as an important basis for determining protein interactions with other substances. The oxidation of thiol groups (-SH) in proteins to form disulfide bonds is often accompanied by changes in molecular conformation, thereby affecting surface charged groups (such as -NH3). + and -COO - The degree of exposure and spatial distribution of ζ-potential affect the zeta potential, which in turn causes changes in the ζ-potential. ζ-potential results indicate (e.g.) Figure 3As shown in the diagram, LF exhibits a high positive potential while TF exhibits a negative potential. After forming the LF-SS-TF complex, the potential remains positive but decreases significantly, indicating that the introduction of TF neutralizes the positive charge on the LF surface. Simultaneously, disulfide bond formation induces protein conformational rearrangement, leading to changes in the exposure and distribution of charged groups. With increasing LF to TF molar ratio, the system's zeta potential gradually decreases and tends to stabilize, indicating a clear ratio-dependent complexation process. pH changes further affect the protein ionization state, causing significant shifts in the zeta potential under different conditions, reflecting the system's sensitivity to environmental factors. In contrast, the zeta potential changes of NLF and NTF are smaller and closer, indicating their surface charge properties are more stable. In summary, LF and TF not only exhibit electrostatic interactions but also undergo disulfide bond cross-linking and conformational changes, resulting in a structurally stable complex system with reconfigured electrical properties.

[0038] (4) Particle size and PDI analysis The polydispersity index (PDI) is an important parameter characterizing the breadth and uniformity of particle size distribution in a particulate system. A lower PDI value indicates a more concentrated particle size distribution and better dispersibility. Particle size, as another key structural parameter, directly affects the physicochemical properties of the system, including solubility, emulsification, and foaming properties. Generally, smaller particle size helps increase specific surface area, thereby improving the system's solubility and interfacial activity. Therefore, combining PDI with changes in particle size allows for a more comprehensive and accurate assessment of the dispersion stability and functional characteristics of particulate systems. Figure 4 As shown, LF-SS-TF has a larger particle size and higher PDI than single-protein groups, with a wider distribution and limited dispersion stability, while TF has a smaller particle size, lower PDI, and a more homogeneous system. Molar ratio adjustment shows that optimal dispersibility and functionality can be achieved under conditions of moderate particle size and low PDI; increasing pH can cause particle size to increase, but PDI decreases and dispersibility increases in the pH range of 5-7, indicating that particle performance is synergistically regulated by protein composition, pH, and ratio.

[0039] Preferred Implementation The preparation method of LF-SS-TF is as follows: (1) Dissolve LF and TF in 10mM phosphate buffer solution (pH=8.0) respectively to obtain LF solution with a concentration of 2mg / mL and TF solution with a concentration of 2mg / mL; (2) Mix the LF solution and TF solution at a molar ratio of 1.0, add GSH (final concentration of 5mM), and incubate in a water bath at 37°C with shaking for 2 h; (3) The reaction was terminated by adding EDTA to a final concentration of 0.05%; (4) Dialysis at 4 ℃ (the molecular cutoff of the dialysis membrane is 10 kDa), and free GSH is removed after 24 h to obtain LF-SS-TF.

[0040] TEM analysis Scanning electron microscopy observations were performed on LF, TF, NLF, NTF, and LF-SS-TF (prepared in the optimal embodiment), as follows: Figure 5 As shown, LF exhibits well-dispersed near-spherical nanoparticles with a relatively uniform particle size distribution and clear boundaries; TF also shows a near-spherical structure, but the particle dispersion is slightly poor, with slight local aggregation. In the control group, NLF particles are smaller and more densely distributed, exhibiting a tightly packed state, while NTF shows a loosely dispersed structure with relatively blurred particle outlines. In contrast, it is difficult to observe clear, independent spherical particles in the LF-SS-TF complex; the overall structure presents a continuous, blurred aggregate or network-like state, with the boundaries between particles disappearing. These differences indicate that LF and TF have undergone significant interactions and structural rearrangement under disulfide bonds, transforming from originally dispersed single protein nanoparticles into a large-scale aggregated composite system, proving the successful formation of the LF-SS-TF complex.

[0041] The LF-SS-TF used in the following performance tests were all prepared using the optimal embodiment.

[0042] (I) Analysis of growth curves of lactoferrin (LF), transferrin (TF), and dynamically covalently cross-linked dual proteins (LF-SS-TF) on Escherichia coli 1917: 1. Resuscitation and culture of microbial strains Escherichia coli 1917, preserved in glycerol tubes from a -80 °C freezer, was streaked onto LB agar and incubated at 37 °C for 12 h. A single colony was picked and inoculated into 3 mL of LB liquid medium, and incubated at 160 r / min and 37 °C for 12 h. Colony counting was performed using the dilution plating method, with 100 μL of the plate taken at this time.

[0043] Bacterial count (CFU / mL) = Number of single colonies × Dilution factor × 10 2. Growth curve experiment This study used an ELISA reader to detect the effects of LF, TF, and LF-SS-TF on the growth of *Escherichia coli* 1917. The specific steps were as follows: The concentration of the bacterial culture (logarithmic growth phase) was adjusted to 1×10⁻⁶. 5CFU / mL; 100 μL LLF-SS-TF solution (0.5 mg / mL) and 100 μL bacterial culture were added to a 96-well plate and incubated together. LB solution was used as a negative control, and 100 μL LF solution and 100 μL TF solution (0.5 mg / mL) were used as positive controls. The plates were incubated at 37 ℃ for 12 h, and the OD value was measured every 2 h. 600 Absorbance in nm.

[0044] Results and Analysis: from Figure 6 The growth curves show that all treatment groups (LF, TF, and LF-SS-TF) and the control group (LB) exhibited typical bacterial growth trends, with no obvious growth inhibition, but some growth retardation was observed at different time stages. During the adaptation and early logarithmic growth phase (0-4 h), the OD values ​​of each group were... 600 The small differences in values ​​indicate that different treatments have limited impact on the initial growth of Escherichia coli 1917; during the rapid growth phase of 4-8 h, the OD values ​​of the LF, TF, and LF-SS-TF groups were relatively high. 600 The values ​​were all lower than those of the LB control group, indicating that all three protein systems were less effective at promoting bacterial growth than the LB liquid medium. Specifically, the overall OD values ​​of the LF-SS-TF group were lower than those of the LB control group. 600 The values ​​were slightly lower than those of the LF and TF groups, indicating that the combined effect of LF and TF on delaying bacterial growth was slightly enhanced, but the difference was not significant; during the 8-12 h stationary period, the OD values ​​of each experimental group were... 600 The values ​​gradually approach each other, indicating that the bacteria will eventually be able to resume growth and enter a stable phase.

[0045] Comprehensive analysis shows that OD 600 As the concentration increased over time, LF, TF, and their complex LF-SS-TF all exhibited growth-promoting effects. This indicates that LF, TF, and their complex LF-SS-TF not only did not inhibit bacterial cell growth, but also served as nutrient sources or functional carriers, providing available nitrogen sources, amino acids, or trace iron binding environments for bacterial growth, thereby promoting their proliferation.

[0046] (II) Determination of the antioxidant capacity of lactoferrin (LF), transferrin (TF), and the dual protein (LF-SS-TF): LF, TF, and LF-SS-TF solutions with concentrations of 0.5, 1.0, 1.5, and 2.0 mg / mL were prepared to determine their antioxidant properties.

[0047] 1. Determination of ABTS+ free radical scavenging rate Reaction solution: Equal volumes of 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution were mixed and reacted in the dark for 12 h. Before the experiment, the reaction solution was diluted with anhydrous ethanol to adjust the absorbance to 0.70 ± 0.02. 200 μL of sample was placed in a test tube, 200 μL of ABTS solution was added, and the mixture was thoroughly mixed. The mixture was allowed to stand at room temperature for 1 h, and then the absorbance was measured at 734 nm. Distilled water was used as the blank control group instead of the sample. The clearance rate was calculated using the following formula:

[0048] In the formula: A1 — absorbance of the sample; A0 — Absorbance with distilled water added.

[0049] 2. DPPH free radical scavenging rate determination A 0.04 mg / mL DPPH solution was prepared using anhydrous ethanol and stored in the dark. 20 μL of the sample solution was accurately transferred, and 200 μL of DPPH solution was added. After mixing, the mixture was reacted at room temperature in the dark for 1 h. The absorbance A2 was then measured at 517 nm.

[0050]

[0051] Where: A0—absorbance with distilled water added; A1 – Absorbance of anhydrous ethanol as a substitute for DPPH solution; A2 — Sample absorbance.

[0052] 3.OH - Free radical scavenging rate determination Working solution: Mix 10 mmol / L salicylic acid-ethanol solution, 10 mmol / L FeSO4 solution and 8.8 mmol / L H2O2 solution in a 1:1:1 ratio. Take 50 μL of sample solution and add 150 μL of working solution. Mix well and react at room temperature for 1 h. Measure the absorbance at 510 nm and record it as A2.

[0053]

[0054] Where: A0—absorbance with distilled water added; A1—Absorbance of H2O-substituted H2O2 solution; A2 — Sample absorbance.

[0055] Results and Analysis: 1. Analysis of ABTS⁺ free radical scavenging ability To investigate the antioxidant properties of different samples, the ABTS⁺ free radical scavenging rates (e.g., LF, TF, and LF-SS-TF at different concentrations (0.5-2.0 mg / mL) were compared. Figure 7 (As shown in Figure A). The results showed that the clearance rate of all three samples decreased with increasing concentration, but there were significant differences between the different samples.

[0056] The LF-SS-TF complex exhibits the strongest ABTS⁺ free radical scavenging ability, achieving a scavenging rate of approximately 82% at 0.5 mg / mL. This scavenging rate stabilizes at around 85%–86% with increasing concentrations of 1.0–2.0 mg / mL, demonstrating extremely high and stable antioxidant activity. LF shows moderate scavenging ability, at approximately 58% at a low concentration (0.5 mg / mL), with a significant upward trend, reaching approximately 83% at 2.0 mg / mL. In contrast, TF exhibits the lowest free radical scavenging ability, at only around 18% at 0.5 mg / mL, and even at 2.0 mg / mL, it only rises to approximately 37%, indicating significantly weaker overall antioxidant performance compared to both the LF monomer and the complex.

[0057] This result indicates that after LF and TF bind via disulfide bonds (-SS-), their antioxidant groups may undergo conformational masking or chemical inactivation, leading to a decrease in free radical scavenging efficiency. Possible reasons include: ① the active thiol groups participate in the bonding process during disulfide bond formation, weakening the electron donor capacity; ② the protein conformation changes after complexation, reducing the exposure of some antioxidant active sites; ③ intermolecular interactions affect the electron transfer process, inhibiting the reduction reaction of ABTS⁺ free radicals.

[0058] In summary, the antioxidant capacity of the three samples followed the order LF-SS-TF > LF > TF. The results indicate that while the formation of a disulfide bond complex between LF and TF may improve its structural stability, it also weakens its free radical scavenging activity to some extent. Therefore, future studies need to seek a balance between maintaining the structural stability of the complex and preserving its antioxidant activity.

[0059] 2. Analysis of DPPH free radical scavenging ability like Figure 7As shown in Figure B, LF, TF, and LF-SS-TF all exhibited certain scavenging effects on DPPH free radicals at different concentrations (0.5-2.0 mg / mL), and the scavenging rate increased significantly with increasing concentration, indicating that all three have concentration-dependent antioxidant activity. Among all samples, LF-SS-TF showed the highest DPPH free radical scavenging ability, followed by TF, with LF showing the lowest. This difference is closely related to the protein structure and its antioxidant mechanism. Transferrin molecules are rich in tyrosine and tryptophan residues; these aromatic amino acids can donate electrons or hydrogen atoms to DPPH free radicals, thereby reducing the free radicals to their stable forms. Therefore, TF showed higher scavenging ability in the DPPH system dominated by electron transfer (ET). In contrast, the main antioxidant mechanism of lactoferrin is iron chelation, which binds Fe... 3+ It inhibits the generation of hydroxyl radicals, but its performance is weaker in the DPPH model (which is mainly electron-donating). In addition, due to the disulfide bond connection, some active sites of the LF-SS-TF complex may be spatially restricted, but the combination of the two proteins makes its overall antioxidant capacity slightly higher than that of LF alone, showing some functional retention but no significant synergistic enhancement.

[0060] In summary, the antioxidant capacity of the three samples in the DPPH free radical system was in the following order: LF-SS-TF > LF > TF. This indicates that transferrin's strong electron transfer ability plays a major role in the DPPH free radical scavenging process, while lactoferrin mainly participates in the antioxidant reaction through the iron chelation pathway, and its contribution to this system is relatively small.

[0061] 3. OH - Free radical scavenging ability analysis like Figure 7 As shown in C, LF, TF, and LF-SS-TF inhibit hydroxyl radicals (OH-). - The scavenging rates of all three antioxidants increased significantly with increasing sample concentration, indicating that they all possessed certain antioxidant activity in this system. The overall trend was: LF-SS-TF > TF > LF. At 2.0 mg / mL, LF-SS-TF exhibited the highest scavenging rate, significantly higher than LF and TF, suggesting that LF, after binding with TF, effectively inhibited OH-. - It exhibits an enhancing effect on free radicals.

[0062] This enhancement is mainly related to the iron ion chelating properties of LF and TF and the synergistic effect of disulfide bonds. Hydroxyl radicals are primarily generated through the Fenton reaction (Fe... 2+ + H2O2→Fe 3+ The reaction ⇌ Fe(OH)⁻ is formed by the reaction of Fe(OH)⁻ and Fe(OH)⁻. Both LF and TF have strong iron-binding capabilities and can bind to Fe. 3+ Binding to block Fe2+ Participating in the reaction, thereby inhibiting OH - The formation of Fe. Because the iron binding constant of TF is higher than that of LF, its effect on Fe... 3+ It has a stronger chelation efficiency, and therefore is more effective in inhibiting OH-. - The effect on generation is more significant. In LF-SS-TF, LF and TF are bound by disulfide bonds, which not only retains the high iron chelating ability of LF, but also enhances the efficiency of electron transfer between protein molecules, thus further improving its scavenging ability in the hydroxyl radical system.

[0063] In addition, both LF and TF molecules contain residues such as tyrosine, tryptophan, and cysteine, which have hydrogen-donating or electron-donating capabilities and can directly react with OH groups. - The reaction generates stable molecules, thereby exerting a direct scavenging effect. After recombination, the increased steric accessibility of some active residues also contributes to improved antioxidant capacity. In summary, the order of antioxidant capacity among the three in the hydroxyl radical scavenging system is: LF-SS-TF > TF > LF.

[0064] The results show that the combination of LF and TF can achieve a dual enhancement of iron ion chelation and electron donor mechanisms, thus exhibiting a strong synergistic antioxidant effect in the hydroxyl radical system.

[0065] Example 3 A method for preparing red yeast rice liposomes modified with lactoferrin (LF), transferrin (TF), and dual proteins (Lf-SS-TF) includes the following steps: 1. Preparation of the standard curve for red yeast rice yellow pigment Prepare gradient dilutions of red yeast rice pigment (0.05, 0.1, 0.2, 0.25, 0.3, 0.45, 0.5 mg / mL) using PBS solution. Measure the absorbance at 500 nm. Plot the red yeast rice pigment concentration on the x-axis and the absorbance on the y-axis to create a standard curve. Measure the absorbance of the red yeast rice pigment dilutions at 500 nm at room temperature. Subtract the average absorbance of the blank standard at 500 nm from the absorbance of each standard at 500 nm (for correction). Plot the average absorbance against the concentration (mg / mL) to create a standard curve (e.g., [image of standard curve]). Figure 8 (as shown) 2. Preparation method of liposomes Weigh out 50 mg of soybean lecithin and cholesterol in a mass ratio of 4:1, add 30 mL of chloroform solution, and sonicate for 20 min. Then, rotary evaporate at 45℃ and 100 r / min under normal pressure for 20 min, followed by rotary evaporation at 30℃ and 100 r / min under reduced pressure for 60 min until a uniform film forms on the flask wall. Then, purge with nitrogen for 5 min. Take 30 mL of red yeast rice pigment PBS solution (concentration 0.4 mg / ml), preheat to 45℃, place in a rotary evaporation flask, and rotary evaporate at 120 r / min under normal pressure in a water bath for 30 min to ensure complete hydration, obtaining a liposome suspension. Sonicate for 10 min using an ultrasonic homogenizer (30% amplitude, 5 s on, 5 s off) to obtain uniform red yeast rice pigment liposomes (Lipo).

[0066] 3. Determination of encapsulation efficiency Take an appropriate amount of Lipo and add it to a 50 kDa ultrafiltration centrifuge tube. Centrifuge at 4000 r / min for 15 min until the volume of the supernatant no longer changes. Measure the absorbance of the Lipo effluent at a wavelength of 500 nm. Calculate the concentration C1 of free red yeast rice pigment according to the standard curve. Under the same conditions, without centrifugation, determine the total red yeast rice pigment content C2 in the Lipo and calculate the encapsulation efficiency using the formula.

[0067]

[0068] In the formula: C1—concentration of red yeast rice pigment in the effluent, mg / mL; C2 – Concentration of Lipo red yeast rice pigment before centrifugation, mg / mL.

[0069] 4. Preparation of LF-Lipo, TF-Lipo, and LF-SS-TF-Lipo Take 2 mL of the Lipo suspension prepared above and mix it with 2 mL of LF, TF, and LF-SS-TF double protein solutions with a concentration of 1 mg / mL. Stir magnetically in an oil bath at 25°C for 30 min to obtain LF, TF, and LF-SS-TF modified Lipo.

[0070] Results and Analysis: Table 1 Encapsulation efficiency of LF, TF, and LF-SS-TF liposomes

[0071] Note: C1 is the concentration of red yeast rice pigment in the effluent, mg / mL; C2 is the concentration of added red yeast rice yellow pigment, mg / mL.

[0072] As shown in Table 1 above, the encapsulation efficiency of ordinary liposomes for red yeast rice yellow pigment was 55.25%. Under the same preparation conditions, 1 mg / ml of LF, TF, and LF-SS-TF were added to the liposome suspension at a 1:1 ratio to obtain protein liposomes with encapsulation efficiencies of 57.00%, 62.25%, and 76.25%, respectively. The encapsulation efficiency of liposomes improved after the addition of proteins, which may be because the proteins modify the liposome surface, forming a coating and enhancing the stability of the liposomes. The liposomes with the highest encapsulation efficiency and the most significant improvement after the addition of two proteins indicate that LF-SS-TF, formed by the combination of LF and TF, has a better modification effect on liposomes.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A dynamically covalently cross-linked dual protein, characterized in that... The dynamically covalently cross-linked dual protein is constructed by using lactoferrin and transferrin as raw materials and reduced glutathione as a tool to induce a disulfide bond exchange reaction between lactoferrin and transferrin.

2. The method for preparing a dynamically covalently cross-linked dual protein according to claim 1, characterized by the following steps: (1) Dissolve lactoferrin and transferrin in phosphate buffer solution to obtain lactoferrin solution and transferrin solution respectively; (2) Mix lactoferrin solution and transferrin solution, add reduced glutathione, and incubate in a water bath at 35-40 ℃ with shaking for 1-4 h; (3) Add EDTA to terminate the reaction; (4) Dialyze at 0-4 ℃ for 24-48 h to remove free reduced glutathione and obtain dynamically covalently cross-linked double protein.

3. The method for preparing a dynamically covalently cross-linked biprotein according to claim 2, characterized in that, The phosphate buffer solution in step (1) has a concentration of 5-20 mM and a pH of 6.0-8.

0.

4. The method for preparing a dynamically covalently cross-linked biprotein according to claim 2, characterized in that, The concentration of the lactoferrin solution in step (1) is 1-4 mg / mL; the concentration of the transferrin solution is 1-4 mg / mL.

5. The method for preparing a dynamically covalently cross-linked dual protein according to claim 2, characterized in that, In step (2), the molar ratio of lactoferrin to transferrin is 0.33-4.

0.

6. The method for preparing a dynamically covalently cross-linked biprotein according to claim 2, characterized in that, The final concentration of reduced glutathione in step (2) is 5-30 mM.

7. The method for preparing a dynamically covalently cross-linked biprotein according to claim 2, characterized in that, The amount of EDTA added in step (3) is 0.05-0.20% of the total solution volume.

8. The method for preparing a dynamically covalently cross-linked biprotein according to claim 2, characterized in that, The dialysis in step (4) uses a dialysis membrane with a molecular cutoff of 10 kDa.

9. The application of a dynamically covalently cross-linked dual protein according to claim 1 in loading red yeast rice yellow pigment.

10. The application according to claim 9, characterized in that, The application method is as follows: S1. Weigh out soybean lecithin and cholesterol, add chloroform solution, and dissolve by ultrasonication; S2. At atmospheric pressure, rotary evaporate at 50-45℃ and 100-300 r / min for 10-30 min, then at reduced pressure, rotary evaporate at 25-35℃ and 100-300 r / min for 30-100 min until a uniform film is formed on the wall of the tank. Then, nitrogen gas is introduced for protection for 5-20 min. S3. Take red yeast rice pigment PBS solution, preheat to 30-50℃, place in a rotary evaporation flask, and rotary evaporate in a normal pressure water bath at 50-200 r / min for 10-60 min to ensure complete hydration and obtain liposome suspension. S4. Take the liposome suspension and mix it with the dynamic covalently cross-linked double protein solution. Stir magnetically in an oil bath at 20-35℃ for 10-60 min to obtain red yeast rice liposomes modified with dynamic covalently cross-linked double proteins.

Images