Protein-polysaccharide-polyphenol ternary complex used as Pickering emulsifier and preparation method of protein-polysaccharide-polyphenol ternary complex

Abstract

The invention provides a protein-polysaccharide-polyphenol ternary complex used as a Pickering emulsifier and a preparation method of the protein-polysaccharide-polyphenol ternary complex. Specifically, the ternary complex is a complex formed by whey protein nanofibers and a pectin-polyphenol covalent complex. The ternary complex provided by the invention has very excellent interface stabilizer / active agent performance, and can be used as an emulsifier / interface stabilizer / interface active agent of Pickering emulsion.

Description

Technical Field

[0001] This invention relates to the food industry, and more specifically to a protein-polysaccharide-polyphenol ternary complex that can be used as a Pickering emulsifier and its preparation method. Background Technology

[0002] Pickering emulsions have garnered increasing attention due to their high stability, low cost, and ease of preparation. Unlike traditional emulsions where small-molecule emulsifiers are dynamically adsorbed at the interface, the solid particles in stable Pickering emulsions are typically immobilized at the interface in an irreversible manner. Therefore, Pickering emulsions exhibit greater tolerance to adverse environmental conditions. Furthermore, using natural biopolymers as emulsifiers avoids the potential health risks associated with small-molecule chemical emulsifiers. Consequently, Pickering emulsions have attracted widespread attention in the food and pharmaceutical industries.

[0003] On the other hand, Pickering emulsions face challenges in long-term stability, such as droplet coalescence or phase separation with prolonged storage. Background technologies include optimizing the surface chemistry, size, and shape of particles, as well as employing composite materials or hierarchical structures to enhance the robustness and durability of the interfacial film, aiming to address these issues and improve the long-term stability and applicability of the emulsions. These technological advancements have driven the application of Pickering emulsions in multiple industrial sectors. Summary of the Invention

[0004] The purpose of this invention is to provide a protein-polysaccharide-polyphenol ternary complex with excellent emulsifying properties, its preparation method, and its use as a surfactant in Pickering emulsions.

[0005] In a first aspect, the present invention provides a protein-polysaccharide-polyphenol ternary complex, wherein the ternary complex is a complex formed by whey protein nanofibers and a pectin-polyphenol covalent complex.

[0006] The polyphenols are selected from the group consisting of gallic acid, chlorogenic acid, catechin, epigallocatechin gallate, or combinations thereof.

[0007] In another preferred embodiment, the whey protein nanofiber-pectin-polyphenol complex has a mass ratio of whey protein nanofiber to pectin-polyphenol of 1:0.2-1, preferably 1:0.3-0.8, and more preferably 1:0.4-0.6.

[0008] In another preferred embodiment, the pectin-polyphenol covalent complex is a pectin-gallic acid covalent complex.

[0009] In another preferred embodiment, the degree of grafting of the polyphenols in the pectin-polyphenol covalent complex is 1.5-3.5 mg / g, preferably 2-3 mg / g, more preferably 2.4-2.8 mg / g, and even more preferably 2.6-2.7 mg / g.

[0010] In another preferred embodiment, the degree of gallic acid grafting in the pectin-gallic acid covalent complex is 1.5-3.5 mg / g, preferably 2-3 mg / g, more preferably 2.4-2.8 mg / g, and even more preferably 2.69±0.13 mg / g.

[0011] In another preferred embodiment, the pectin-polyphenol covalent complex is connected to polyphenols via phenolic hydroxyl groups, carboxyl groups, and / or methyl ester groups in the pectin.

[0012] In another preferred embodiment, the content of gallic acid in the pectin-gallic acid covalent complex is 4.61 ± 0.05 mg / g (determined using the Folin-Ciocalteu method).

[0013] In another preferred embodiment, the pectin-polyphenol covalent complex is prepared by a method comprising the following steps:

[0014] (i) Provide an aqueous solution containing pectin and polyphenols;

[0015] (ii) Add ascorbic acid and hydrogen peroxide to initiate a free radical reaction, and react at room temperature under inert gas protection;

[0016] (iii) After the reaction is complete, add ethanol to the reaction solution and let it stand to precipitate;

[0017] (iv) Collect the precipitate, wash away the unbound polyphenols, reconstitute the precipitate and freeze-dry it to obtain the pectin-polyphenol covalent complex.

[0018] In another preferred embodiment, step (i) includes: providing an aqueous solution of pectin and an aqueous solution of polyphenols and mixing them thoroughly to obtain the aqueous solution containing pectin and polyphenols.

[0019] In another preferred embodiment, step (ii) includes: adding ascorbic acid and mixing thoroughly before adding hydrogen peroxide solution.

[0020] In another preferred embodiment, the ratio of pectin to ascorbic acid is 1:0.15-0.35, preferably 1:0.2-0.3, and more preferably 1:0.23-0.27.

[0021] In another preferred embodiment, the ratio of pectin to hydrogen peroxide is 1g:10-30mmol, preferably 1g:15-25mmol, and more preferably 1g:18-22mmol.

[0022] In another preferred embodiment, in step (ii), the reaction temperature is 4-40°C, preferably 15-35°C, more preferably 20-30°C, such as 25°C.

[0023] In another preferred embodiment, in step (ii), the reaction time is 12-48 h, preferably 18-36 h, more preferably 20-30 h, such as 24 h.

[0024] In another preferred embodiment, in step (iii), the amount of ethanol used is 3-6 times that of the reaction solution, preferably 3.5-5 times, more preferably 3.8-4.5 times, such as 4 times.

[0025] In another preferred embodiment, in step (iv), the unbound polyphenols are washed with ethanol, such as 95% ethanol.

[0026] In another preferred embodiment, step (iv) includes: redissolving the precipitate in water and then freeze-drying it.

[0027] In another preferred embodiment, the whey protein nanofibers are prepared by a method comprising the following steps:

[0028] (a-1) Provides an aqueous solution of whey protein;

[0029] (a-2) Adjust the pH of the above whey protein aqueous solution to 1.5-2.5, and then heat it at 75-95℃ for 3-8 hours;

[0030] (a-3) After heating, the reaction solution is cooled to room temperature to obtain whey protein nanofibers.

[0031] In another preferred embodiment, in step (a-1), the concentration of whey protein in the aqueous solution is 1-8 wt%, preferably 2-6 wt%, more preferably 3-5 wt%, such as 4 wt%.

[0032] In another preferred embodiment, step (a-2) includes adjusting the pH of the aqueous solution of the whey protein to 1.8-2.2 and then heating it at 80-90°C for 5-6 hours.

[0033] In another preferred embodiment, in step (a-3), the cooling is rapid cooling in a water bath.

[0034] In another preferred embodiment, the ternary composite is prepared by a method comprising the following steps:

[0035] (i) Provide an aqueous solution containing whey protein nanofibers and a pectin-polyphenol complex;

[0036] (ii) The pH of the solution is adjusted to 3-5 and the reaction is stirred to obtain the ternary complex.

[0037] In another preferred embodiment, step (i) includes mixing an aqueous solution of whey protein nanofibers and an aqueous solution of pectin-polyphenol complex to obtain the aqueous solution containing whey protein nanofibers and pectin-polyphenol complex.

[0038] In another preferred embodiment, in step (i), the mass ratio of whey protein nanofibers to pectin-polyphenol complex in the aqueous solution is 1:0.2-1, preferably 1:0.3-0.8, and more preferably 1:0.4-0.6.

[0039] In another preferred embodiment, in step (i), the concentration of whey protein nanofibers in the aqueous solution containing whey protein nanofibers and pectin-polyphenol complex is 0.5-4 wt%, preferably 0.8-5 wt%, and more preferably 1-2 wt%.

[0040] In another preferred embodiment, in step (i), the concentration of the pectin-polyphenol complex in the aqueous solution containing whey protein nanofibers and pectin-polyphenol complex is 0.2-1 wt%, preferably 0.3-0.8 wt%, and more preferably 0.4-0.6 wt%.

[0041] In another preferred embodiment, in step (ii), the reaction temperature is 4-40°C, preferably 15-35°C, more preferably 20-30°C, such as 25°C.

[0042] In another preferred embodiment, in step (ii), the reaction time is 0.5-2 h, preferably 45 min-1.5 h, such as 1 h.

[0043] In another preferred embodiment, in step (ii), the pH is 3.5-4.5, preferably 4-4.2. (Under this pH condition, the protein nanofibers are positively charged, and the pectin is negatively charged, and the two combine through non-covalent interactions dominated by electrostatic interactions.)

[0044] A second aspect of the present invention provides a method for preparing a protein-polysaccharide-polyphenol ternary complex as described in the first aspect of the present invention, comprising the steps of:

[0045] (i) Provide an aqueous solution comprising whey protein nanofibers and a pectin-gallic acid complex;

[0046] (ii) The pH of the solution is adjusted to 3-5 and the reaction is stirred to obtain the ternary complex.

[0047] In another preferred embodiment, step (i) includes mixing an aqueous whey protein solution and an aqueous pectin-gallic acid complex solution to obtain the aqueous solution containing whey protein and the pectin-gallic acid complex.

[0048] In another preferred embodiment, in step (i), the concentration of whey protein nanofibers in the aqueous solution is 0.5-2 wt%, preferably 0.8-1.5 wt%, and more preferably 1-1.2 wt%.

[0049] In another preferred embodiment, in step (i), the concentration of the pectin-gallic acid complex in the aqueous solution is 0.1-1 wt%, preferably 0.2-0.8 wt%, and more preferably 0.4-0.6 wt%.

[0050] In another preferred embodiment, in step (ii), the reaction temperature is 4-40°C, preferably 15-35°C, more preferably 20-30°C, such as 25°C.

[0051] In another preferred embodiment, in step (ii), the reaction time is 0.5-2 h, preferably 45 min-1.5 h, such as 1 h.

[0052] In another preferred embodiment, in step (ii), the pH is 3.5-4.5, preferably 4-4.2.

[0053] In a third aspect, the present invention provides a Pickering emulsion, characterized in that the Pickering emulsion uses the protein-polysaccharide-polyphenol ternary complex of the first aspect of the present invention as an interface stabilizer / activator.

[0054] In another preferred embodiment, the Pickering emulsion further includes water and edible oil.

[0055] In another preferred embodiment, the edible oil is selected from the group consisting of soybean oil, peanut oil, corn oil, rapeseed oil, sunflower oil, olive oil, coconut oil, sesame oil, or combinations thereof.

[0056] In another preferred embodiment, the content of the protein-polysaccharide-polyphenol ternary complex in the Pickering emulsion is 0.1-2 wt%, more preferably 0.2-1 wt%, such as 0.3 wt%, 0.5 wt%, 0.6 wt%, or 0.8 wt%.

[0057] In another preferred embodiment, the volume ratio of water to edible oil in the Pickering emulsion is 1:1-2, preferably 1:1.2-1.8, and more preferably 1:1.4-1.6.

[0058] The Pickering emulsion of the present invention can be used as a fat substitute to prepare low-fat foods, such as low-fat meat products (e.g., sausages, canned goods, etc.).

[0059] In a fourth aspect, the invention provides the use of the protein-polysaccharide-polyphenol ternary complex of the first aspect as an interface stabilizer / activator.

[0060] In another preferred embodiment, the protein-polysaccharide-polyphenol ternary complex is used as an interface stabilizer / activator for Pickering emulsions.

[0061] In another preferred embodiment, the protein-polysaccharide-polyphenol ternary complex is used as an interface stabilizer / activator in the preparation of Pickering emulsion.

[0062] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0063] Figure 1 The three-phase contact angles of different composites are shown;

[0064] Figure 2 The microstructures of different complexes are shown;

[0065] Figure 3 The storage modulus (A) and loss modulus (B) of different Pickering emulsions are shown as a function of angular velocity;

[0066] Figure 4 The microstructures of different Pickering emulsions are shown;

[0067] Figure 5 The storage stability (A) and centrifugal stability (B) of different Pickering emulsions are shown. From left to right in the figure, they are the WE, WPE and WPF-PG groups. Detailed Implementation

[0068] Through extensive and in-depth research, including numerous screenings and tests, the inventors have developed a protein-polysaccharide-polyphenol ternary complex with excellent emulsifying properties, its preparation method, and its application as a surfactant in Pickering emulsions. This invention utilizes whey protein, pectin, and polyphenols to develop a fibrous protein-polysaccharide-polyphenol ternary complex for the preparation of Pickering emulsions. First, whey protein is modified to prepare protein nanofibers, and a covalent complex of polyphenols and pectin is synthesized. Subsequently, the whey protein nanofibers are combined with the pectin-polyphenol covalent complex via non-covalent interactions to form the final product. Experiments have demonstrated that the ternary complex possesses excellent interfacial stability. This invention is based on these findings.

[0069] the term

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0071] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0072] As used herein, the term “room temperature” or “normal temperature” refers to a temperature of 4–40°C, preferably 25 ± 5°C.

[0073] Polyphenols

[0074] The polyphenols of this invention refer to natural polyphenols produced by plants that have biological safety.

[0075] Preferably, the polyphenol is a natural plant polyphenol with beneficial physiological functions such as anti-oxidation and anti-inflammation.

[0076] Preferably, the polyphenol is selected from the group consisting of gallic acid. chlorogenic acid Catechins Epigallocatechin gallate Or a combination thereof, preferably gallic acid.

[0077] Polyphenols are rich in phenolic hydroxyl groups and benzene rings, which can bind to proteins and polysaccharides in a covalent or non-covalent manner.

[0078] pectin

[0079] In this invention, there are no special requirements for pectin, and food-grade pectin from various sources (such as fruit peel) commonly used in the art can be used.

[0080] Preferably, the relative molecular mass of the pectin is 20,000 to 400,000.

[0081] In another preferred embodiment, the degree of esterification of the pectin is 60-75%.

[0082] In another preferred embodiment, the pectin has a galacturonic acid content of 50-100%, more preferably 60-90%, and even more preferably 70-80%.

[0083] Pectin-polyphenol covalent complex

[0084] In this invention, polyphenols are covalently linked to the polysaccharide chains of pectin through an induced free radical reaction. The free radical-induced reaction has multiple binding sites; the phenolic hydroxyl groups, carboxyl groups, and / or methyl ester groups in the pectin molecule can all be linked to polyphenols.

[0085] In another preferred embodiment, the degree of grafting of the polyphenols in the pectin-polyphenol covalent complex is 1.5-3.5 mg / g, preferably 2-3 mg / g, more preferably 2.4-2.8 mg / g, and even more preferably 2.6-2.7 mg / g.

[0086] In another preferred embodiment, the pectin-polyphenol covalent complex is prepared by a method comprising the following steps:

[0087] (i) Provide an aqueous solution containing pectin and polyphenols;

[0088] (ii) Add ascorbic acid and hydrogen peroxide to initiate a free radical reaction, and react at room temperature under inert gas protection;

[0089] (iii) After the reaction is complete, add ethanol to the reaction solution and let it stand to precipitate;

[0090] (iv) Collect the precipitate, wash away the unbound polyphenols, reconstitute the precipitate and freeze-dry it to obtain the pectin-polyphenol covalent complex.

[0091] In another preferred embodiment, step (i) includes: providing an aqueous solution of pectin and an aqueous solution of polyphenols and mixing them thoroughly to obtain the aqueous solution containing pectin and polyphenols.

[0092] In another preferred embodiment, step (ii) includes: adding ascorbic acid and mixing thoroughly before adding hydrogen peroxide solution.

[0093] In another preferred embodiment, the ratio of pectin to ascorbic acid is 1:0.15-0.35, preferably 1:0.2-0.3, and more preferably 1:0.23-0.27.

[0094] In another preferred embodiment, the ratio of pectin to hydrogen peroxide is 1g:10-30mmol, preferably 1g:15-25mmol, and more preferably 1g:18-22mmol.

[0095] In another preferred embodiment, in step (ii), the reaction temperature is 4-40°C, preferably 15-35°C, more preferably 20-30°C, such as 25°C.

[0096] In another preferred embodiment, in step (ii), the reaction time is 12-48 h, preferably 18-36 h, more preferably 20-30 h, such as 24 h.

[0097] In another preferred embodiment, in step (iii), the amount of ethanol used is 3-6 times that of the reaction solution, preferably 3.5-5 times, more preferably 3.8-4.5 times, such as 4 times.

[0098] In another preferred embodiment, in step (iv), the unbound polyphenols are washed with ethanol, such as 95% ethanol.

[0099] In another preferred embodiment, step (iv) includes: redissolving the precipitate in water and then freeze-drying it.

[0100] In another preferred embodiment, the pectin-polyphenol complex is a pectin-gallic acid covalent complex.

[0101] In another preferred embodiment, the degree of gallic acid grafting in the pectin-gallic acid covalent complex is 1.5-3.5 mg / g, preferably 2-3 mg / g, more preferably 2.4-2.8 mg / g, and even more preferably 2.69±0.13 mg / g.

[0102] Whey protein nanofibers

[0103] Whey protein nanofibers refer to whey protein with a slender nanofiber morphology and a diameter of approximately 10-20 nanometers.

[0104] Preferably, the whey protein nanofibers are prepared by a method comprising the following steps:

[0105] (a-1) Provides an aqueous solution of whey protein;

[0106] (a-2) Adjust the pH of the above whey protein aqueous solution to 1.5-2.5, and then heat it at 75-95℃ for 3-8 hours;

[0107] (a-3) After heating, the reaction solution is cooled to room temperature to obtain whey protein nanofibers.

[0108] In another preferred embodiment, in step (a-1), the concentration of whey protein in the aqueous solution is 1-8 wt%, preferably 2-6 wt%, more preferably 3-5 wt%, such as 4 wt%.

[0109] In another preferred embodiment, step (a-2) includes adjusting the pH of the aqueous solution of the whey protein to 1.8-2.2 and then heating it at 80-90°C for 5-6 hours.

[0110] In another preferred embodiment, in step (a-3), the cooling is rapid cooling in a water bath.

[0111] Prolonged heating under acidic conditions can cause whey protein to self-assemble into nanofibers, thereby exposing more hydrophobic groups.

[0112] whey protein nanofibers-pectin-polyphenol complex

[0113] The whey protein nanofiber-pectin-polyphenol complex of this invention is a composite fiber network formed by the entanglement of whey protein nanofibers and a pectin-polyphenol covalent complex. The whey protein nanofibers and the pectin-polyphenol covalent complex are typically bonded in a non-covalent manner. For example, under low pH conditions, the protein nanofibers are positively charged, and the pectin is negatively charged; the two are bonded through non-covalent interactions, primarily electrostatic interactions.

[0114] Proteins and polysaccharides have good biocompatibility and degradability. Proteins can enhance the interfacial adsorption capacity of polysaccharides, while polysaccharides can give proteins higher stability.

[0115] The interfacial properties of polyphenols and their ability to alter the structure of proteins and polysaccharides can significantly improve the interfacial activity of protein-polysaccharide complexes. Therefore, protein-polysaccharide-polyphenol ternary complexes show broad application prospects in the preparation of food-grade Pickering emulsions.

[0116] Preparation method

[0117] This invention also provides a method for preparing the protein-polysaccharide-polyphenol ternary complex as described herein, comprising the steps of:

[0118] (i) Provide an aqueous solution comprising whey protein nanofibers and a pectin-gallic acid complex;

[0119] (ii) The pH of the solution is adjusted to 3-5 and the reaction is stirred to obtain the ternary complex.

[0120] In another preferred embodiment, step (i) includes mixing an aqueous whey protein solution and an aqueous pectin-gallic acid complex solution to obtain the aqueous solution containing whey protein and the pectin-gallic acid complex.

[0121] In another preferred embodiment, in step (i), the concentration of whey protein nanofibers in the aqueous solution is 0.5-2 wt%, preferably 0.8-1.5 wt%, and more preferably 1-1.2 wt%.

[0122] In another preferred embodiment, in step (i), the concentration of the pectin-gallic acid complex in the aqueous solution is 0.1-1 wt%, preferably 0.2-0.8 wt%, and more preferably 0.4-0.6 wt%.

[0123] In another preferred embodiment, in step (ii), the reaction temperature is 4-40°C, preferably 15-35°C, more preferably 20-30°C, such as 25°C.

[0124] In another preferred embodiment, in step (ii), the reaction time is 0.5-2 h, preferably 45 min-1.5 h, such as 1 h.

[0125] In another preferred embodiment, in step (ii), the pH is 3.5-4.5, preferably 4-4.2.

[0126] use

[0127] The present invention also provides the use of the aforementioned protein-polysaccharide-polyphenol ternary complex as an interface stabilizer / surfactant, particularly as an interface stabilizer / surfactant in Pickering emulsions.

[0128] The use of the aforementioned protein-polysaccharide-polyphenol ternary complex as an interface stabilizer / activator for Pickering emulsions is provided.

[0129] The use of the aforementioned protein-polysaccharide-polyphenol ternary complex as an interface stabilizer / activator in the preparation of Pickering emulsions is provided.

[0130] Furthermore, the present invention also provides a Pickering emulsion, wherein the Pickering emulsion uses the protein-polysaccharide-polyphenol ternary complex described in the present invention as an interface stabilizer / activator.

[0131] In another preferred embodiment, the Pickering emulsion is a water- and edible oil-based Pickering emulsion.

[0132] In another preferred embodiment, the edible oil is selected from the group consisting of soybean oil, peanut oil, corn oil, rapeseed oil, sunflower oil, olive oil, coconut oil, sesame oil, or combinations thereof.

[0133] In another preferred embodiment, the content of the protein-polysaccharide-polyphenol ternary complex in the Pickering emulsion is 0.1-2 wt%, more preferably 0.2-1 wt%, such as 0.3 wt%, 0.5 wt%, 0.6 wt%, or 0.8 wt%.

[0134] In another preferred embodiment, the volume ratio of water to edible oil in the Pickering emulsion is 1:1-2, preferably 1:1.2-1.8, and more preferably 1:1.4-1.6.

[0135] The Pickering emulsion of the present invention can be used as a fat substitute to prepare low-fat foods, such as low-fat meat products.

[0136] In another preferred embodiment, the Pickering emulsion is prepared by mixing an aqueous solution of a ternary complex with a vegetable oil phase. The mixture is then homogenized (e.g., at 10,000-20,000 rpm for 2-8 min) to obtain the Pickering emulsion.

[0137] Preferably, the stable storage time (without stratification) of the Pickering emulsion is ≥10 days, ≥20 days, or even ≥30 days.

[0138] The main advantages of this invention include:

[0139] This invention provides a novel whey protein nanofiber-pectin-polyphenol complex that surprisingly exhibits excellent interfacial stabilizer / surfactant properties, making it suitable as an emulsifier / interfacial stabilizer / surfactant for Pickering emulsions.

[0140] Furthermore, a Pickering emulsion comprising the whey protein nanofiber-pectin-polyphenol complex of the present invention as an emulsifier / interface stabilizer / surfactant is also provided, wherein the Pickering emulsion can maintain the emulsion state better and for a longer period of time, thereby extending the shelf life.

[0141] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0142] Example 1

[0143] (1) Preparation of pectin-gallic acid covalent complex: 1 g of pectin and 1 g of gallic acid were dissolved in 50 mL of distilled water respectively, and the two solutions were mixed. 0.25 g of ascorbic acid was added to 100 mL of the mixed solution. After stirring for 10 min, 4 mL of 5 mol / L hydrogen peroxide solution was added to initiate the reaction. The reaction was carried out under a nitrogen atmosphere for 24 h. After the reaction was completed, 4 volumes of 95% ethanol were added to the mixed solution. After standing for 8 h to precipitate, the precipitate was collected and washed with 95% ethanol to remove unbound gallic acid. The precipitate was reconstituted and lyophilized. The gallic acid content was 2.69 ± 0.13 mg / g (determined using the Folin-Ciocalteu method).

[0144] (2) Preparation of whey protein nanofibers: 4g of whey protein was dissolved in 100mL of distilled water. The pH of the whey protein solution was adjusted to 2.0 and then heated in a water bath at 85℃ for 5h. After heating, the solution was rapidly cooled to room temperature in a water bath to obtain the whey protein nanofiber solution.

[0145] (3) Preparation of the ternary complex: 100 mL of 2% whey protein nanofiber solution and 100 mL of 1% pectin-gallic acid complex were mixed. The pH of the mixed solution was adjusted to 4.0 at room temperature and stirred for 1 h.

[0146] (4) Preparation of Pickering emulsion: 200 mL of the ternary complex solution prepared in (3) was mixed with 300 mL of vegetable oil (sunflower seed oil). The mixture was homogenized at 16000 rpm for 3 min to obtain the Pickering emulsion.

[0147] Example 2

[0148] To fully illustrate the effect of fibrinization on the emulsifying ability of proteins and the enhancing effect of gallic acid on the emulsifying ability of whey protein nanofiber-pectin complexes, whey protein, whey protein nanofibers, and unmodified pectin-whey protein nanofiber complexes were used as controls, and their physicochemical properties were characterized. For ease of representation, whey protein was named WP, ​​whey protein nanofibers were named WPF, whey protein nanofiber-pectin complexes were named WPF-P, and whey protein nanofiber-pectin-gallic acid complexes were named WPF-PG. Based on this, Pickering emulsions were prepared using WPF, WPF-P, and WPF-PG, and their physicochemical properties were characterized. The WPF-stabilized emulsion was named WE, the WPF-P-stabilized emulsion was named WPE, and the WPF-PG-stabilized emulsion was named WPGE.

[0149] (1) Interfacial wettability of the complex

[0150] The three-phase contact angle (θ) of WP, WPF, WPF-P and WPF-PG are as follows: Figure 1 As shown. The contact angle reflects the hydrophilicity or hydrophobicity of a material. When θ < 90°, a smaller θ indicates a more hydrophilic material. When θ > 90°, a larger θ indicates a more hydrophobic material. It is generally believed that the closer θ is to 90°, the stronger the adsorption capacity of the complex at the interface. WP exhibits strong hydrophilicity. During the fibrosis process, the hydrophobic groups inside WP are exposed, thus enhancing the surface hydrophobicity of WPF. The interaction between pectin and WPF further enhances the hydrophobicity of WPF-P. Among the four samples, WPF-PG has the closest θ to 90°. One possible reason is that the interfacial properties of polyphenols themselves, as well as their binding with WPF and pectin, give the ternary complex the best emulsifying activity.

[0151] (2) Microstructure of the complex

[0152] Microstructures of different complexes, such as Figure 2 As shown, WP exhibits granular structures of varying sizes. WPF formed by the aggregation of WP consists of elongated fibers with a diameter of approximately 10-20 nanometers. There are significant morphological differences between WPF-P and WPF-PG. In WPF-P, pectin and WPF first aggregate in an orderly manner to form fiber bundles, which further aggregate to form a composite fiber network. In WPF-PG, GA groups hinder the orderly arrangement between pectin and GA and bind to hydrophobic amino acid residues on the WPF surface. Therefore, pectin-GA and WPF are directly entangled, forming a denser composite fiber network.

[0153] (3) Rheological properties of Pickering emulsion

[0154] Figure 3The storage modulus (G')(A) and loss modulus (G”)(B) of WE, WPE, and WPGE are shown as a function of angular velocity. The addition of pectin and the pectin-gallic acid complex improved the viscoelasticity of the emulsion, with pectin-GA showing a more significant reinforcing effect. The G' and G” values ​​of WPE and WPGE are significantly higher than those of WE, because the composite fiber network has higher strength than the single fiber network. The composite fiber network forms a stronger continuous layer at the interface. Both G' and G” values ​​of WPGE are higher than those of WPE, indicating that the composite fiber network formed in a direct interlacing mode can form a more robust three-dimensional network outside the droplet. The increased inter-fiber entanglement tightness may be the reason for the improved network strength around the oil droplet.

[0155] (4) Microstructure of Pickering emulsion

[0156] Microstructures of different emulsions, such as Figure 4 As shown, WE has a higher droplet diameter and greater inhomogeneity than WPE and WPGE. WPF's hydrophobicity and weaker network strength make it difficult to effectively prevent the aggregation of WE droplets. WPF-P forms a strong and ordered three-dimensional network around the droplets, resulting in smaller and more uniform droplet diameters in WPE. Compared to WPF-P, WPF-PG has a more compact network structure and stronger adsorption and fixation capabilities at the oil-water interface. Therefore, WPGE has the smallest droplet diameter and the most uniform distribution.

[0157] (5) Stability of Pickering emulsion

[0158] Figure 5 The storage stability (A) and centrifugal stability (B) of different emulsions are shown. After 10 days of storage at room temperature and centrifugation at 2000 rpm for 10 min, both WE and WPE showed significant phase separation. However, the WPGE group did not show significant phase separation, indicating that WPF-PG emulsion exhibited the strongest emulsion stability.

[0159] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A protein-polysaccharide-polyphenol ternary complex, characterized in that, The ternary complex is a complex formed by whey protein nanofibers and a pectin-polyphenol covalent complex; The polyphenols are selected from the group consisting of gallic acid, chlorogenic acid, catechin, epigallocatechin gallate, or combinations thereof.

2. The complex according to claim 1, characterized in that, In the whey protein nanofiber-pectin-polyphenol complex, the mass ratio of whey protein nanofiber to pectin-polyphenol complex is 1:0.2-1, preferably 1:0.3-0.8, and more preferably 1:0.4-0.

6.

3. The complex according to claim 1, characterized in that, The pectin-polyphenol covalent complex is a pectin-gallic acid covalent complex.

4. The complex according to claim 1, characterized in that, In the pectin-polyphenol covalent complex, the degree of polyphenol grafting is 1.5-3.5 mg / g, preferably 2-3 mg / g, more preferably 2.4-2.8 mg / g, and even more preferably 2.6-2.7 mg / g.

5. The complex according to claim 1, characterized in that, The whey protein nanofibers are prepared by a method comprising the following steps: (a-1) Provides an aqueous solution of whey protein; (a-2) Adjust the pH of the above whey protein aqueous solution to 1.5-2.5, and then heat it at 75-95℃ for 3-8 hours; (a-3) After heating, the reaction solution is cooled to room temperature to obtain whey protein nanofibers.

6. The complex according to claim 1, characterized in that, The ternary complex is prepared by a method comprising the following steps: (i) Provide an aqueous solution containing whey protein nanofibers and a pectin-polyphenol complex; (ii) The pH of the solution is adjusted to 3-5 and the reaction is stirred to obtain the ternary complex.

7. The method for preparing the protein-polysaccharide-polyphenol ternary complex as described in claim 1, comprising the steps of: (i) Provide an aqueous solution comprising whey protein nanofibers and a pectin-gallic acid complex; (ii) The pH of the solution is adjusted to 3-5 and the reaction is stirred to obtain the ternary complex.

8. A Pickering emulsion, characterized in that, The Pickering emulsion uses the protein-polysaccharide-polyphenol ternary complex of the first aspect of the present invention as an interface stabilizer / activator.

9. The use of the protein-polysaccharide-polyphenol ternary complex as described in claim 1 as an interface stabilizer / activator for Pickering emulsions.

10. The use of the protein-polysaccharide-polyphenol ternary complex as described in claim 1 as an interface stabilizer / activator in the preparation of Pickering emulsion.

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