A beta-lactoglobulin-pyrroloquinoline quinone non-covalent complex and its use in emulsions

Abstract

The application discloses a beta-lactoglobulin-pyrrolquinoline quinone non-covalent complex and application of the beta-lactoglobulin-pyrrolquinoline quinone non-covalent complex in an emulsifying system. The beta-lactoglobulin-pyrrolquinoline quinone non-covalent complex is formed by non-covalent combination of beta-lactoglobulin and pyrrolquinoline quinone or a pharmaceutically acceptable salt of pyrrolquinoline quinone, and is prepared by the following steps: mixing a beta-lactoglobulin solution with a pyrrolquinoline quinone or salt solution, stirring, placing the mixed solution in a dialysis bag with a molecular weight cut-off of 8-14 kDa, removing free pyrrolquinoline quinone by dialysis at 0-10 DEG C for 24-72 h, and pre-freezing and freeze-drying. The beta-lactoglobulin-pyrrolquinoline quinone non-covalent compound prepared in the application realizes the synergistic optimization of the comprehensive functional characteristics of a substantial increase in emulsifying activity index, a significant reduction in surface hydrophobicity, a significant reduction in interfacial tension and a significant enhancement of antioxidant activity, and provides a new technical scheme for modifying food protein by quinone small molecules.

Description

Technical Field

[0001] This invention belongs to the fields of food science and biotechnology, specifically relating to a non-covalent complex constructed using β-lactoglobulin as a carrier and pyrroloquinoline quinone as a bioactive small molecule, and its application in emulsification systems. Background Technology

[0002] β-Lactoglobulin (β-LG) is a major component of whey protein in ruminant animals, with a molecular weight of approximately 18 kDa. Under physiological conditions, it mainly exists in dimer form. Its molecular structure includes a hydrophobic endogenous cavity and polar groups distributed on its surface. This amphiphilic structure endows β-LG with excellent emulsifying properties, making it widely applicable in food processing fields such as dairy products, baked goods, and meat products.

[0003] However, the high surface hydrophobicity of β-LG makes it highly sensitive to environmental factors such as temperature, pH, and ionic strength. Under common food processing conditions, β-LG is prone to conformational changes, aggregation, and even precipitation, which not only leads to a significant decrease in its functional properties but also greatly limits its practical application in complex food matrices (such as high-protein beverages and acidic milk systems).

[0004] Previous studies have reported that β-LG can form non-covalent complexes with various small-molecule bioactive substances (such as polyphenols, vitamins, and fatty acids). These interactions can simultaneously regulate the structural and functional properties of β-LG and improve the stability and bioavailability of the bound small molecules. For example, polyphenolic small molecules such as resveratrol, proanthocyanidins B2, and quercetin have been shown to alter the secondary structure of β-LG through non-covalent binding, thereby improving the emulsifying and other functional properties of β-LG.

[0005] However, existing β-LG-small molecule non-covalent composite technologies still have the following shortcomings: First, the improvement in emulsifying performance is limited. The improvement effect of existing composite systems on the emulsifying activity of β-LG is not ideal. The adsorption capacity of β-LG at the oil-water interface after non-covalent composite modification is not significantly enhanced, and its ability to reduce oil-water interfacial tension remains insufficient, making it difficult to meet the requirements of high emulsifying performance in food processing scenarios. Second, the improvement effect on surface hydrophobicity is poor. Surface hydrophobicity is one of the key factors determining the protein interface anchoring ability and emulsifying performance. However, the existing small molecule ligands, when combined with β-LG, have limited effect on regulating the surface hydrophobicity of β-LG, failing to effectively improve its spreading and stabilizing ability at the oil-water interface. Furthermore, there is a disconnect between the imparting of antioxidant properties and the improvement of protein function. Although existing composite systems can introduce antioxidant activity to a certain extent, this improvement in antioxidant properties often fails to form an effective linkage with the protein's own interfacial properties (emulsifying properties, surface hydrophobicity), making it difficult to achieve simultaneous optimization of the multi-dimensional functions of the composite system.

[0006] Therefore, there is an urgent need in this field to develop a novel non-covalent composite system that can simultaneously improve the emulsifying activity, surface hydrophobicity, and antioxidant properties of β-LG, and in particular, significantly reduce the oil-water interfacial tension, in order to solve the technical problems existing in the prior art. Summary of the Invention

[0007] To address the aforementioned technical problems, the purpose of this invention is to provide a non-covalent complex of β-lactoglobulin-pyrroloquinoline quinone and its preparation method. By utilizing the binding of pyrroloquinoline quinone (PQQ) with β-lactoglobulin (β-LG) to form a non-covalent complex, the interfacial properties and antioxidant activity of β-lactoglobulin are significantly improved, while also promoting the application of PQQ in food.

[0008] To achieve the above objectives, the present invention first provides a β-lactoglobulin-pyrroloquinoline quinone non-covalent complex, wherein the complex is formed by the non-covalent interaction between β-lactoglobulin and pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof, wherein the molar ratio of β-lactoglobulin to pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof is 1 to 1.05:1, wherein the molar ratio referred to herein is a binding stoichiometric ratio.

[0009] The non-covalent complex of this invention is formed by the non-covalent interaction between β-lactoglobulin and pyrroloquinoline quinone. The pyrroloquinoline quinone binds to the hydrophobic cavity of β-lactoglobulin through hydrogen bonds and / or hydrophobic interactions. The secondary structure of β-lactoglobulin in the complex undergoes rearrangement, characterized by a decrease in α-helix content and an increase in β-sheet content, while simultaneously exhibiting a significant decrease in surface hydrophobicity.

[0010] In one embodiment of the present invention, the α-helix content of β-lactoglobulin in the non-covalent complex is 10%-16%, and the β-sheet content is 40%-46%.

[0011] In one embodiment of the present invention, the key amino acid residues for the binding of pyrroloquinoline quinone to β-lactoglobulin include Leu10, Ile12, Gln13, Pro50, Gly52, Glu51, Lys75, and Pro79.

[0012] In one embodiment of the present invention, the complex exhibits superior overall functional properties, wherein the emulsifying activity index reaches 108.64 ± 1.48 m at 2.0 mg / mL. 2 / g, the surface hydrophobicity is reduced by more than 51.36% compared with natural β-lactoglobulin, the DPPH free radical scavenging rate is increased by more than 2 times compared with natural β-lactoglobulin, and the initial tension of the air-water interface is reduced to 40.68mN / m.

[0013] The present invention also provides a method for preparing the above-mentioned β-lactoglobulin-pyrroloquinoline quinone non-covalent complex, comprising the following steps: S1. Mix the β-lactoglobulin solution with a solution of pyrroloquinoline quinone or its salt, and stir to form a mixed solution. S2. Dialyze the mixed solution obtained in step S1 to remove unbound free pyrroloquinoline quinone molecules, pre-freeze and freeze-dry the dialyzed solution, and collect the freeze-dried powder.

[0014] In one embodiment of the present invention, the β-lactoglobulin solution is prepared by dissolving β-lactoglobulin in a buffer solution, and the mass concentration of β-lactoglobulin in the mixed solution is 10-40 mg / mL.

[0015] In one embodiment of the present invention, the pyrroloquinoline quinone or its salt solution is prepared by dissolving pyrroloquinoline quinone or its salt in a buffer solution, and the mass concentration of pyrroloquinoline quinone or its salt is 0.5-4.0 mg / mL.

[0016] In one embodiment of the present invention, the buffer solution is a phosphate buffer solution with a pH of 6.5-7.5 and a concentration of 0.005-0.02 mol / L.

[0017] In one embodiment of the present invention, the volume ratio of the β-lactoglobulin solution to the pyrroloquinoline quinone or its salt solution is 1 to 5:1.

[0018] In one embodiment of the present invention, the stirring temperature is 20-35℃, the stirring speed is 100-600 r / min, and the stirring time is 0.5-6 h.

[0019] In one embodiment of the present invention, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialysis is performed at 0-10°C for 24-72 hours.

[0020] In one embodiment of the present invention, the pre-freezing temperature is -196℃ to -40℃, and the pre-freezing time is 2-24h; the freeze-drying temperature is -60℃ to -30℃, the vacuum degree is 0.01-1mbar, and the freeze-drying time is 8-48h.

[0021] The present invention also provides an application of the above-mentioned β-lactoglobulin-pyrroloquinoline quinone non-covalent complex in the food field, the application including use as a bioactive carrier in functional foods, an emulsifier and stabilizer in emulsified beverages, or a nutritional fortification additive in functional foods.

[0022] In one embodiment of the present invention, the application is as an emulsifier for preparing a functional emulsion encapsulating a fat-soluble active ingredient, the emulsion having good physical stability and storage stability.

[0023] In one embodiment of the present invention, the food includes emulsified beverages, fortified foods, or sports nutrition foods.

[0024] The beneficial effects are: (1) This invention utilizes pyrroloquinoline quinone or its pharmaceutically acceptable salts to react with β-lactoglobulin to form a non-covalent complex. Because pyrroloquinoline quinone binds to the endogenous hydrophobic cavity of β-lactoglobulin in an approximately 1:1 stoichiometric ratio, it forms hydrogen bonds and hydrophobic interactions with residues such as Leu10, Ile12, Gln13, Pro50, and Gly52. This binding triggers a conformational rearrangement of β-lactoglobulin from α-helix to β-sheet, significantly enhancing the adsorption capacity and interfacial membrane stability of the complex at the oil-water interface. The β-lactoglobulin-pyrroloquinoline quinone non-covalent complex prepared by this invention also achieves a significant increase in the emulsifying activity index (reaching 108.64 ± 1.48 m). 2 The synergistic optimization of comprehensive functional properties, including significantly reduced surface hydrophobicity (by 51.36%), significantly reduced interfacial tension (equilibrium interfacial tension of approximately 10 mN / m), and significantly enhanced antioxidant activity, provides a novel technical solution for modifying food proteins with quinone small molecules.

[0025] (2) In this invention, the encapsulation of pyrroloquinoline quinone by the hydrophobic cavity of β-lactoglobulin can protect the active structure of pyrroloquinoline quinone to a certain extent. At the same time, the antioxidant activity of pyrroloquinoline quinone and the exposure of endogenous antioxidant residues of β-lactoglobulin (such as Tyr, Cys, etc.) produce a synergistic effect, thereby enhancing the overall free radical scavenging ability of the complex.

[0026] (3) This invention employs a mild non-covalent bonding process, which requires no chemical cross-linking agents or high-temperature treatment. The preparation process is green and safe, simple to operate, and the conditions are controllable, making it suitable for industrial-scale production. The non-covalent bonding method avoids the irreversible destruction of the protein's natural conformation by chemical modification, preserves the safety of β-lactoglobulin as a food protein, and has good prospects for industrial application.

[0027] (4) Compared with other non-covalent complexes such as β-lactoglobulin-quercetin and β-lactoglobulin-ascorbic acid, the β-lactoglobulin-pyrroloquinoline quinone complex of the present invention exhibits superior comprehensive functional properties under the same preparation conditions. This advantage is closely related to the directional conformational rearrangement (α-helix → β-sheet) induced by the insertion of pyrroloquinoline quinone into the hydrophobic cavity of β-lactoglobulin. Attached Figure Description

[0028] Figure 1 The circular dichroism spectral analysis results of the samples from Examples 1-5 and Comparative Example 1 are shown; where A is the CD spectrum and B is the graph showing the change in secondary structure content. Figure 2 The intrinsic fluorescence spectrum changes of β-lactoglobulin in the β-lactoglobulin-pyrroloquinoline quinone complexes of Examples 1-5 are shown in (A), and the curves showing the changes in the maximum fluorescence emission wavelength (λmax) and fluorescence intensity of β-lactoglobulin in the complexes with increasing pyrroloquinoline quinone concentration are shown in (B). Figure 3 The temperature-dependent fluorescence spectra (A-C) of the β-lactoglobulin-pyrroloquinoline quinone complexes of Examples 1-5 at 298K, 308K, and 318K, as well as the Stern-Volmer plot (F0 / F vs [Q]) (D), the double logarithmic plot (log[(F0-F) / F] vs log[Q]) (E), and the Van't Hoff plot (lnKa vs 1 / T) (F) are shown. Figure 4 The results show the molecular docking between pyrroloquinoline quinone and β-lactoglobulin; where A is a two-dimensional binding mode diagram and B is a three-dimensional binding mode diagram. Figure 5 The results are from a 100 ns molecular dynamics simulation of the β-lactoglobulin-pyrroloquinoline quinone complex; where A is the RMSD plot, B is the RMSF plot, C is the Rg plot, and D is the SASA plot. Figure 6 The graph shows a comparison of the emulsifying activity index (EAI) and emulsifying stability index (ESI) of the complexes of Examples 1-5 and Comparative Examples 1-3. Figure 7 The graph shows a comparison of the surface hydrophobicity (H0) results of the composites in Examples 1-5 and Comparative Examples 1-3. Figure 8The diagram shows the dynamic changes in the gas-water interfacial tension of the complexes in Examples 1-5 and Comparative Examples 1-3. Figure 9 DPPH and ABTS of the complexes of Examples 1-5 and Comparative Examples 1-3 + Free radical scavenging rate comparison chart; Figure 10 This diagram illustrates the binding mechanism, structural features, and application areas of the β-lactoglobulin-pyrroloquinoline quinone non-covalent complex of the present invention. Detailed Implementation

[0029] This invention discloses a β-lactoglobulin-pyrroloquinoline quinone non-covalent complex and its application in emulsion systems. The invention is further illustrated below with specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, all raw materials are commercially available, and all testing methods are conventional methods.

[0030] The β-lactoglobulin (purity ≥95%, CAS: 9045-23-2) used in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; disodium pyrroloquinoline quinone (purity ≥99%, CAS: 122628-50-6) was purchased from Huaxi Biotechnology Co., Ltd.; phosphate buffer (0.01 mol / L, pH 7.0) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; L-ascorbic acid (vitamin C, CAS: 50-81-7, purity ≥99%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; quercetin (CAS: 117-39-5, purity ≥98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; all other reagents were of analytical grade.

[0031] The dialysis bags with a molecular weight cutoff of 8-14 kDa used in the embodiments and comparative examples of this invention are dialysis bags MD44, purchased from Beijing Solarbio Technology Co., Ltd.

[0032] Structural characterization: 1. Circular dichroism spectroscopy analysis The secondary structure changes of β-lactoglobulin and the β-lactoglobulin-pyrroloquinoline quinone complex were detected using a Jasco J-810 circular dichroism spectrometer (JASCO Corporation, Japan). The β-lactoglobulin concentration in the sample solution was 0.1 mg / mL, and the solution was placed in a quartz cuvette with a 1 mm path length. The scanning wavelength range was 190–260 nm. The secondary structure content of β-lactoglobulin was calculated using CDNN software.

[0033] 2. Fluorescence spectroscopy and thermodynamic analysis: Fluorescence quenching behavior was detected using an FS-4600 fluorescence spectrophotometer (Hitachi Shimadzu Corporation, Japan). The excitation wavelength was 290 nm, and the emission scanning range was 310–450 nm. Detection was performed at three temperatures: 298 K, 308 K, and 318 K.

[0034] 3. Molecular docking and molecular dynamics simulation Molecular docking was performed using AutoDock Vina software. The β-lactoglobulin crystal structure (PDB ID: 1BEB) was preprocessed with PyMOL, and a global search for docking was conducted with grid center coordinates of x=-11.03, y=12.62, z=-8.24 (Å). A 100 ns molecular dynamics simulation of the β-lactoglobulin-pyrroloquinolinequinone complex was performed using Gromacs 2024.2 software. The force field parameters for pyrroloquinolinequinone were represented by the GAFF force field, for β-lactoglobulin by the Amber99sb force field, and the solvent was represented by the TIP3P water model.

[0035] Functional characteristics evaluation: 1. Emulsification performance testing 15 mL of a 0.1% protein solution was mixed with 5 mL of soybean oil and homogenized at 12000 rpm for 1 min to prepare an emulsion. Samples were taken at 0 min and 10 min after homogenization, mixed with 0.1% SDS solution, and the absorbance was measured at 500 nm. The emulsifying activity index (EAI) and emulsifying stability index (ESI) were calculated.

[0036] 2. Surface hydrophobicity test Surface hydrophobicity (H0) was determined using the ANS fluorescent probe method. Samples were prepared into solutions with concentrations ranging from 0.005 to 0.5 mg / mL. 4 mL of each solution was added to 20 μL of 8.0 mM ANS solution (PBS, pH 7.0), and the mixture was reacted in the dark for 15 min. Excitation wavelength was 390 nm, and emission wavelength was 470 nm to measure fluorescence intensity. A graph was plotted between relative fluorescence intensity and protein concentration; the slope of the initial straight line represents the H0 value.

[0037] 3. Antioxidant activity detection Using DPPH and ABTS + Antioxidant activity was evaluated using a free radical scavenging assay. DPPH free radical scavenging rate: An equal volume of sample solution was mixed with DPPH ethanol solution, and the mixture was reacted at 25°C in the dark for 30 min. The absorbance was then measured at 517 nm. ABTS + Free radical scavenging rate: An equal volume of ABTS stock solution and potassium persulfate solution were mixed and allowed to stand at room temperature in the dark for 15 hours to prepare a stable free radical working solution. A small amount of the sample solution was then mixed with ABTS. +After mixing the working solutions and reacting in the dark for 6 minutes, the absorbance was measured at 734 nm, and the final removal efficiency was calculated using the corresponding formula.

[0038] 4. Interfacial tension detection The interfacial tension between air and water was measured using an OCA-25 optical contact angle meter.

[0039] Example 1: A method for preparing a β-lactoglobulin-pyrroloquinoline quinone nonvalent complex includes the following steps: S1. Dissolve β-lactoglobulin in 0.01 mol / L, pH 7.0 PBS buffer to prepare β-lactoglobulin stock solution with a concentration of 40 mg / mL; dissolve disodium pyrroloquinoline quinone in the same buffer to prepare pyrroloquinoline quinone stock solution with a concentration of 4.0 mg / mL.

[0040] S2. Mix the β-lactoglobulin stock solution and the pyrroloquinoline quinone stock solution at a 1:1 volume ratio to obtain a mixed solution, wherein the final concentration of β-lactoglobulin is 20 mg / mL and the final concentration of pyrroloquinoline quinone is 2.0 mg / mL. Stir magnetically at 300 rpm for 2 hours at 25°C to ensure thorough mixing of pyrroloquinoline quinone and β-lactoglobulin and the formation of a non-covalent complex.

[0041] S3. Transfer the mixed solution obtained in step S3 into a dialysis bag with a molecular weight cutoff of 8-14 kDa. After sealing the bag, immerse it in PBS buffer (0.01 mol / L, pH 7.0) and dialyze at 4°C for 48 h. Replace the external solution every 12 h during this period to fully remove unbound free pyrroloquinoline quinone molecules.

[0042] S4. The dialyzed solution was pre-frozen at -80℃ for 12 hours, and then freeze-dried in a vacuum freeze dryer at -50℃ and 0.1mbar for 24 hours. The freeze-dried powder was then collected.

[0043] Example 2 The difference between Example 2 and Example 1 is that the final concentration of pyrroloquinoline quinone in step S2 is 0.5 mg / mL.

[0044] Example 3 The difference between Example 3 and Example 1 is that the final concentration of pyrroloquinoline quinone in step S2 is 1.0 mg / mL.

[0045] Example 4 The difference between Example 4 and Example 1 is that the final concentration of pyrroloquinoline quinone in step S2 is 1.5 mg / mL.

[0046] Example 5 The difference between Example 5 and Example 1 is that the final concentration of pyrroloquinoline quinone in step S2 is 2.5 mg / mL.

[0047] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that, in step S2, pyrroloquinoline quinone stock solution is not added; instead, β-lactoglobulin stock solution is mixed with an equal volume of PBS buffer. The remaining steps are the same as in Example 1.

[0048] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that pyrroloquinoline quinone disodium salt was replaced with quercetin. Quercetin was dissolved in the same buffer solution to prepare a quercetin stock solution with a concentration of 4.0 mg / mL, which was then mixed with β-lactoglobulin stock solution at a 1:1 volume ratio, resulting in a final concentration of quercetin of 2.0 mg / mL.

[0049] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the disodium pyrroloquinoline quinone was replaced with L-ascorbic acid. L-ascorbic acid was dissolved in the same buffer solution to prepare an L-ascorbic acid stock solution with a concentration of 4.0 mg / mL. This stock solution was then mixed with β-lactoglobulin stock solution at a 1:1 volume ratio, resulting in a final concentration of 2.0 mg / mL for L-ascorbic acid.

[0050] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that, in step S2, β-lactoglobulin stock solution was not added; instead, pyrroloquinoline quinone stock solution was mixed with an equal volume of PBS buffer, resulting in a final concentration of pyrroloquinoline quinone of 2.0 mg / mL. The remaining steps were the same as in Example 1.

[0051] Figure 1 The circular dichroism spectral analysis results of the samples from Examples 1-5 and Comparative Example 1 are shown below. Figure 1 It can be seen that the α-helix content of natural β-lactoglobulin is 17.00%, and when the concentration of pyrroloquinoline quinone reaches 2.5 mg / mL, this value decreases to 13.01%. Figure 1 (B) Conversely, with increasing pyrroloquinoline quinone concentration, the β-sheet content increased from an initial 40.25% to 43.10%, while the β-turn and random coil contents remained relatively unchanged. These results indicate that pyrroloquinoline quinone binding triggers a rearrangement of the secondary structure of β-lactoglobulin, resulting in a transition from α-helices to β-sheets, which is attributed to changes in the intramolecular hydrogen bonding and hydrophobic interaction network.

[0052] Figure 2 The intrinsic fluorescence spectra of β-lactoglobulin in the β-lactoglobulin-pyrroloquinoline quinone complex in Examples 1-5 are presented, along with curves showing the changes in the maximum fluorescence emission wavelength (λmax) and fluorescence intensity of β-lactoglobulin in the complex with increasing pyrroloquinoline quinone concentration. Figure 2 As can be seen, under 290 nm excitation, native β-lactoglobulin exhibits a strong fluorescence emission peak at 333 nm. At 298 K, 308 K, and 318 K, the fluorescence intensity gradually decreases with increasing pyrroloquinoline quinone concentration. With the addition of pyrroloquinoline quinone, the maximum fluorescence emission wavelength of β-lactoglobulin redshifts to 341 nm. Figure 2 A) indicates that the fluorescence quenching of β-lactoglobulin by pyrroloquinoline quinone is a static quenching mechanism, and that the microenvironment of hydrophobic amino acid residues in β-lactoglobulin becomes more polar, with more residues exposed to the aqueous phase.

[0053] Figure 3 (AF) presents the temperature-dependent fluorescence spectra of the non-covalent complex β-lactoglobulin-pyrroloquinolinequinone complex at 298 K, 308 K, and 318 K, along with Stern-Volmer plots (F0 / F vs [Q]), double logarithmic plots (log[(F0-F) / F] vs log[Q]), and Van't Hoff plots (lnKa vs 1 / T). Figure 3 As can be seen from the data, the quenching rate constant Kq is 1.677 × 10⁻⁶ at 298 K, 308 K, and 318 K, respectively. 11 1.432×10 11 and 1.134×10 11 L / mol·s, both are much higher than the maximum dynamic quenching constant (2.0×10). 10 The concentration of β-lactoglobulin (L / mol·s) confirmed the formation of a ground-state non-covalent complex. Double logarithmic curve fitting confirmed that the binding stoichiometry (n) between β-lactoglobulin and pyrroloquinoline quinone was 1.02 (approximately 1:1).

[0054] Table 1. Fluorescence quenching and thermodynamic parameters of the β-lactoglobulin-pyrroloquinoline quinone complex at different temperatures.

[0055] from Figure 3 The thermodynamic results in F and Table 1 show that the non-covalent complex has ΔH = -1.84 kJ / mol and ΔS = 0.0418 kJ / (mol·K). At 298 K, 308 K, and 318 K, ΔG is -14.09, -14.51, and -14.93 kJ / mol, respectively, proving that the bonding process is spontaneously exothermic, with hydrophobic interactions as the main driving force.

[0056] Molecular docking results show ( Figure 4The binding energy of pyrroloquinoline quinone to β-lactoglobulin is -6.4 kcal / mol. Pyrroloquinoline quinone binds to the hydrophobic cavity of β-lactoglobulin, forming multiple hydrogen bonds with Leu10, Ile12, Gln13, Gly52, and Pro50, and undergoing hydrophobic interactions with Pro79, Lys75, Ile12, Gly52, and Pro50. Among these, Ile12, Gly52, and Pro50 are bifunctional residues, participating in both types of interactions, thus enhancing binding stability.

[0057] Molecular dynamics simulation results ( Figure 5 The RMSD of the β-lactoglobulin-pyrroloquinoline quinone complex fluctuated in the range of 0.18–0.33 nm. Figure 5 A) indicates increased conformational flexibility, which is beneficial for structural rearrangement. RMSF analysis shows that the complex exhibits structural flexibility fluctuations in loop regions such as residues 30-40 and 110-120. Figure 5 (B) This phenomenon is consistent with the inherent flexibility of the N-terminal and C-terminal loop regions of β-LG; identifiable local fluctuations also occurred at residues 13, 52, and 75 of the PQQ binding site, indicating that ligand binding triggered a local conformational adjustment of the binding pocket. The cyclotron radius (Rg) stabilized at approximately 1.42 nm. Figure 5 C) Solvent-accessible surface area (SASA) fluctuates between 88 and 92 nm. 2 ( Figure 5 D) indicates that the complex has a compact and stable structure. The binding free energy calculated by MM-PBSA is -16.97 kcal / mol, and the number of hydrogen bonds remained stable at about 10 throughout the simulation, confirming the dynamic stability of the complex.

[0058] Figure 6 The experimental results showed that the EAI of natural β-lactoglobulin was low, and the EAI increased significantly with increasing pyrroloquinoline quinone concentration, reaching a maximum of 108.64 ± 1.48 mg / mL at pyrroloquinoline quinone. 2 / g, ESI also showed a trend of first increasing and then decreasing. The improved emulsifying performance is attributed to the enhanced adsorption capacity of the β-lactoglobulin-pyrroloquinoline quinone complex at the oil-water interface and the improved interfacial film stability.

[0059] Figure 6 The emulsifying properties and emulsifying stability of the complexes from Example 1 and Comparative Examples 1-3 were also compared. The results showed that the EAI of native β-lactoglobulin was 59.08 ± 3.38 m. 2 / g, ESI was 35.78±2.21min; β-lactoglobulin-ascorbic acid ESI was 75.42±2.33m 2 / g, ESI was 43.81±2.36min; β-lactoglobulin-quercetin ESI was 86.72±1.17m 2 / g, ESI was 56.35±2.75min; β-lactoglobulin-pyrroloquinoline quinone's ESI reached 108.64±1.48m. 2 / g, ESI reached 62.76±2.24min. The ESI of β-lactoglobulin-pyrroloquinoline quinone was 1.25 times that of β-lactoglobulin-quercetin, 1.44 times that of β-lactoglobulin-ascorbic acid, and 1.84 times that of natural β-lactoglobulin, respectively. The ESI also showed the same trend, and the differences were all statistically significant. p<0.01 Free pyrroloquinoline quinone itself lacks emulsifying activity and cannot form stable emulsions, proving that the improvement in EAI and ESI does not originate from the physicochemical properties of pyrroloquinoline quinone itself. These results further indicate that after pyrroloquinoline quinone binds to β-lactoglobulin in a 1:1 molar ratio and embeds into the hydrophobic cavity, it induces a directional conformational rearrangement of β-lactoglobulin (α-helix → β-sheet), significantly enhancing the protein molecule's adsorption capacity at the oil-water interface and the stability of the interfacial membrane.

[0060] Figure 7 The hydrophobicity results for each sample are presented, from... Figure 7 It can be seen that the H0 value of natural β-lactoglobulin is 8870.67±286.33, which gradually decreases with increasing pyrroloquinoline quinone concentration, decreasing to 4314.37±128.67 at 2.0 mg / mL pyrroloquinoline quinone, a decrease of 51.36% compared to natural β-lactoglobulin. The significant reduction in surface hydrophobicity indicates that pyrroloquinoline quinone is embedded inside the hydrophobic cavity of β-lactoglobulin, effectively shielding internal hydrophobic sites and reducing hydrophobic aggregation between protein molecules.

[0061] from Figure 7 The experimental results in Table 2 also show that the H0 value of natural β-lactoglobulin was 8870.67±286.33, the H0 value of β-lactoglobulin-ascorbic acid was 6406.53±176.73, the H0 value of β-lactoglobulin-quercetin was 5537.93±252.43, and the H0 value of β-lactoglobulin-pyrroloquinoline quinone was 4314.37±128.67. The H0 reduction of β-lactoglobulin-pyrroloquinoline quinone was 51.36%, significantly higher than that of β-lactoglobulin-quercetin (37.57%) and β-lactoglobulin-ascorbic acid (27.78%), and the differences were all statistically significant. p<0.01Ascorbic acid, being highly hydrophilic, primarily binds to the hydrophilic regions of β-lactoglobulin, having limited influence on sites within the hydrophobic cavity. While quercetin is hydrophobic, its binding site differs from that of pyrroloquinoline quinone, failing to effectively shield the core hydrophobic sites within the cavity. Pyrroloquinoline quinone embeds itself within the hydrophobic cavity of β-lactoglobulin, specifically binding to residues such as Leu10, Ile12, Gln13, Pro50, and Gly52, resulting in thorough encapsulation of internal hydrophobic groups and a significant reduction in the exposed hydrophobic surface area. These results further confirm that the specific binding of pyrroloquinoline quinone to the hydrophobic cavity sites of β-lactoglobulin and its directional conformational rearrangement are the reasons for the significant reduction in surface hydrophobicity of the β-lactoglobulin-pyrroloquinoline quinone complex.

[0062] Figure 8 The interfacial tension results for each sample are presented. The initial interfacial tension of natural β-lactoglobulin was 50.18 mN / m, which decreased to 40.68 mN / m at a pyrroloquinoline quinone concentration of 2.0 mg / mL, indicating that the adsorption rate of the β-lactoglobulin-pyrroloquinoline quinone complex at the gas-liquid interface is accelerated, which is beneficial to improving emulsifying performance. Figure 8 The experimental results in Table 2 also show that the initial interfacial tension of natural β-lactoglobulin was 50.18 mN / m, which gradually decreased over time, reaching equilibrium after 3600 s, with an equilibrium interfacial tension of approximately 18 mN / m. The initial interfacial tensions of β-lactoglobulin-quercetin and β-lactoglobulin-ascorbic acid were 44.16 mN / m and 46.67 mN / m, respectively, with equilibrium interfacial tensions of approximately 13 mN / m and 12 mN / m, respectively. The initial interfacial tension of β-lactoglobulin-pyrroloquinoline quinone was 40.68 mN / m, and its rate of decrease within 0-500 s was significantly higher than that of other samples. After 3600 s, the equilibrium interfacial tension decreased to approximately 10 mN / m, significantly lower than that of natural β-lactoglobulin, β-lactoglobulin-quercetin, and β-lactoglobulin-ascorbic acid. p<0.01 The interfacial tension of the free pyrroloquinoline quinone solution is close to that of pure water (approximately 71.8 mN / m), proving that it does not possess surface activity. These results indicate that the adsorption rate and amount of the β-lactoglobulin-pyrroloquinoline quinone complex at the gas-liquid interface are significantly superior to those of natural β-lactoglobulin and other non-covalent complexes, with the largest reduction in interfacial tension. This is closely related to the significant decrease in surface hydrophobicity, increased molecular flexibility, and conformational rearrangement of the β-lactoglobulin-pyrroloquinoline quinone complex, further confirming that the β-lactoglobulin-pyrroloquinoline quinone complex possesses optimal interfacial activity and emulsification potential.

[0063] Figure 9 Antioxidant activity results for Examples 1-5 and Comparative Examples 1-3 are presented. The DPPH radical scavenging rate of natural β-lactoglobulin (Comparative Example 1) was 17.09 ± 0.65%, and the ABTS... +The free radical scavenging rate was 26.94 ± 0.99%. The free radical scavenging rates of β-lactoglobulin-quercetin (Comparative Example 2) and β-lactoglobulin-ascorbic acid (Comparative Example 3) were higher than those of natural β-lactoglobulin, but the increase was related to the antioxidant activity of the ligands themselves, and did not show a continuous upward trend with increasing ligand concentration. The free radical scavenging rate of the β-lactoglobulin-pyrroloquinoline quinone complex (Examples 1-5) increased monotonically with increasing pyrroloquinoline quinone concentration. At 2.0 mg / mL pyrroloquinoline quinone, the DPPH scavenging rate was 79.61 ± 0.36%, ABTS + The scavenging rate was 83.80 ± 0.56%. This antioxidant activity is not a simple addition of pyrroloquinoline quinone: the Tyr20 and Tyr42 residues exposed by the conformational rearrangement of β-lactoglobulin synergistically interact with pyrroloquinoline quinone, and its redox stability is enhanced after embedding into the hydrophobic cavity. Although free pyrroloquinoline quinone solutions possess free radical scavenging capabilities, their water solubility limits their application in oil-phase systems, restricting their use in food. These results indicate that the β-lactoglobulin-pyrroloquinoline quinone complex confers antioxidant activity while maintaining protein carrier function, and this antioxidant activity is closely related to the complex structure, rather than being caused by the simple release of free ligands.

[0064] Table 2. Emulsifying properties, emulsion stability, hydrophobicity, gas-water interfacial tension, and antioxidant data of samples from Example 1 and Comparative Examples 1-3.

[0065] Note: Free PQQ is a water-soluble small molecule with no interfacial activity and cannot form a stable emulsion, so EAI and ESI are meaningless.

[0066] Application Example 1: Application of the β-lactoglobulin-pyrroloquinoline quinone complex in O / W emulsions Medium-chain triglycerides (MCTs) were used as the oil phase. The β-lactoglobulin-pyrroloquinoline quinone complex prepared in Example 1 was dissolved in 10 mM phosphate buffer (pH 7.0) and swollen overnight to form an aqueous phase with a complex concentration of 20 g / L. The oil phase was slowly added to the aqueous phase (water to oil volume ratio 10:1) under stirring in a high-speed emulsifier and sheared at 10,000 r / min for 6 min to form a crude emulsion. The crude emulsion was further homogenized using a high-pressure homogenizer with two-stage homogenization, three times. The first homogenization pressure was 60 MPa, and the second homogenization pressure was 6 MPa, to obtain an O / W type emulsion.

[0067] Application Comparative Example 1: Preparation of Natural β-Lactoglobulin Emulsion The β-lactoglobulin-pyrroloquinoline quinone complex in Application Example 1 was replaced with natural β-lactoglobulin prepared in Comparative Example 1, and all other conditions were the same as in Application Example 1.

[0068] Comparative Example 2: Preparation of β-lactoglobulin-ascorbic acid emulsion The complex in Application Example 1 was replaced with the β-lactoglobulin-quercetin complex prepared in Comparative Example 2, and the other conditions were the same as in Application Example 1.

[0069] Comparative Example 3: Preparation of β-lactoglobulin-quercetin emulsion The complex in Application Example 1 was replaced with the β-lactoglobulin-ascorbic acid complex prepared in Comparative Example 3, and the other conditions were the same as in Application Example 1.

[0070] The four emulsions were quickly transferred to brown bottles and stored under nitrogen cover in the dark. The average particle size, PDI, and Zeta potential of the freshly prepared emulsions and those stored at 25°C in the dark for 30 days were measured using a laser particle size analyzer, and the particle size change rate was calculated. Each sample was measured in triplicate.

[0071] The results are shown in Table 3. The stable emulsion of the β-lactoglobulin-pyrroloquinoline quinone complex had the smallest fresh particle size (239.6±0.9 nm), the lowest PDI (0.142±0.002), and the largest absolute value of the Zeta potential (-31.6±0.2 mV). After 30 days of storage, the particle size of the β-lactoglobulin-pyrroloquinoline quinone group increased to 251.2±0.8 nm, with a particle size change rate of only 4.84±0.33%, significantly lower than that of the natural β-lactoglobulin group (19.85±0.27%), the β-lactoglobulin-ascorbic acid group (15.10±0.35%), and the β-lactoglobulin-quercetin group (10.14±0.34%). p<0.01 ).

[0072] Table 3 Physicochemical properties and storage stability of O / W emulsions prepared with different emulsifiers

[0073] The results of the above application examples demonstrate that the β-lactoglobulin-pyrroloquinoline quinone complex still exhibits the best emulsifying stability in emulsion systems. Its fresh emulsion has the smallest particle size and most uniform distribution, and its particle size change rate after 30 days of storage is significantly lower than that of other groups, proving that the interfacial film formed by this complex is the most dense and stable. This further verifies the macroscopic physical stability advantage of the conformational rearrangement induced by pyrroloquinoline quinone embedding into the hydrophobic cavity of β-lactoglobulin, which can be directly converted into a real emulsion system.

[0074] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A β-lactoglobulin-pyrroloquinoline quinone nonvalent complex, characterized in that, The complex is formed by the non-covalent interaction between β-lactoglobulin and pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof, wherein the molar ratio of β-lactoglobulin to pyrroloquinoline quinone or a pharmaceutically acceptable salt thereof is 1 to 1.05:1, and the pyrroloquinoline quinone is bound to the hydrophobic cavity of β-lactoglobulin through hydrogen bonds and / or hydrophobic interactions.

2. The β-lactoglobulin-pyrroloquinoline quinone nonvalent complex according to claim 1, characterized in that, The non-covalent complex contains 10-16% α-helix and 40-46% β-sheet of β-lactoglobulin. The key amino acid residues for the binding of pyrroloquinoline quinone to β-lactoglobulin include Leu10, Ile12, Gln13, Pro50, Gly52, Glu51, Lys75, and Pro79.

3. A method for preparing the β-lactoglobulin-pyrroloquinoline quinone non-covalent complex according to claim 1 or 2, characterized in that, Includes the following steps: S1. Mix the β-lactoglobulin solution with a solution of pyrroloquinoline quinone or its salt, and stir to form a mixed solution. S2. Dialyze the mixed solution obtained in step S1 to remove unbound free pyrroloquinoline quinone molecules, pre-freeze and freeze-dry the dialyzed solution, and collect the freeze-dried powder.

4. The preparation method according to claim 3, characterized in that, In step S1, the β-lactoglobulin solution is prepared by dissolving β-lactoglobulin in a buffer solution, and the mass concentration of β-lactoglobulin in the mixed solution is 10-40 mg / mL. The pyrroloquinoline quinone or its salt solution is prepared by dissolving pyrroloquinoline quinone or its salt in a buffer solution, and the mass concentration of pyrroloquinoline quinone or its salt is 0.5-4.0 mg / mL. The buffer solution is a phosphate buffer with a pH of 6.5-7.5 and a concentration of 0.005-0.02 mol / L.

5. The preparation method according to claim 3, characterized in that, In step S1, the volume ratio of the β-lactoglobulin solution to the pyrroloquinoline quinone or its salt solution is 1 to 5:

1.

6. The preparation method according to claim 3, characterized in that, In step S1, the stirring temperature is 20-35℃, the stirring speed is 100-600 r / min, and the stirring time is 0.5-6 h.

7. The preparation method according to claim 3, characterized in that, In step S2, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialysis is performed at 0-10℃ for 24-72 hours.

8. The preparation method according to claim 3, characterized in that, In step S2, the pre-freezing temperature is -196℃ to -40℃, and the pre-freezing time is 2-24h; the freeze-drying temperature is -60℃ to -30℃, the vacuum degree is 0.01-1mbar, and the freeze-drying time is 8-48h.

9. The application of the β-lactoglobulin-pyrroloquinoline quinone non-covalent complex according to claim 1 or 2 in the food field, characterized in that, The applications include use as a bioactive carrier in functional foods, an emulsifier and stabilizer in emulsified beverages, or a nutritional fortification additive in functional foods.

10. The application according to claim 9, characterized in that, The application is as an emulsifier for preparing functional emulsions that encapsulate fat-soluble active ingredients.

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