Bovine lactoferrin-phosvitin compound and preparation method thereof

Abstract

The invention provides a bovine lactoferrin-phosvitin compound and a preparation method thereof. The phosvitin provided by the invention contains a large amount of phosphorylated serine, so that the phosvitin has relatively high negative charges, and forms a compound with a structure taking LF as a core and PSV as a shell with bovine lactoferrin through the synergistic effect of electrostatic interaction and hydrogen-bond interaction, so that the stability coefficient of the phosvitin is increased, the denaturation temperature is increased by 1-16 DEG C, and the denaturation time is shortened by 1-16 DEG C; the adverse effect of heat treatment on the lactoferrin is reduced, the heat stability of the lactoferrin is enhanced, the oxidation resistance is not affected, and the application range of the lactoferrin is expanded.

Description

Technical Field

[0001] This application belongs to the fields of food technology and food ingredients, specifically relating to a bovine lactoferrin-egg yolk phosphoprotein complex and its preparation method. Background Technology

[0002] Lactoferrin (LF) is a globular iron-binding glycoprotein mainly found in milk and some mucosal secretions. LF consists of a 700-amino acid polypeptide chain folded into two symmetrical homologous globular lobes (N-lobes and C-lobes), with a molecular weight of approximately 80 kDa and an isoelectric point between 8.4 and 9.0. Numerous studies have shown that LF has higher nutritional value and broader biological activities than other proteins in milk, offering numerous health benefits to humans, including anti-tumor, antioxidant, immunomodulatory, antiviral, anti-inflammatory, antimicrobial, and iron metabolism-regulating effects. Therefore, lactoferrin is an ideal functional food ingredient and has been used in functional food formulations and dietary supplements in commercial foods, nutritional supplements, and cosmetics, showing great potential in the food and healthcare fields. However, LF is sensitive to heat treatment and is prone to denaturation or aggregation, leading to structural changes and loss of biological function, which greatly limits its applications.

[0003] Currently, methods to improve the thermal stability of bovine lactoferrin mainly involve polysaccharide complexation and glycosylation modification. However, these methods have drawbacks such as complex reaction processes and impact on biological activity. Utilizing polysaccharides to form complexes with bovine lactoferrin can improve its thermal stability; commonly used polysaccharides include carrageenan and xanthan gum. Considering its application in infant formula, food additive regulations restrict the use of most polysaccharides, allowing only carrageenan and xanthan gum. However, their use as emulsifiers and thickeners affects the processing application and sensory properties of lactoferrin. Furthermore, the complexation of some polysaccharides with lactoferrin only occurs at lower pH levels, limiting the suitable food range and restricting its widespread application. The Maillard reaction, which causes a carbonyl-amine reaction between sugar molecules and the free amino groups of lactoferrin to form a glycoprotein complex, is also a common method to improve the thermal stability of bovine lactoferrin. However, this reaction process is very complex, and the properties of the resulting glycosylated products are affected by various factors, including the type of sugar, leading to unpredictable results. Simultaneously, the temperature of traditional glycosylation methods may denature lactoferrin, destroying its biological activity. Furthermore, the Maillard reaction may form harmful byproducts, limiting its applications. Therefore, while these methods have some effect on improving the thermal stability of lactoferrin, it is still necessary to find safe and effective methods based on the molecular structure characteristics of lactoferrin to significantly enhance its thermal stability and functional properties.

[0004] Lactoferrin is one of the few proteins that carries a positive surface charge under neutral conditions (isoelectric point pI ≈ 8.5), which allows it to bind to casein and form a relatively stable micelle structure to maintain thermal stability. Its basic amino acid-rich regions (such as lysine and arginine residues) are key sites for binding with casein. However, the thermal stability and functional properties of extracted lactoferrin decrease after it is separated from the system coexisting with casein. The strong binding affinity between casein and lactoferrin is due to the negative charge of phosphoserine residues on casein's surface, which can form an electrostatic interaction with the positively charged regions of lactoferrin, promoting their binding. Therefore, exploring a natural protein similar to casein, carrying a negative charge and possessing good thermal stability, theoretically has the potential to improve the thermal stability and functional properties of lactoferrin.

[0005] Phosvtin (PSV) is a high-phosphorus protein found in egg yolks, accounting for about 4% of the dry weight of the yolk. It consists of 216 amino acid residues, of which 56% are serine, and 80% of the serine is phosphorylated. At pH 7, all phosphoserine residues in the PSV molecule are negatively charged. In addition, PSV is a heat-resistant protein. Studies have shown that heating a PSV solution at 100°C for several hours within the pH range of 4–8 will not result in precipitation or any other changes. PSV has similar phosphorylated residues to casein, but its phosphorylation degree is higher than that of casein, making it one of the most phosphorylated proteins in nature. Therefore, phosvtin meets the two conditions mentioned above: (1) it carries a large number of negative charges, enabling it to have a strong electrostatic interaction with lactoferrin under neutral conditions, and the two have a natural compatibility; (2) it has good thermal stability, and after binding with lactoferrin, it can give the protein complex system good stability and avoid the thermal denaturation of lactoferrin. A literature review revealed no reports on the use of egg yolk phosphatase to improve the thermal stability of lactoferrin. Therefore, this study aimed to construct lactoferrin-egg yolk phosphatase complexes with varying mass ratios to enhance the thermal stability and functional properties of lactoferrin. Summary of the Invention

[0006] This application describes the construction of a bovine lactoferrin-egg yolk phosphatase complex using natural bovine lactoferrin as a raw material, aiming to improve the thermal stability of natural bovine lactoferrin. Furthermore, this method is simple to operate, produces no byproducts, and operates under mild reaction conditions, showing considerable promise for large-scale production applications.

[0007] This application provides a method for preparing bovine lactoferrin-egg yolk phosphoprotein complex, the preparation method specifically including the following steps:

[0008] Step S1 Preparation of lactoferrin stock solution: Bovine lactoferrin is dissolved in distilled water and magnetically stirred at room temperature to fully hydrate it. After stirring, the solution is filtered using a syringe filter to obtain the lactoferrin stock solution.

[0009] Step S2 Preparation of egg yolk high phosphoprotein stock solution: Dissolve egg yolk high phosphoprotein in distilled water, stir magnetically at room temperature to fully hydrate, and filter with a syringe filter after stirring to obtain egg yolk high phosphoprotein stock solution;

[0010] Step S3: pH adjustment of the protein stock solution. The pH of the lactoferrin stock solution and the egg yolk high-phosphorus protein stock solution is adjusted using 0.1 M NaOH solution or 0.1 M HCl solution, respectively.

[0011] Step S4 incubation treatment: The lactoferrin reserve solution and the egg yolk high phosphoprotein reserve solution, whose pH has been adjusted in step S3, are mixed in a certain mass ratio and then incubated at room temperature.

[0012] Step S5 involves preparing bovine lactoferrin-egg yolk high-phosphorus protein complex. The bovine lactoferrin-egg yolk high-phosphorus protein solution incubated in step S4 is freeze-dried under vacuum to obtain the bovine lactoferrin-egg yolk high-phosphorus protein complex.

[0013] This application also provides a lactoferrin-egg yolk phosphatase complex prepared according to the preparation method, characterized in that the mass ratio of bovine lactoferrin to egg yolk phosphatase is 1-10:1.

[0014] This application also provides the use of the bovine lactoferrin-egg yolk phosphoprotein complex.

[0015] Beneficial effects

[0016] This application provides a bovine lactoferrin-egg yolk phosphatase complex and its preparation method, as well as the application of egg yolk phosphatase in improving the thermal stability of bovine lactoferrin, thus expanding its application scope. Multiple methods have confirmed that the synergistic effect of electrostatic interactions and hydrogen bonding is the main driving force for the formation of the bovine lactoferrin-egg yolk phosphatase complex, resulting in a structure with LF as the core and PSV as the shell. The addition of PSV significantly reduces the turbidity of the bovine lactoferrin solution, eliminating obvious turbidity, increasing its stability coefficient, raising the denaturation temperature by 1-16℃, effectively reducing the aggregation degree of bovine lactoferrin under heat treatment, enhancing its overall thermal stability, and improving its antioxidant properties. Furthermore, this method is simple to operate and has mild reaction conditions, showing considerable promise for large-scale production applications.

[0017] The egg yolk high-phosphorus protein provided in this application is derived from eggs, has no adverse effects on the human body, and is suitable for all population groups. The egg yolk high-phosphorus protein provided in this application contains a large amount of phosphorylated serine, giving it a high negative charge, which can form a complex with bovine lactoferrin, reducing the adverse effects of heat treatment on lactoferrin, enhancing its thermal stability, and maintaining its activity.

[0018] This application demonstrates that phosphoprotein in egg yolk can form a complex with bovine lactoferrin, and improves the thermal stability of lactoferrin in terms of turbidity, stability coefficient, and denaturation temperature. The results achieved in this application are significantly better than those reported in existing literature. For example, in the prior art, when bovine lactoferrin is complexed with polysaccharides such as carrageenan and xanthan gum, heating still leads to an increase in turbidity and a cloudy system, indicating that thermal denaturation of lactoferrin still occurs. In this application, however, the turbidity of the bovine lactoferrin-phosphoprotein complex solution decreases significantly before and after heating, with no obvious turbidity, indicating that the complex solution has excellent stability. Attached Figure Description

[0019] Figure 1 The thermoprotective effect of PSV on bovine lactoferrin, including (A) appearance images, where 1-6 are LF, PSV, LF:PSV = 1:1, LF:PSV = 2:1, LF:PSV = 5:1, and LF:PSV = 10:1 respectively; (B) turbidity changes before and after heat treatment; (C) stability coefficient; and (D) differential scanning calorimetry curves.

[0020] Figure 2 SDS-PAGE analysis results, where lane 1 is the marker, and lanes 2-6 are LF, PSV, LF : PSV = 1 : 1, LF : PSV = 2 : 1, LF : PSV = 5 : 1, and LF : PSV = 10 : 1, respectively.

[0021] Figure 3 (A) Zeta potentials of LF-PSV complexes with different mass ratios before and after heat treatment; (B) Fourier transform infrared spectra of LF-PSV complexes with different mass ratios.

[0022] Figure 4 The curve showing the change of heat flux (μJ / s) over time (s) is shown above, and the graph showing the change of integrated peak area (kJ / mol) over the LF / PSV molar ratio is shown below.

[0023] Figure 5 Antioxidant properties of LF-PSV complexes with different mass ratios. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0025] One embodiment of this application provides a method for preparing a bovine lactoferrin-egg yolk phosphoprotein complex, the preparation method specifically including the following steps:

[0026] Step S1 Preparation of lactoferrin stock solution: Bovine lactoferrin is dissolved in distilled water and magnetically stirred at room temperature to fully hydrate it. After stirring, the solution is filtered using a syringe filter to obtain the lactoferrin stock solution.

[0027] Step S2 Preparation of egg yolk high phosphoprotein stock solution: Dissolve egg yolk high phosphoprotein in distilled water, stir magnetically at room temperature to fully hydrate, and filter with a syringe filter after stirring to obtain egg yolk high phosphoprotein stock solution;

[0028] Step S3: pH adjustment of the protein stock solution. The pH of the lactoferrin stock solution and the egg yolk high-phosphorus protein stock solution is adjusted using 0.1 M NaOH solution or 0.1 M HCl solution, respectively.

[0029] Step S4 incubation treatment: The lactoferrin reserve solution and the egg yolk high phosphoprotein reserve solution, whose pH has been adjusted in step S3, are mixed in a certain mass ratio and then incubated at room temperature.

[0030] Step S5 involves preparing bovine lactoferrin-egg yolk high-phosphorus protein complex. The bovine lactoferrin-egg yolk high-phosphorus protein solution incubated in step S4 is freeze-dried under vacuum to obtain the bovine lactoferrin-egg yolk high-phosphorus protein complex.

[0031] In one embodiment, in step S1, the stirring time of the magnetic stirring is 10-14 h.

[0032] In one embodiment, in step S1, the needle filter is a 0.22 μm needle filter.

[0033] In one embodiment, in step S3, the pH of the protein stock solution is adjusted to 7.0 ± 0.05.

[0034] In one embodiment, in step S4, the lactoferrin reserve solution and the egg yolk phosphatase reserve solution are mixed in a certain mass ratio of 1-10:1.

[0035] In one embodiment, in step S4, the incubation conditions are magnetic stirring for 1-4 hours.

[0036] One embodiment of this application provides a lactoferrin-egg yolk phosphatase complex prepared according to the preparation method, characterized in that the mass ratio of bovine lactoferrin to egg yolk phosphatase is 1-10:1.

[0037] One embodiment of this application provides the use of the bovine lactoferrin-egg yolk phosphoprotein complex.

[0038] In one embodiment, the application is that the bovine lactoferrin-egg yolk phosphoprotein complex is used as a functional food ingredient.

[0039] In one embodiment, the application is that the bovine lactoferrin-egg yolk phosphoprotein complex is used as a cosmetic ingredient.

[0040] One embodiment of this application provides a method for preparing a bovine lactoferrin-egg yolk phosphatase complex, wherein the mass ratio of bovine lactoferrin to egg yolk phosphatase is 1-10:1, comprising the following steps:

[0041] (1) Dissolve bovine lactoferrin in distilled water and stir magnetically at room temperature to fully hydrate it. After stirring, filter the solution using a syringe filter to obtain a lactoferrin stock solution.

[0042] (2) Dissolve egg yolk high phosphoprotein in distilled water, stir magnetically at room temperature to fully hydrate, and filter with a syringe filter after stirring to obtain egg yolk high phosphoprotein stock solution.

[0043] (3) Adjust the pH of the above protein stock solution using 0.1 M NaOH solution or 0.1 M HCl solution respectively;

[0044] (4) Mix the two protein stock solutions with appropriate pH at a certain mass ratio and incubate at room temperature;

[0045] (5) After the incubated bovine lactoferrin-egg yolk high phosphoprotein solution is freeze-dried under vacuum, bovine lactoferrin-egg yolk high phosphoprotein complex can be obtained.

[0046] The working principle of this application is to confirm, through SDS-PAGE, Zeta potential, Fourier transform infrared spectroscopy, and isothermal titration calorimetry, that the formation of bovine lactoferrin-egg yolk phosphatase complex and the driving force of the complexation are the synergistic effect of electrostatic and hydrophobic interactions. The two molecules first bind initially by reducing intermolecular repulsion through electrostatic interactions, and then form a highly stable complex through hydrophobic interactions. Furthermore, the addition of PSV effectively reduces the aggregation degree of bovine lactoferrin under heat treatment, increasing its denaturation temperature by 1-16℃ and enhancing its thermal stability.

[0047] Extraction of egg yolk phosphoprotein

[0048] Carefully crack open the shell of a fresh egg and pour the egg liquid into an egg yolk separator. Transfer the yolk from the separator to filter paper and gently rotate it to remove any remaining egg white and egg bands from the yolk surface. After the yolk membrane has dried, puncture it with a toothpick or other sharp instrument and collect the yolk liquid in a beaker. Dilute 200 mL of yolk liquid with 1 L of water and adjust the pH to 5.0. Place the diluted yolk solution in an ice-water bath (4°C) and stir magnetically for 6 hours. Then, centrifuge the diluted yolk solution at 15°C and 8000 g for 60 minutes to remove soluble protein. Add 400 mL of 0.05 M NaCl solution to the collected precipitate and dissolve and stir in an ice-water bath (4°C) for 4 hours.

[0049] The solution was centrifuged at 15℃ and 11000 g for 30 min, the precipitate was collected and 400 mL of n-hexane-ethanol mixture (n-hexane: ethanol = 3:1) was added, and lipids were extracted at about 4℃ for 6 h. The precipitate was then filtered into a cake-like shape, and 200 mL of 1.74 M NaCl solution was added and extracted overnight at 4℃.

[0050] After centrifuging the solution at 15℃ and 8000 g for 20 min, the supernatant was filtered, the filtrate was dialyzed for 48 h, and then freeze-dried into powder and stored at -20℃ for later use.

[0051] Preparation of bovine lactoferrin-egg yolk phosphatidylcholine complex

[0052] Prepare LF and PSV stock solutions separately: Dissolve LF and PSV separately in distilled water to prepare a 10 mg / mL protein solution. Stir at room temperature for 12 h to fully hydrate. After stirring, filter the solutions using a 0.22 μm syringe filter to obtain the protein stock solutions.

[0053] pH adjustment: Following the pH first method (the method of adjusting the protein stock solution to the target pH before mixing), adjust the pH of the above protein stock solution to 7.0 ± 0.05 using 0.1 M NaOH solution or 0.1 M HCl solution.

[0054] Construction of LF-PSV complexes: LF and PSV solutions with appropriate pH were mixed at ratios of 1:1, 2:1, 5:1, and 10:1 (w / w) and stirred at room temperature for 1 h. The mixtures were then freeze-dried into powder to obtain LF-PSV complexes of different mass ratios, which were stored at -20℃ for later use.

[0055] Comparative example:

[0056] Control group 1 (LF group): Bovine lactoferrin solutions of a certain concentration that were not heat-treated and those that were heat-treated (heated in a 90℃ water bath for 10 min);

[0057] Control group 2 (PSV group): Egg yolk high phosphoprotein solutions of a certain concentration that were not heat-treated and those that were heat-treated (heated in a water bath at 90°C for 10 min).

[0058] The obtained complex and the above control group were characterized as follows:

[0059] Example 1: Effect of PSV on the thermal stability of bovine lactoferrin

[0060] The thermoprotective effect of PSV on bovine lactoferrin was investigated by measuring the turbidity of the bovine lactoferrin-egg yolk high-phosphorus protein complex before and after heat treatment.

[0061] Figure 1 Images showing the turbidity and appearance of the LF-PSV complex before and after heat treatment are presented. Before heat treatment, the LF solution in control group 1 was a characteristic pale pink, while the PSV solution in control group 2 was pale yellow. With increasing PSV content, the LF-PSV complex solution became increasingly pale yellow, and all solutions were clear and transparent. All complexes existed in the system as soluble compounds, indicating an interaction between LF and PSV at pH 7.0. In contrast, after heat treatment, the solution became significantly turbid, appearing milky white, due to the thermal denaturation of LF leading to molecular aggregation. Solutions with higher PSV additions remained clear, while the complex solution with a mass ratio of 10:1 (LF:PSV) became slightly turbid after heat treatment, but light could still pass through. This may be because the lower PSV addition resulted in weaker protective effects; therefore, higher PSV concentrations provide some thermal protection for LF.

[0062] like Figure 1As shown in Figure B, the turbidity of the LF solution in control group 1 was only 0.0403, which increased to 0.217 after heat treatment, indicating that LF underwent denaturation and a significant increase in aggregation. The addition of PSV in different proportions caused changes in turbidity, attributed to the formation of complexes between LF and PSV molecules. Heat treatment had almost no effect on the turbidity of PSV in control group 2, indicating that PSV has good thermal stability. The turbidity of the LF-PSV complex decreased significantly after heat treatment, and heat treatment had almost no effect on the turbidity of the complex, indicating that LF and PSV self-assemble into a complex under electrostatic interaction, which is beneficial for enhancing the thermal stability of LF and significantly reducing LF aggregation during heat treatment. However, at a ratio of 10:1 (LF:PSV), the difference in the complex before and after heat treatment was significant, compared to... Figure 1 The answer is A.

[0063] like Figure 1 As shown in Figure C, the stability coefficient of the control group 1 LF was only 61.77%, while the stability coefficients of the LF-PSV complexes (1:1, 2:1, 5:1) were 75.40%, 64.93%, and 64.90%, respectively, which is basically consistent with the DSC results. Notably, the stability coefficient of the LF:PSV = 1:1 complex (82.35%) was higher than that of the control group 2 PSV complex (68.84%), indicating that the complexes at this ratio have higher stability than LF and PSV alone. The increased stability coefficient of the LF-PSV complex may be due to the effective binding between LF and PSV inhibiting the thermal aggregation of the protein.

[0064] The denaturation temperatures of control group 1 LF, control group 2 PSV, and the LF-PSV complex were determined using differential scanning calorimetry (DSC). Figure 1 As shown in Figure D, the single endothermic peak temperatures of control group 1 LF and control group 2 PSV are 102℃ and 118.8℃, respectively, representing their denaturation temperatures. Compared with control group 1 LF, the DSC spectrum of the LF-PSV complex is similar to that of control group 1 LF, with obvious endothermic peaks and significantly increased thermal denaturation temperatures, increasing by 16.7℃, 10.19℃, 7.2℃, and 1.5℃, respectively. The LF to PSV mass ratios of 1:1, 2:1, and 5:1 showed better improvement. The denaturation temperature of the complex increased with increasing PSV addition, indicating that the addition of PSV helps improve the thermal stability of LF.

[0065] Example 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0066] To further understand whether the addition of PSV affects the stoichiometry and subunit composition of lactoferrin, SDS-PAGE analysis was performed on LF-PSV complexes with different ratios. Figure 3 (A). SDS-PAGE results showed that the major molecular weight of control group 1 LF was around 80 kDa, and the molecular weight of control group 2 PSV was around 35 kDa, consistent with results reported by other researchers. In contrast, LF-PSV complexes of different proportions showed some new bands, indicating that LF and PSV interacted to form new subunits. The complexes showed distinct bands at 16 kDa and 21 kDa, possibly indicating the formation of new small molecules from LF and PSV. Simultaneously, some large molecular bands between 100 and 250 kDa were observed, indicating the formation of some soluble aggregates.

[0067] Example 3: Effect of PSV on the Zeta potential of bovine lactoferrin

[0068] The zeta potentials of control group 1 LF, control group 2 PSV, and LF-PSV complexes with different mass ratios were measured before and after heat treatment. Figure 2 As shown, the isoelectric point of control group 1 LF is approximately 8.9, carrying a positive charge at pH 7.0. After LF and PSV recombine, the zeta potential changes from positive to negative. This is because PSV exhibits a negative potential due to its unique structure. The change in zeta potential from positive to negative indicates that PSV molecules tend to bind to the surface of LF molecules and form a complex. A reasonable hypothesis is that this results in a structure with LF as the core and PSV as the shell, similar to casein micelles. The zeta potential of the complex further decreases with increasing PSV concentration, meaning that more PSV molecules bind to the LF surface, making the system relatively more stable. The ITC results show that LF and PSV have reached saturation binding when the molar ratio of LF to PSV is 0.6:1. Therefore, at mass ratios of 2:1, 5:1, and 10:1 (corresponding to molar ratios of 1:1, 2.5:1, and 5:1), the potential of the complex gradually decreases as the proportion of PSV increases; it reaches saturation at 0.6:1. However, when the mass ratio is 1:1 (molar ratio of 0.5:1), the potential of the complex increases slightly due to the excess of LF.

[0069] Heat treatment increased the zeta potential of the control group 1 LF because protein unfolding and aggregation, with heat treatment exposing more charged groups, leading to an increase in the zeta potential. In contrast, the complex underwent conformational folding during heat treatment, causing charged amino acid residues previously exposed on the protein surface to be encapsulated within the complex. This reduced the surface charge of the protein, resulting in a decrease in the absolute value of the zeta potential, further protecting the LF molecule surrounding the center. After heat treatment, the absolute values ​​of the zeta potentials of the LF-PSV complexes were all above 25 mV. The higher electrostatic repulsion prevented intermolecular aggregation, thus increasing thermal stability.

[0070] Example 4: Fourier Transform Infrared Spectroscopy (FTIR)

[0071] FTIR spectroscopy can be used to obtain information about amide bands in proteins, including the amide I band (1600-1700 cm⁻¹). -1 Amide II band (1500 - 1600 cm) -1 This allows for further research into changes in protein conformation.

[0072] like Figure 3 As shown in Figure B, the FTIR spectra of control group 1 LF and control group 2 PSV contain the characteristic band (3000 cm⁻¹) of stretching vibrations of the OH group from free amino acids. −1 - 3300 cm −1 ), and 2800 cm – 1 - 3000 cm – 1 The absorption band is associated with the asymmetric and symmetric stretching vibrations of the CH group, but the complex shows an absorption band at 3000 cm⁻¹. – 1 The peak at -3300 cm⁻¹ showed varying degrees of shift, possibly due to hydrogen bonding between LF and PSV. The peak at 1658 cm⁻¹ can be attributed to the stretching vibration of the C=O group, but a redshift occurred in the complex; this shift can be attributed to the -COO group in the control group 2PSV. - -NH4 and control group 1 LF + Electrostatic interaction. At 1539 cm - 1 The characteristic peak at [location] originates from the bending of the N-H group and the stretching of the CN group, but a slight blue shift occurs in the LF-PSV complex, indicating a change in protein structure. Control group 1: 1066 cm⁻¹ in LF. - 1 The characteristic peak at the location is attributed to the bending of the C-H group and the stretching vibration of the N≡C or C=C group of the Trp residue, which red-shifts upon binding to PSV. This may be due to conformational rearrangement and changes in secondary structure of the protein.

[0073] Example 5: Isothermal titration calorimetry (ITC)

[0074] Thermodynamic data can be used to explore the potential interaction between LF and PSV and to predict energy exchange processes between the two substances. These results can be obtained using the isothermal titration calorimetry (ITC) method. A heatmap of heat flux versus time (obtained by titrating LF with PSV at 25 °C and pH 7.0) is shown below. Figure 5 As shown, all titration curves for the injections were exothermic. Table 1 presents the thermodynamic parameters ΔG and ΔS derived through isotherm fitting and derivation. The enthalpy change of -70.04 kJ / mol indicates that the binding of LF to PSV is an exothermic reaction, consistent with the exothermic binding behavior of large protein molecules. The Gibbs free energy of -25.489 kJ / mol indicates that the reaction is spontaneous. The interaction mode of the binding force was determined based on thermodynamic constants. A negative ΔH may be related to electrostatic interactions, while a negative ΔS indicates that other interactions (such as hydrogen bonding) and conformational changes may have occurred between LF and PSV. Therefore, when ΔH < 0 and ΔS < 0, hydrogen bonding and van der Waals forces play a dominant role. Furthermore, the dissociation constant (Kd) and binding stoichiometry n were obtained from the fitted equations. The dissociation constant is 3.463 × 10⁻⁶. -9 M, 1 / Kd (Ka) represents the binding constant (binding affinity) between the ligand and the protein; therefore, LF and PSV have a certain affinity under neutral conditions. n = 0.608 indicates that one PSV molecule binds to 0.6 LF molecules, consistent with the result of the complex's zeta potential.

[0075] Table 1. Thermodynamic parameters.

[0076] Variable Value Kd(M) 3.463E-5 ΔH (kJ / mol) -70.04 ΔS (J / mol·K) -149.5 n 0.608

[0077] Example 6 Antioxidant activity of LF-PSV complex

[0078] like Figure 5 As shown, to investigate whether the PSV complexation affects the bioactivity of LF, the antioxidant activity of the complex was determined using the DPPH free radical scavenging method. The ABTS scavenging rate of the control group 1 LF was 44.30%, while the highest ABTS scavenging rate (53.83%) was observed in the complex at a ratio of 5:1 (w / w). The ABTS scavenging rate was positively correlated with antioxidant activity, indicating that the PSV complexation does not affect the antioxidant activity of the complex. This is because the interaction between LF and PSV increases the number of amino acid residues related to antioxidant function on the surface, such as aromatic side chains. Stronger antioxidant capacity also helps maintain the structural integrity and functional properties of the protein by minimizing oxidative degradation, thus contributing to improved overall stability of the complex.

[0079] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, numerous improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a bovine lactoferrin-egg yolk phosphatase complex, characterized in that, The preparation method specifically includes the following steps: Step S1 Preparation of lactoferrin stock solution: Bovine lactoferrin is dissolved in distilled water and magnetically stirred at room temperature to fully hydrate it. After stirring, the solution is filtered using a syringe filter to obtain the lactoferrin stock solution. Step S2 Preparation of egg yolk high phosphoprotein stock solution: Dissolve egg yolk high phosphoprotein in distilled water, stir magnetically at room temperature to fully hydrate, and filter with a syringe filter after stirring to obtain egg yolk high phosphoprotein stock solution; Step S3: pH adjustment of the protein stock solution. The pH of the lactoferrin stock solution and the egg yolk high-phosphorus protein stock solution is adjusted using 0.1 M NaOH solution or 0.1 M HCl solution, respectively. Step S4 incubation treatment: The lactoferrin reserve solution and the egg yolk high phosphoprotein reserve solution, whose pH has been adjusted in step S3, are mixed in a certain mass ratio and then incubated at room temperature. Step S5 involves preparing bovine lactoferrin-egg yolk high-phosphorus protein complex. The bovine lactoferrin-egg yolk high-phosphorus protein solution incubated in step S4 is freeze-dried under vacuum to obtain the bovine lactoferrin-egg yolk high-phosphorus protein complex.

2. The method for preparing the lactoferrin-egg yolk phosphatase complex according to claim 1, characterized in that, In step S1, the stirring time of the magnetic stirrer is 10-14 h.

3. The method for preparing the lactoferrin-egg yolk high-phosphorus protein complex according to claim 1, characterized in that, In step S1, the needle filter is a 0.22 μm needle filter.

4. The method for preparing the lactoferrin-egg yolk high-phosphorus protein complex according to claim 1, characterized in that, In step S3, the pH of the protein stock solution is adjusted to 7.0 ± 0.

05.

5. The method for preparing the lactoferrin-egg yolk phosphatase complex according to claim 1, characterized in that, In step S4, the lactoferrin reserve solution and the egg yolk phosphatase reserve solution are mixed in a certain mass ratio of 1-10:

1.

6. The method for preparing the lactoferrin-egg yolk phosphatase complex according to claim 1, characterized in that, In step S4, the incubation conditions are magnetic stirring for 1-4 hours.

7. The lactoferrin-egg yolk phosphatidylcholine complex prepared by the method according to any one of claims 1-6, characterized in that, The mass ratio of bovine lactoferrin to egg yolk phosphatidylcholine is 1-10:

1.

8. The application of the bovine lactoferrin-egg yolk phosphatase complex prepared according to claim 7.

9. The application according to claim 8, characterized in that, The bovine lactoferrin-egg yolk phosphatase complex is used as a functional food ingredient.

10. The application according to claim 8, characterized in that, The bovine lactoferrin-egg yolk phosphatase complex is used as a cosmetic ingredient.

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