Protein-polyphenol-polysaccharide ternary covalent complex as well as preparation method and application thereof

Abstract

The invention relates to the technical field of preparation of Pickering emulsion stabilizers, in particular to a protein-polyphenol-polysaccharide ternary covalent complex as well as a preparation method and application thereof. The method comprises the following steps: step 1, preparing a whey protein isolate aqueous solution, completely hydrating the whey protein isolate aqueous solution, compounding a grape seed oligomeric proanthocyanidins aqueous solution and a hydrated protein aqueous solution, adjusting the pH value, and carrying out stirring reaction and dialysis freeze-drying to prepare a protein-polyphenol binary compound; and 2, compounding the binary compound solution with the lycium barbarum polysaccharide solution, uniformly stirring, carrying out Maillard reaction to obtain a ternary compound solution, and freeze-drying to obtain the protein-polyphenol-polysaccharide ternary compound. The invention provides a non-toxic, harmless and pollution-free natural emulsifier and stabilizer, and simultaneously prepares a Chinese wolfberry seed oil Pickering emulsion capable of loading nutritional ingredients and active substances, thereby providing reference for screening research of the natural emulsifier and protection of nutritional ingredients of Chinese wolfberry seed oil, which are easy to photodecompose and oxidize; the problem that the effect of protein serving as a Pickering emulsion stabilizer in the prior art is poor is solved.

Description

Technical Field

[0001] This invention relates to the field of Pickering emulsion stabilizer preparation technology, specifically to a whey protein isolate-grape seed oligomeric proanthocyanidins-goji berry polysaccharide ternary covalent complex, its preparation method and application. Background Technology

[0002] Whey protein isolate is a protein extracted from milk and processed. It contains many essential amino acids and possesses natural safety while also exhibiting good nutritional value and functional properties, such as excellent emulsifying ability, thermal stability, and gelling properties. However, when used as a pickering emulsion stabilizer, whey protein isolate still has certain limitations in terms of stability and functionality. After adsorption at the oil-water interface, proteins undergo structural unfolding and lateral attraction, inevitably causing thinning of the interfacial film and flocculation of emulsion droplets. Therefore, it is necessary to improve the properties of the protein to enhance the stability of the constructed delivery system and optimize the bioavailability of functional factors within the delivery system.

[0003] Polyphenols are plant secondary metabolites. Based on the natural affinity of protein molecules for polyphenols, they can form covalent interactions with proteins under alkaline conditions, thereby improving the protein's structure, function, and emulsifying properties. Compared to pure proteins, protein-polyphenol complexes provide additional hydrophobic sites, increasing the flexibility of protein molecules and improving their interfacial activity, thus forming a thicker and more viscoelastic interfacial layer. However, emulsions prepared from protein-polyphenol complexes have poor tolerance to harsh environments (temperature, ionic strength, pH, etc.). Furthermore, high concentrations of polyphenols may induce protein aggregation, thereby reducing the stability of the emulsion. Therefore, further design of stable modified materials is needed to overcome the shortcomings of the interfacial layer composed of protein-polyphenol complexes.

[0004] Studies have shown that, under the same preparation conditions, emulsions stabilized by protein-polysaccharide covalent complexes tend to have smaller particle sizes compared to natural proteins, thus improving emulsion stability. Furthermore, due to the inherent stability of polysaccharides, the mechanical properties of the interfacial layer can be further enhanced by modifying the binary complex with polysaccharides. Simultaneously, proteins complexed with polysaccharides can provide higher viscosity, stronger electrostatic repulsion, and steric hindrance to the aqueous phase, thereby delaying droplet aggregation and providing better emulsion stability. Lycium barbarum polysaccharides are bioactive polysaccharides extracted from Lycium barbarum, possessing various physiological functions such as immunomodulation and antioxidant effects. Currently, research on the application of Lycium barbarum polysaccharides in Pickering emulsion systems is limited. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a protein-polyphenol-polysaccharide ternary covalent complex, its preparation method and application. It is a method for preparing a novel protein-polyphenol-polysaccharide emulsifier and its application in the field of Pickering emulsions. The aim is to solve the problems of poor stability and single function of traditional emulsifiers by proposing a natural and efficient ternary covalent complex, and to use this ternary covalent complex as an emulsifier to prepare wolfberry seed oil Pickering emulsions.

[0006] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0007] The first technical objective of this invention is to provide a method for preparing a protein-polyphenol-polysaccharide ternary covalent complex, comprising the following steps:

[0008] (1) Mix the fully hydrated protein with an equal volume of polyphenol solution, adjust the pH and react for 24 h, then freeze dry to complete the preparation of the protein-polyphenol binary complex.

[0009] (2) The freeze-dried binary complex was prepared into a solution, mixed with an equal volume of a certain concentration of wolfberry polysaccharide solution, the pH value was adjusted, and the protein-polyphenol-polysaccharide ternary complex was prepared by freeze drying after water bath heating.

[0010] Based on the above technical solution, the present invention can be further improved as follows:

[0011] Furthermore, in step (1), the binary complex is prepared by the following method:

[0012] S1: Dissolve whey protein isolate powder in pure water, stir at 400 rpm and hydrate at 4℃ for more than 8 hours, centrifuge, and take the supernatant as whey protein isolate solution. Adjust the pH value and stir for later use. Grape seed oligomeric proanthocyanidins are completely dissolved after pH adjustment to obtain polyphenol solution. Mix the whey protein isolate solution and polyphenol solution in equal volumes, stir, dialyze and freeze dry to obtain protein-polyphenol binary complex.

[0013] The whey protein isolate was mixed with pure water at a mass-to-volume ratio of 1 g to 50 mL. Centrifugation was performed at 4000 rpm for 3 min to remove insoluble precipitates. The pH of both the protein solution and the polyphenol solution was adjusted to 9-10. The concentration of the grape seed oligomeric proanthocyanidins solution was 0.2%-0.4 (w / v). The mixture was stirred for 24 h under aerobic conditions at room temperature to ensure complete reaction. Dialysis was performed at 8000-14000 Da for 48 h, with water changed every 6 h.

[0014] Furthermore, in step (2), the concentration of the binary complex is 2% (w / v), the concentration of the Lycium barbarum polysaccharide aqueous solution is 0.1%-0.25% (w / v), the water bath heating temperature is 75℃-85℃, and the heating time is 20-25 min.

[0015] The second technical objective of this invention is to provide a protein-polyphenol-polysaccharide ternary covalent complex prepared by the method described above.

[0016] The third technical objective of this invention is to provide an application of the protein-polyphenol-polysaccharide ternary complex prepared by the method described above in the preparation of Pickering emulsions.

[0017] Specifically, the preparation method of Pickering emulsion includes the following steps: dissolving the protein-polyphenol-polysaccharide ternary complex described in claim 6 in distilled water to obtain an aqueous phase, adding wolfberry seed oil in a certain proportion, forming a primary emulsion by high-speed shearing, and then ultrasonically emulsifying to obtain Pickering emulsion.

[0018] Furthermore, the concentration of the ternary complex in the aqueous phase was 2% (w / v), and the ratio of oil to water phase was 2:8-5:5; the high-speed shearing rate was 10,000-15,000 rpm, and the time was 2-5 min; the ultrasonic cell disruptor used in the ultrasonic emulsification method had a power of 300W, a time of 5-20 min, and an interval of 3 s and 5 s (on, off).

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. A complex formed through the interaction of whey protein isolate, grape seed oligomeric proanthocyanidins, and wolfberry polysaccharides integrates the functional advantages of all three. Through covalent cross-linking of whey protein isolate and grape seed oligomeric proanthocyanidins under alkaline conditions, synergistically utilizing the steric hindrance effect of wolfberry polysaccharides, a novel emulsifier with both high emulsifying activity and antioxidant capacity is formed. This facilitates the preparation of stable Pickering emulsions rich in various active ingredients, providing new insights into the preparation and selection of natural emulsifiers.

[0021] 2. Goji berry seeds are the main component of goji berry waste, accounting for 60%–70% of the dry weight of the waste. Goji berry seeds contain approximately 12%–17% seed oil, which includes natural active ingredients such as unsaturated fatty acids, vitamin E, carotenoids, and phospholipids. These ingredients can lower plasma cholesterol, prevent atherosclerosis, and promote brain development in children, making it a nutritional and health-promoting oil with great development potential. However, the unsaturated double bonds in its active ingredients are easily oxidized, resulting in low solubility and bioavailability in the aqueous environment of the gastrointestinal system. The prepared goji berry seed oil Pickering emulsion exhibits excellent stability and antioxidant properties, which helps to improve the application value of goji berry seed oil in the food and health product fields.

[0022] 3. This invention provides a non-toxic, harmless, and pollution-free natural emulsifier and stabilizer, and simultaneously prepares a Pickering emulsion with effective nutritional components and health benefits. This provides a reference for the screening and research of natural emulsifiers, basic research, and the development of wolfberry seed oil products, and also provides a new raw material option for the research and development of functional foods. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The results of molecular dynamics simulations of binary and ternary complexes are shown; schematic diagram of ternary complex binding (A); changes in the root mean square deviation (RMSD) (B), radius of gyration (C), root mean square fluctuation of residues (RMSF) (D), and number of hydrogen bonds (E) of the complex over time.

[0025] Figure 2 This is a flowchart illustrating the preparation method of the protein-polyphenol-polysaccharide ternary covalent complex according to the present invention.

[0026] Figure 3 The diagram shows the particle size and potential distribution of the composite in the embodiments of the present invention and the composite in the comparative examples.

[0027] Figure 4 The images show the ultraviolet and fluorescence spectra of the complexes in the embodiments of the present invention and the comparative examples.

[0028] Figure 5 These are SEM images of the complex from the embodiments of the present invention and the complex from the comparative examples.

[0029] Figure 6The results of free radical scavenging experiments are shown for the complexes of the embodiments of the present invention and the comparative complexes.

[0030] Figure 7 The images show the appearance and microstructure of the Pickering emulsions prepared from the complexes of the present invention and the comparative examples.

[0031] Figure 8 The image shows the Pickering emulsion CLSM diagrams of the complexes prepared in the embodiments of the present invention and the comparative examples. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0034] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0035] In the description of this invention, it should be understood that the terms "middle", "upper", "lower", "rise", "fall", "vertical", "surface", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0036] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0037] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0038] This invention discloses a protein-polyphenol-polysaccharide ternary covalent complex, its preparation method, and its application.

[0039] Furthermore, the binary complex in the following examples was prepared by the following method:

[0040] A whey protein isolate with a concentration of 20 mg / mL was prepared by dissolving in ultrapure water and swelling at 4°C for more than 6 hours. After centrifugation at 4000 rpm for 2 minutes, the supernatant was collected, and the pH of the protein solution was adjusted to 9.0 using 0.1 mol / L NaOH solution. An equal volume of the protein solution was mixed with a 2 mg / mL grape seed oligomeric proanthocyanidin solution (prepared with ultrapure water, pH=9), and the pH was adjusted to 9.0 again. The mixture was stirred at room temperature for 24 hours, and then transferred to a dialysis bag (molecular weight cutoff 8000-14000 Da) and dialyzed in ultrapure water at 4°C for 48 hours (with the ultrapure water changed every 6 hours). The dialyzed solution was freeze-dried to obtain the protein-polyphenol covalent graft.

[0041] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0042] Example 1:

[0043] A protein-polyphenol-polysaccharide covalent ternary complex, the preparation method of which includes the following steps:

[0044] (1) Prepare an aqueous solution of protein-polyphenol binary complex, and after full hydration, adjust the pH to 7 with 0.1 mol / L NaOH to obtain a binary complex solution (concentration of 0.02 g / mL);

[0045] (2) Dissolve the Lycium barbarum polysaccharide in distilled water and adjust the pH to 7 with 0.1 mol / L NaOH to obtain a Lycium barbarum polysaccharide solution (concentration of 0.001 g / mL);

[0046] (3) The aqueous solution of the binary complex obtained in step (1) and the solution of Lycium barbarum polysaccharide obtained in step (2) are mixed in a volume ratio of 1:1, heated in a water bath at 85°C for 20 min, and freeze-dried at -76°C and 0 Pa for 36 h to obtain a protein-polyphenol-polysaccharide ternary covalent complex.

[0047] A Pickering emulsion, the preparation method of which includes the following steps:

[0048] The above ternary complex was dissolved in distilled water at a mass-to-volume ratio of 0.2 g × 10 mL to obtain an aqueous phase. Goji berry seed oil was added at a volume ratio of 2:8 (oil to aqueous phase). The mixture was homogenized at 12,000 rpm for 2 min using a high-speed homogenizer to obtain a pre-emulsion. The pre-emulsion was then subjected to ultrasonic treatment with a power of 300 W using an ultrasonic probe, with an interval of 3 s followed by a 5 s pause, for a total ultrasonic treatment time of 10 min. During the ultrasonic treatment, the emulsion was kept in an ice bath, with the probe immersed to a depth of 2 cm below the liquid surface, resulting in a Pickering emulsion.

[0049] Example 2:

[0050] A protein-polyphenol-polysaccharide covalent ternary complex, the preparation method of which includes the following steps:

[0051] (1) Prepare an aqueous solution of protein-polyphenol binary complex, and after full hydration, adjust the pH to 7 with 0.1 mol / L NaOH to obtain a binary complex solution (concentration of 0.02 g / mL);

[0052] (2) Dissolve the Lycium barbarum polysaccharide in distilled water and adjust the pH to 7 with 0.1 mol / L NaOH to obtain a Lycium barbarum polysaccharide solution (concentration of 0.002 g / mL);

[0053] (3) The aqueous solution of the binary complex obtained in step (1) and the solution of Lycium barbarum polysaccharide obtained in step (2) are mixed in a volume ratio of 1:1, heated in a water bath at 85 °C for 20 min, and freeze-dried at -76 °C and 0 Pa for 36 h to obtain a protein-polyphenol-polysaccharide ternary covalent complex.

[0054] A Pickering emulsion, the preparation method of which includes the following steps:

[0055] The above ternary complex was dissolved in distilled water at a mass-to-volume ratio of 0.2 g × 10 mL to obtain an aqueous phase. Goji berry seed oil was added at a volume ratio of 2:8 (oil to aqueous phase). The mixture was homogenized at 12,000 rpm for 2 min using a high-speed homogenizer to obtain a pre-emulsion. The pre-emulsion was then subjected to ultrasonic treatment with a power of 300 W using an ultrasonic probe, with an interval of 3 s followed by a 5 s pause, for a total ultrasonic treatment time of 10 min. During the ultrasonic treatment, the emulsion was kept in an ice bath, with the probe immersed to a depth of 2 cm below the liquid surface, resulting in a Pickering emulsion.

[0056] Example 3:

[0057] A protein-polyphenol-polysaccharide covalent ternary complex, the preparation method of which includes the following steps:

[0058] (1) Prepare an aqueous solution of protein-polyphenol binary complex, and after full hydration, adjust the pH to 7 with 0.1 mol / L NaOH to obtain a binary complex solution (concentration of 0.02 g / mL);

[0059] (2) Dissolve the wolfberry polysaccharide in distilled water and adjust the pH to 7 with 0.1 mol / L NaOH to obtain a wolfberry polysaccharide solution (concentration of 0.003 g / mL);

[0060] (3) The aqueous solution of the binary complex obtained in step (1) and the solution of Lycium barbarum polysaccharide obtained in step (2) are mixed in a volume ratio of 1:1, heated in a water bath at 85 °C for 20 min, and freeze-dried at -76 °C and 0 Pa for 36 h to obtain a protein-polyphenol-polysaccharide ternary covalent complex.

[0061] A Pickering emulsion, the preparation method of which includes the following steps:

[0062] The above ternary complex was dissolved in distilled water at a mass-to-volume ratio of 0.2 g × 10 mL to obtain an aqueous phase. Goji berry seed oil was added at a volume ratio of 2:8 (oil to aqueous phase). The mixture was homogenized at 12,000 rpm for 2 min using a high-speed homogenizer to obtain a pre-emulsion. The pre-emulsion was then subjected to ultrasonic treatment with a power of 300 W using an ultrasonic probe, with an interval of 3 s followed by a 5 s pause, for a total ultrasonic treatment time of 10 min. During the ultrasonic treatment, the emulsion was kept in an ice bath, with the probe immersed to a depth of 2 cm below the liquid surface, resulting in a Pickering emulsion.

[0063] Example 4:

[0064] A protein-polyphenol-polysaccharide covalent ternary complex, the preparation method of which includes the following steps:

[0065] (1) Prepare an aqueous solution of protein-polyphenol binary complex, and after full hydration, adjust the pH to 7 with 0.1 mol / L NaOH to obtain a binary complex solution (concentration of 0.02 g / mL);

[0066] (2) Dissolve the Lycium barbarum polysaccharide in distilled water and adjust the pH to 7 with 0.1 mol / L NaOH to obtain a Lycium barbarum polysaccharide solution (concentration of 0.004 g / mL);

[0067] (3) The aqueous solution of the binary complex obtained in step (1) and the solution of Lycium barbarum polysaccharide obtained in step (2) are mixed in a volume ratio of 1:1, heated in a water bath at 85°C for 20 min, and freeze-dried at -76°C and 0 Pa for 36 h to obtain a protein-polyphenol-polysaccharide ternary covalent complex.

[0068] A Pickering emulsion, the preparation method of which includes the following steps:

[0069] The above ternary complex was dissolved in distilled water at a mass-to-volume ratio of 0.2 g × 10 mL to obtain an aqueous phase. Goji berry seed oil was added at a volume ratio of 2 × 8 for the oil phase to the aqueous phase. The mixture was homogenized at 12,000 rpm for 2 min using a high-speed homogenizer to obtain a pre-emulsion. The pre-emulsion was then subjected to ultrasonic treatment with a power of 300 W using an ultrasonic probe, with an interval of 3 s followed by a 5 s pause, for a total ultrasonic treatment time of 10 min. During the ultrasonic treatment, the emulsion was kept in an ice bath, and the probe was immersed to a depth of 2 cm below the liquid surface to obtain a Pickering emulsion.

[0070] Example 5:

[0071] A protein-polyphenol-polysaccharide covalent ternary complex, the preparation method of which includes the following steps:

[0072] (1) Prepare an aqueous solution of protein-polyphenol binary complex, and after full hydration, adjust the pH to 7 with 0.1 mol / L NaOH to obtain a binary complex solution (concentration of 0.02 g / mL);

[0073] (2) Dissolve the Lycium barbarum polysaccharide in distilled water and adjust the pH to 7 with 0.1 mol / L NaOH to obtain a Lycium barbarum polysaccharide solution (concentration of 0.005 g / mL);

[0074] (3) The aqueous solution of the binary complex obtained in step (1) and the solution of Lycium barbarum polysaccharide obtained in step (2) are mixed in a volume ratio of 1:1, heated in a water bath at 85°C for 20 min, and freeze-dried at -76°C and 0 Pa for 36 h to obtain a protein-polyphenol-polysaccharide ternary covalent complex.

[0075] A Pickering emulsion, the preparation method of which includes the following steps:

[0076] The above ternary complex was dissolved in distilled water at a mass-to-volume ratio of 0.2 g × 10 mL to obtain an aqueous phase. Goji berry seed oil was added at a volume ratio of 2:8 (oil to aqueous phase). The mixture was homogenized at 12,000 rpm for 2 min using a high-speed homogenizer to obtain a pre-emulsion. The pre-emulsion was then subjected to ultrasonic treatment with a power of 300 W using an ultrasonic probe, with an interval of 3 s followed by a 5 s pause, for a total ultrasonic treatment time of 10 min. During the ultrasonic treatment, the emulsion was kept in an ice bath, with the probe immersed to a depth of 2 cm below the liquid surface, resulting in a Pickering emulsion.

[0077] To further demonstrate the beneficial effects of the present invention and to better understand it, the following comparative examples further illustrate the technical features disclosed in the present invention, but should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above description of the invention, without inventive effort, are also considered to fall within the protection scope of the present invention.

[0078] Comparative Example 1:

[0079] Step (1): Preparation of protein aqueous solution

[0080] A whey protein isolate solution with a mass concentration of 20 mg / mL was prepared by dissolving in ultrapure water, swelling at 4 ℃ for more than 6 h, centrifuging at 4000 rpm for 2 min, and then collecting the supernatant. The pH of the protein solution was adjusted to 7.0 using 0.1 mol / L NaOH solution.

[0081] Step (2): Preparation of Pickering emulsion of wolfberry seed oil

[0082] Following the principle of using the protein solution as the aqueous phase in step (1), wolfberry seed oil was added at a volume ratio of 2:8 (oil to aqueous phase). The mixture was homogenized using a high-speed homogenizer at 12000 rpm for 2 minutes to obtain the colostrum. The colostrum was then subjected to ultrasonic treatment with a 300 W power probe, with an alternating interval of 3 seconds and 5 seconds pause, for a total ultrasonic treatment time of 10 minutes. During the ultrasonic treatment, the emulsion was kept in an ice bath, with the probe immersed to a depth of 2 cm below the liquid surface, resulting in a Pickering emulsion.

[0083] Comparative Example 2:

[0084] Step (1): Same as the preparation method of the binary complex in the example.

[0085] Step (2): Using the protein-polyphenol complex solution as the aqueous phase in Step (1), add wolfberry seed oil at a volume ratio of 2:8 (oil to aqueous phase). Homogenize the mixture using a high-speed homogenizer at 12000 rpm for 2 minutes to obtain the colostrum. Use an ultrasonic probe at 300 W with alternating intervals of 3 seconds and 5 seconds, for a total ultrasonic treatment time of 10 minutes. During the ultrasonic process, the emulsion is kept in an ice bath, with the probe immersed 2 cm below the liquid surface, resulting in a Pickering emulsion.

[0086] It should be noted that the technical route of this invention, which first obtains a protein-polyphenol binary complex and then performs a Maillard reaction to obtain a protein-polyphenol-polysaccharide ternary complex, is significantly different. The technical advantages or advancements of this method compared to the prior art method of first obtaining the polysaccharide-polyphenol binary complex will now be discussed.

[0087] According to the literature review, the following problems exist in forming polysaccharide-polyphenol first: (1) Limited reaction selectivity. After polysaccharide and polyphenol form a binary complex, its structure may produce steric hindrance, which hinders the subsequent binding of proteins and leads to poor structural uniformity of the ternary complex; (2) Structural instability and risk of phase separation. Polyphenol-polysaccharide binary complexes often rely on weak interactions, which makes the binary complex prone to dissociation or aggregation when it is subsequently complexed with proteins, reducing the stability of the complex.

[0088] The preparation method mentioned in this invention first prepares a protein-polyphenol binary complex through an alkaline treatment process. This simple process exposes more hydrophobic regions and reactive groups of the protein, enhancing binding efficiency and polyphenol loading without affecting subsequent binding efficiency. Furthermore, the polyphenol, as a small molecule, enters the binding "pocket" inside the protein, forming a more uniform and compact complex structure through strong hydrogen bonding, reducing the risk of dissociation during subsequent processing or storage. We further validate this through experimental data from molecular dynamics simulations.

[0089] The stereoconfiguration of the binary complex and the binding configuration of Lycium barbarum polysaccharides are as follows: Figure 1 As shown in Figure A. Figure 1Simulation results showed that the RMSD of the binary complex protein backbone stabilized after 10 ns, indicating that the binary complex rapidly reached dynamic equilibrium during the simulation. The polyphenol remained stable within the protein-binding pocket without significant displacement or dissociation. The radius of gyration (Rg) remained stable during the simulation, averaging around 1.5 Å, without significant fluctuations. This indicates that the binary complex did not undergo significant conformational expansion or contraction, maintaining a compact overall structure, consistent with the RMSD results and verifying the system stability of the protein-polyphenol binary complex system. Furthermore, the RMSD and Rg of the ternary complex showed similar trends. Although the local flexibility analysis (RMSF) of the ternary complex structure fluctuated significantly, residues with low RMSF values ​​indicated strong structural rigidity, possibly related to stable polysaccharide binding. Regions with high RMSF values ​​showed greater flexibility, possibly related to functional conformational changes in the protein. Hydrogen bond quantity results showed that both the protein-polyphenol and protein-polyphenol-polysaccharide ternary complexes formed strong hydrogen bond interactions, indicating that a stable hydrogen bond network plays a crucial role in maintaining the complex structure.

[0090] In summary, proteins preferentially bind to polyphenols (through hydrogen bonds, hydrophobic interactions, or π-π stacking) to form stable binary complexes, providing ordered templates for subsequent Maillard reactions. Furthermore, Maillard grafting reactions conducted under mild heating conditions can also prepare protein-polyphenol-polysaccharide ternary complexes without damaging the polyphenol structure. This pathway optimizes the reaction sequence and avoids unnecessary competitive binding, thereby improving the overall performance of the complex.

[0091] Efficacy testing

[0092] Figure 3 The particle size potential results are shown for Comparative Examples 1 and 2, and Examples 1-3. The particle size results show a gradual decrease from the Comparative Examples to the Examples. Smaller particle sizes allow for effective adsorption at the oil-water interface, forming a denser interfacial film and effectively reducing droplet aggregation. Furthermore, it increases the interfacial area of ​​the particles, improving emulsification efficiency and enhancing the stability of the Pickering emulsion. Compared to the smaller absolute potential values ​​of the Comparative Examples, the absolute potential values ​​of Examples 1-3 are significantly increased, enhancing the electrostatic repulsion between particles, effectively inhibiting droplet aggregation, and further reducing the risk of Ostwald ripening. Figure 3 The technical advantages of ternary covalent complexes as natural emulsifiers were verified, providing theoretical support for their application in Pickering emulsions.

[0093] Figure 4The images show the UV and fluorescence spectra of the comparative and exemplary examples. Compared to Comparative Example 1, Examples 1-3 showed a significant increase in absorbance at 280 nm, indicating that the covalent binding of polyphenols to proteins altered the protein conformation, exposing more aromatic amino acids (such as tyrosine and tryptophan). Furthermore, intermediate products (such as reductones and melanoidins) generated by the water bath heating reaction (i.e., Maillard reaction) of the complex and polysaccharides exhibited absorption in the UV region, demonstrating the formation of covalent bonds in the protein-polyphenol-polysaccharide complex. Further, the phenolic hydroxyl groups of grape seed proanthocyanidins bound to tryptophan residues in the protein, quenching fluorescence through energy transfer or electron transfer mechanisms. After the formation of the ternary complex, the protein conformation tended to stabilize, the hydrophobic region was encapsulated by the polyphenol-polysaccharide, and the microenvironment of tryptophan shifted from hydrophobic to polar, leading to a decrease in fluorescence intensity. The changes in UV spectra and the decrease in fluorescence intensity are direct evidence of protein structural modification and the covalent binding of polyphenols and polysaccharides, verifying the successful construction of the ternary complex. This structural change endows the complex with stronger interfacial adsorption capacity and antioxidant function, providing a theoretical basis for its application in Pickering emulsions and functional foods.

[0094] Figure 5 The following are scanning electron microscopy results of the composites in the embodiments and comparative examples of this invention. By directly observing the microstructure and morphology of the composite particles, the essential differences between the comparative and exemplary examples are revealed. With the addition of Lycium barbarum polysaccharides, the particle surface exhibits a distinctly rough structure, which is a characteristic of the composite of Maillard reaction products and polysaccharides. This significantly enhances the interfacial adsorption capacity of the composite, forming a stable emulsion.

[0095] Figure 6 In the DPPH and ABTS free radical scavenging experiments, the free radical scavenging rate of Example 1 was significantly higher than that of Comparative Examples 1 and 2, and it exhibited a more efficient scavenging ability over time. This indicates that after the formation of the complex, the hydrophobic region of the whey protein isolate protects the active groups, the phenolic hydroxyl groups of grape seed oligomeric proanthocyanidins can donate electrons, and the wolfberry polysaccharide enhances the antioxidant chain reaction by stabilizing free radical intermediates to form a highly efficient antioxidant network, which can effectively delay lipid oxidation and extend shelf life.

[0096] Figure 7In this study, the appearance and microstructure of the Pickering emulsions formed in the comparative examples and the examples were observed. It was found that with the addition of Lycium barbarum polysaccharides, the number of oil droplets on the emulsion surface significantly decreased, indicating that the formation of the complex effectively enhanced the anchoring of emulsion particles at the oil-water interface, resulting in a Pickering emulsion with better performance and greater uniformity. Furthermore, from the comparative examples to the examples, the oil droplet size gradually decreased, and the distribution uniformity significantly improved. This indicates that the protein-polyphenol-polysaccharide ternary complex can effectively form a rigid interfacial film, delaying droplet aggregation and resisting Ostwald ripening. This verifies the innovative value of complex design in the emulsion field and provides an important reference for the development of high-performance natural emulsifiers.

[0097] Figure 8 In this study, Nile Red (excitation wavelength: 530 nm, emission wavelength: 576 nm) and FITC (excitation wavelength: 490 nm, emission wavelength: 525 nm) were used to label the dispersed and continuous phases of the Pickering emulsion system, respectively. The fluorescence signal of FITC was concentrated in the continuous phase and complemented that of Nile Red, indicating that the dispersed phase was successfully dispersed in the continuous phase. Compared to Comparative Examples 1 and 2, where the boundary between oil droplets and the water phase was blurred and prone to aggregation or stratification, Example 3 showed a significantly reduced droplet size and the oil droplets were tightly wrapped by the complex particles. This further demonstrates that the complex particles are irreversibly adsorbed at the oil-water interface, forming a rigid protective layer. The ternary complex significantly overcomes the instability of Pickering emulsions and the easy oxidation of oils through synergistic interface anchoring, electrostatic-spatial dual stability, and antioxidant protection. This provides key technical support for the development of long-lasting, stable, and multifunctional emulsion products, while also aligning with the development trends of green food and clean labeling.

[0098] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a protein-polyphenol-polysaccharide ternary covalent complex, characterized in that, The method comprises the following steps: (1) mixing whey protein isolate solution and grape seed oligomeric proanthocyanidin solution in equal volume after being fully hydrated, adjusting pH value, dialysis after fully stirring, and preparing protein-polyphenol binary complex by freeze-drying; (2) mixing the fully hydrated protein-polyphenol binary complex and wolfberry polysaccharide aqueous solution in equal volume, adjusting pH value, heating in water bath, cooling to room temperature in ice bath, and preparing protein-polyphenol-polysaccharide ternary complex by freeze-drying.

2. The method for preparing a protein-polyphenol-polysaccharide ternary complex according to claim 1, characterized by, In step (1), the concentration of whey protein isolate solution is 2% (w / v), and the concentration of grape seed oligomeric proanthocyanidin solution is 0.2%-0.4% (w / v).

3. The method for preparing a protein-polyphenol-polysaccharide ternary complex according to claim 1 or 2, characterized in that, In step (1), the fully stirring is stirring at room temperature for 24 h under aerobic condition; the dialysis is dialysis in ultrapure water at 4 ℃ for 48 h, wherein the water is changed every 6 h; and the pH value is 9.0-10.

0.

4. The method for preparing the protein-polyphenol-polysaccharide ternary complex according to claim 1, characterized in that, In step (2), the concentration of binary complex solution is 2% (w / v), and the concentration of wolfberry polysaccharide aqueous solution is 0.1%-0.25% (w / v).

5. The method for preparing the protein-polyphenol-polysaccharide ternary complex according to claim 1, characterized in that, In step (2), the heating in water bath is heating at 75 ℃-85 ℃ for 20-25 min.

6. A protein-polyphenol-polysaccharide ternary covalent complex prepared by the preparation method of any one of claims 1-5.

7. Application of the protein-polyphenol-polysaccharide ternary complex of claim 6 in preparing Pickering emulsion.

8. Use according to claim 7, characterized in that, The preparation method of Pickering emulsion comprises the following steps: dissolving the protein-polyphenol-polysaccharide ternary complex of claim 6 in distilled water to obtain an aqueous phase, adding wolfberry seed oil in a certain proportion, forming a preliminary emulsion by high-speed shearing, and then ultrasonic emulsification to obtain Pickering emulsion.

9. Use according to claim 8, characterized in that, The concentration of ternary complex in the aqueous phase is 2% (w / v), and the addition ratio of oil phase to aqueous phase is 2:8-5:

5.

10. Use according to claim 9, characterized in that, The high-speed shearing rate is 10000-15000 rpm, and the time is 0.5-5 min; the ultrasonic cell disrupter used in ultrasonic emulsification has a power of 300 W, and the time is 5-20 min, with an interval of 3 s, 5 s (on, off).

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