Preparation method of glycosylation modified casein-phenylalanine conjugate

Abstract

The invention provides a preparation method of a glycosylation modified casein-phenylalanine conjugate. The preparation method comprises the following steps: carrying out acid-base modification on casein, preparing glycosylation modified casein, conjugating the glycosylation modified casein and phenylalanine, purifying and freeze-drying. The casein is modified by the N-acetylglucosamine and the phenylalanine together, so that a new compound is formed. The monosaccharide, amino acid and casein composite system can significantly improve the stability and encapsulation efficiency of the Pickering emulsion through interfacial adsorption, steric hindrance, electrostatic repulsion and other mechanisms. Casein has a dissolution-condensation property under different pH values, so that the casein has a pH response switching characteristic. A composite system formed by combining the glycosylated modified casein and the amino acid can realize a responsive behavior through a specific enzyme. According to the invention, the preparation conditions of the Pickering emulsion are optimized, so that the delivery and controlled release of hydrophobic substances including high phenols are improved.

Description

Technical Field

[0001] The present invention belongs to the fields of biochemistry and food technology, and particularly relates to a method for preparing a glycosylated modified casein-phenylalanine conjugate. Background Art

[0002] Microencapsulation technology, especially Pickering emulsion technology, has attracted much attention in the fields of food, medicine, chemical engineering, and biotechnology due to its unique interfacial stability and responsiveness. A Pickering emulsion is an emulsion stabilized by solid particles that form a physical barrier at the oil-water interface, thereby stabilizing the emulsion. Compared with traditional surfactant-stabilized emulsions, Pickering emulsions have better biocompatibility and environmental friendliness, and at the same time provide more functional properties, such as multiple responsiveness and sustained-release performance. Casein is a natural protein present in milk and is widely used in the food industry due to its good nutritional and functional properties. Casein's excellent emulsifying and foam-forming abilities make it an ideal material for preparing Pickering emulsions. In addition, the surface of casein has abundant amino acid residues, and its properties can be further improved through chemical modification, such as enhancing its interaction with sugars or amino acids. Casein adsorbs at the oil-water interface to form an interfacial film, and monosaccharides and amino acids can enhance the stability of the interfacial film through intermolecular interactions. Casein is also a protein with pH responsiveness, which mainly stems from the characteristics of amino acid residues in its molecular structure. Amino acids with ionizable side chains in casein, such as lysine, arginine, and glutamic acid, etc. These amino acid residues will exhibit different charge states at different pH values, thereby affecting the solubility, aggregation behavior of casein, and its interaction with other molecules. Therefore, in drug delivery systems, casein can be designed to release drugs at specific pH values. For example, under the conditions of simulating gastric juice (acidic environment) and intestinal juice (alkaline environment), the casein carrier can respond to pH changes and control the drug release rate. N-acetylglucosamine (GlcNAc) is a chemical substance with a variety of physiological functions and broad application prospects. It is a white powder or crystalline powder, easily soluble in water, and hardly soluble in organic solvents such as ethanol and propanol. Sugar molecules and casein can undergo covalent binding through enzymatic glycosylation or the Maillard reaction to form glycosylated modified casein. The synergistic effect of the two significantly improves the Pickering emulsion in terms of interfacial stability, anti-aggregation ability, and anti-external stress ability, etc. It plays an important role in the fields of medicine, health products, cosmetics, and food. Phenylalanine (Phe) is an essential amino acid with special chemical and physical properties. Due to its aromatic ring structure, phenylalanine can form stable complexes with various substances. The amino group of phenylalanine can covalently bind to the carboxyl group or glycosyl moiety of casein, and can also non-covalently bind to casein through hydrogen bonds, electrostatic interactions or hydrophobic interactions, enhancing the stability of the emulsion. In addition, casein and phenylalanine can undergo a proteoid reaction to form a special type of peptide bond - isopeptide bond. Different from conventional peptide bonds, it involves the reaction between other side-chain amino groups and carboxyl groups to form amide bonds. This bond can be induced by chemical methods, such as using chemical reagents like N-hydroxysuccinimide ester (NHS ester) and dicyclohexylcarbodiimide (DCC). The formation of isopeptide bonds is of great significance in protein engineering and drug design, as it can be used to change the structure and function of proteins, or to create protein conjugates with specific properties. The composite system formed after the combination of glycosylated modified casein and amino acids can exhibit responsive behavior through specific enzymes (such as proteases, glycosidases, etc.). Proteases (such as trypsin, pepsin) can specifically cleave the peptide bonds of casein, disrupting the structure of the composite system and releasing functional molecules to achieve targeted delivery or functional regulation. Glycosidases (such as α-amylase, β-galactosidase) can hydrolyze the sugar chains in glycosylated modified casein, changing its solubility and interfacial properties. At the same time, the hydrolysis of sugar chains leads to the destruction of the structure of the composite system and the release of encapsulated functional molecules. Certain oxidoreductases (such as glucose oxidase) can also catalyze the redox reaction of sugar molecules in glycosylated modified casein, changing its structure and function. In the small intestine, proteases such as trypsin and chymotrypsin will hydrolyze proteins containing phenylalanine into individual amino acids, releasing encapsulated functional molecules in the small intestine. At present, the research on the stabilization of Pickering emulsions by casein composite systems mostly focuses on the interaction between polysaccharides and casein, while there is little research on the use of monosaccharides, amino acids and casein composite systems to stabilize Pickering emulsions. Making Pickering emulsions have better biocompatibility and higher stability, while providing more functional properties, such as multiple responsiveness and slow-release performance, has always been the direction pursued by technicians. Develop an efficient and simple method for modifying casein to increase its solubility, and then use monosaccharide modification and amino acid modification methods to prepare stable food-grade Pickering emulsions. Due to the solubility-aggregation properties of casein at different pH values, as well as the enzymatic hydrolysis characteristics of sugars, proteins and phenylalanine, the modified casein has pH-responsive switching characteristics and enzyme responsiveness. Therefore, it is proposed to use N-acetylglucosamine and phenylalanine together to modify and modify casein to form a new complex, and to control the structure and interfacial properties of the complex by controlling the synthesis conditions, further control the emulsion stability and encapsulation performance, and achieve the purpose of controlled release of the target substance. Summary of the Invention

[0003] Technical problems to be solved: To improve the stability and functional properties of Pickering emulsions and enhance the delivery and controlled release capabilities of hydrophobic substances including high phenols. The object of the present invention is to provide a preparation method of a glycosylated modified casein-phenylalanine conjugate, including casein acid-base modification, preparation of glycosylated protein, conjugation of glycosylated modified casein and phenylalanine, purification and lyophilization. This method not only optimizes the preparation conditions, stability and functionality of its Pickering emulsion, but the glycosylated modified casein-phenylalanine conjugate also has pH-responsive switching characteristics and enzyme responsiveness. By controlling the synthesis conditions to affect the structure and interfacial properties of the complex, further control the emulsion stability and encapsulation performance, and achieve the purpose of controlled release of the target substance.

[0004] Technical solution: A preparation method of a glycosylated modified casein-phenylalanine conjugate, comprising the following steps: S1. Preparation of casein solution: Dissolve casein powder in phosphate buffer solution to obtain a casein solution; S2. Casein acid-base modification: Adjust the pH value of the casein solution to 9.0-12.0 with sodium hydroxide solution, and then adjust the pH of the solution to 7.0 with hydrochloric acid solution, dilute and stir to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: Add N-acetylglucosamine to the acid-base modified casein solution, and heat to react to obtain glycosylated modified casein; Preparation of N-hydroxysuccinimide ester: Add N-Boc-phenylalanine, N-hydroxysuccinimide, and dicyclohexylcarbodiimide to dimethylformamide for reaction, then filter and evaporate, and crystallize in 2-propanol to obtain N-hydroxysuccinimide ester; Activation of glycosylated modified casein: Add the glycosylated modified casein prepared in step S3 to a buffer solution, add the N-hydroxysuccinimide ester solution prepared in S4, and incubate at room temperature to obtain a modified casein solution; Hydrolysis of ester bond: Add the modified casein solution to a phosphate buffer solution containing hydroxylamine and hydrolyze under alkaline conditions; Purification of casein: Purify the modified casein solution by dialysis or gel filtration; Lyophilization: Lyophilize the purified casein to obtain N-Boc-phenylalanine-glycosylated modified casein; Removal of Boc group: Treat the N-Boc-phenylalanine-glycosylated modified casein obtained in S8 with anhydrous trifluoroacetic acid, remove the anhydrous trifluoroacetic acid, disperse the residue in Tris-HCl buffer solution, adjust the pH to 8 - 9 with sodium hydroxide solution, and lyophilize after dialysis to obtain a glycosylated modified casein-phenylalanine conjugate. Further, the concentration of the casein solution in step S1 is (10 - 15) wt%. Further, the pH of the phosphate buffer solution in step S1 is 7.0. Further, the pH value of the casein solution is adjusted to 12.0 with sodium hydroxide solution in step S2. Further, the concentration of the acid-base modified casein solution in step S2 is (2 - 5) wt%. Further, the mass ratio of the acid-base modified casein to N-acetylglucosamine in step S3 is (2 - 1):(1 - 5). Further, the mass ratio of the acid-base modified casein to N-acetylglucosamine is 1:5. Further, the heating reaction temperature in step S3 is 70 - 90 °C and the time is 2 - 4 h. Further, the heating reaction temperature is 90 °C and the time is 4 h. Further, the molar ratio of N-Boc-phenylalanine, N-hydroxysuccinimide, and dicyclohexylcarbodiimide in step S4 is 1:(1.1 - 1.5):(1.1 - 1.5), the reaction time is 10 - 20 h, and the temperature is 0 - 10 °C. Further, the reaction time is 16 hours and the temperature is 4 °C. Further, in step S5, the glycosylated modified casein is added to a Hepes buffer solution or a sodium borate buffer solution with a pH of 8.0 - 9.0. Further, in step S5, the concentration of the N-hydroxysuccinimide ester solution is (8 - 12) wt%. Further, in step S5, the molar ratio of the glycosylated modified casein to the N-hydroxysuccinimide ester is 1:(1.2 - 2.0), and the incubation time at room temperature is 2 - 3 h. Further, in step S6, the concentration of hydroxylamine is 0.4 - 0.6 M, the alkaline condition is pH (8.0 - 9.0), and the hydrolysis time is 2 - 3 h. Further, the concentration of hydroxylamine is 0.5 M, and the alkaline condition is pH 8.0. Further, in step S9, the mass-to-volume ratio of N-Boc-phenylalanine-glycosylated modified casein to anhydrous trifluoroacetic acid is 100:(1 - 2), and the treatment time is 30 - 50 min. Beneficial effects: 1. The present invention uses N-acetylglucosamine and phenylalanine to jointly modify casein. The best binding effect is achieved when the ratio of casein to N-acetylglucosamine is 1:5 and heated for 4 hours. Casein stabilizes the emulsion through interfacial adsorption, steric hindrance, and electrostatic repulsion, while monosaccharides enhance the mechanical strength and stability of the interfacial film through hydrogen bonding, thickening, and antioxidant properties; 2. Compared with polysaccharide-modified proteins, the present invention uses monosaccharides and phenylalanine to jointly modify casein. A thick film is formed at the interface of the composite system of the three, providing steric hindrance. The charged groups of amino acids can also increase the electrostatic repulsion between droplets, preventing droplet coalescence, and significantly improving the stability and encapsulation efficiency of Pickering emulsions; 3. The composite system after the combination of glycosylated modified casein and amino acids can achieve responsive behavior through specific enzymes (such as proteases, glycosidases, etc.), thereby realizing specific functional regulation. The present invention enables Pickering emulsions to achieve functions such as targeted release, controlled release, and intelligent response during the delivery of drugs, polyphenolic substances, and functional substances; 4. The pH-responsive casein in the present invention can be designed to release drugs at a specific pH value. For example, under the conditions of simulating gastric juice (acidic environment) and intestinal juice (alkaline environment), the carrier can respond to pH changes and control the release rate of substances; 5. The composite system after the combination of glycosylated modified casein and amino acids in the present invention is added to a Pickering emulsion prepared by embedding lycopene in tomato seed oil, which can improve the emulsion stability and the antioxidant capacity of lycopene. Description of the drawings Figure 1 Particle size diagram of Pickering emulsions prepared with different ratios and heating times of casein and N-acetylglucosamine Figure 2 Particle size diagrams of Pickering emulsions prepared in Example 1 and Comparative Examples 1-5; Figure 3 Entrapment efficiency diagrams of Pickering emulsions prepared in Example 1 and Comparative Examples 1-5; Figure 4 Creaming index diagrams of Pickering emulsions prepared in Example 1 and Comparative Examples 1-5; Figure 5 Antioxidant results diagrams of Pickering emulsions prepared in Example 1 and Comparative Examples 1-5; Figure 6 Particle size diagrams of Pickering emulsions prepared in Example 1 and Comparative Examples 1-5 at different pH values; Detailed implementation mode The present invention will be further described below in conjunction with the drawings and embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments: Example 1 A preparation method of a glycosylated modified casein-phenylalanine conjugate, comprising the following steps: S1. Preparation of casein solution: 10 g of casein powder is dissolved in 100 mL of 0.01 M phosphate buffer solution with pH 7.0, and magnetically stirred for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH value of the casein solution is adjusted to 12.0 with 2.0 M sodium hydroxide solution, and continuously magnetically stirred for 30 min. Then the pH of the solution is adjusted to 7.0 with hydrochloric acid solution, and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: N-acetylglucosamine is added to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to N-acetylglucosamine is 1:5, and heated and reacted at 90 °C for 4 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide are added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 hours, then filtered and evaporated, and crystallized in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 is added to 0.1 M Hepes buffer solution (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 is added. The molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester is 1:1.5, and incubated at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: Add the modified casein solution to a phosphate buffer of 0.5 M hydroxylamine and hydrolyze it for 2 h at pH 8.0; S7. Casein purification: Purify the modified casein solution by dialysis or gel filtration; S8. Lyophilization: Lyophilize the purified casein to obtain N-Boc-phenylalanine-glycosylated modified casein; S9. Removal of Boc group: Treat 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained in S8 with 1 mL of anhydrous trifluoroacetic acid for 30 min, rotary evaporate to remove anhydrous trifluoroacetic acid, disperse the residue in Tris-HCl buffer, slowly adjust the pH to 8 - 9 with a lower concentration of sodium hydroxide solution, dialyze and then lyophilize to obtain the glycosylated modified casein-phenylalanine conjugate. Example 2 A preparation method of a glycosylated modified casein-phenylalanine conjugate, comprising the following steps: S1. Casein solution preparation: Dissolve 10 g of casein powder in 100 mL of 0.01 M phosphate buffer at pH 7.0, and stir magnetically for 30 min to obtain a casein solution; S2. Acid-base modification of casein: Adjust the pH value of the casein solution to 12.0 with 2.0 M sodium hydroxide solution, continuously stir magnetically for 30 min, then adjust the pH of the solution to 7.0 with hydrochloric acid solution, dilute it to 2 wt% with pure water, and stir to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: Add N-acetylglucosamine to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to N-acetylglucosamine is 1:5, and heat and react at 90 °C for 3 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: Add N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide in a molar ratio of 1:1.5:1.5 to dimethylformamide and react at 4 °C for 16 h, then filter, evaporate, and crystallize in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: Add the glycosylated modified casein prepared in step S3 to 0.1 M Hepes buffer (pH 8.0), add the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4, and the molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester is 1:1.5, incubate at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: Add the modified casein solution to a phosphate buffer of 0.5 M hydroxylamine and hydrolyze it for 2 h at pH 8.0; S7. Casein purification: Purify the modified casein solution by dialysis or gel filtration; S8. Lyophilization: Lyophilize the purified casein to obtain N-Boc-phenylalanine-glycosylated modified casein; S9. Removal of Boc group: Treat 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained in S8 with 1 mL of anhydrous trifluoroacetic acid for 30 min, rotary evaporate to remove anhydrous trifluoroacetic acid, disperse the residue in Tris-HCl buffer, slowly adjust the pH to 8 - 9 with a lower concentration of sodium hydroxide solution, dialyze and then lyophilize to obtain glycosylated modified casein-phenylalanine conjugate. Example 3 A preparation method of glycosylated modified casein-phenylalanine conjugate, comprising the following steps: S1. Casein solution preparation: Dissolve 10 g of casein powder in 100 mL of 0.01 M phosphate buffer at pH 7.0, stir magnetically for 30 min to obtain a casein solution; S2. Acid-base modification of casein: Adjust the pH value of the casein solution to 12.0 with 2.0 M sodium hydroxide solution, continuously stir magnetically for 30 min, then adjust the pH of the solution to 7.0 with hydrochloric acid solution, dilute to 2 wt% with pure water, and stir to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: Add N-acetylglucosamine to the acid-base modified casein solution, the mass ratio of acid-base modified casein to N-acetylglucosamine is 1:5, heat and react at 90 °C for 2 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: Add N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide in a molar ratio of 1:1.5:1.5 to dimethylformamide, react at 4 °C for 16 hours, then filter and evaporate, and crystallize in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: Add the glycosylated modified casein prepared in step S3 to 0.1 M Hepes buffer (pH 8.0), add the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4, the molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester is 1:1.5, incubate at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: Add the modified casein solution to 0.5 M hydroxylamine phosphate buffer, and hydrolyze at pH 8.0 for 2 h; S7. Casein purification: Purify the modified casein solution by dialysis or gel filtration; S8. Lyophilization: Lyophilize the purified casein to obtain N-Boc-phenylalanine-glycosylated modified casein; Removal of the Boc group: 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained by treating S8 with 1 mL of anhydrous trifluoroacetic acid was treated for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8-9 with a lower concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-phenylalanine conjugate. Example 4 A preparation method of a glycosylated modified casein-phenylalanine conjugate, comprising the following steps: S1. Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and magnetically stirred for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH value of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution, and the mass ratio of the acid-base modified casein to N-acetylglucosamine was 1:4, and heated and reacted at 90 °C for 4 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 hours, then filtered and evaporated, and crystallized in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 was added to 0.1 M Hepes buffer (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 was added. The molar ratio of the glycosylated modified casein to the N-hydroxysuccinimide ester was 1:1.5, and incubated at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: The modified casein solution was added to 0.5 M hydroxylamine phosphate buffer and hydrolyzed at pH 8.0 for 2 h; S7. Purification of casein: The modified casein solution was purified by dialysis or gel filtration; S8. Freeze-drying: The purified casein was freeze-dried to obtain N-Boc-phenylalanine-glycosylated modified casein; Removal of the Boc group: 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained from S8 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a lower concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-phenylalanine conjugate. Example 5 A preparation method of a glycosylated modified casein-phenylalanine conjugate, comprising the following steps: S1. Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and magnetically stirred for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution, and the mass ratio of the acid-base modified casein to N-acetylglucosamine was 1:3. The reaction was carried out by heating at 90 °C for 4 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 h, then filtered and evaporated, and crystallized in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 was added to 0.1 M Hepes buffer (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 was added. The molar ratio of the glycosylated modified casein to the N-hydroxysuccinimide ester was 1:1.5, and incubated at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: The modified casein solution was added to 0.5 M hydroxylamine phosphate buffer and hydrolyzed at pH 8.0 for 2 h; S7. Purification of casein: The modified casein solution was purified by dialysis or gel filtration; S8. Freeze-drying: The purified casein was freeze-dried to obtain N-Boc-phenylalanine-glycosylated modified casein; Removal of Boc group: 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained from S8 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a low concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-phenylalanine conjugate. Comparative Example 1 The difference between this comparative example and Example 1 is that phenylalanine is not added, and the specific steps are as follows: S1. Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and stirred magnetically for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to N-acetylglucosamine was 1:5. The reaction was carried out by heating at 90 °C for 4 h to obtain glycosylated modified casein; S4. Purification of casein: The modified casein solution was purified by dialysis or gel filtration; S5. Freeze-drying: The purified casein was freeze-dried. Comparative Example 2 The difference between this comparative example and Example 1 is that polysaccharide-modified casein is used, and the specific steps are as follows: S1. Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and stirred magnetically for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: Dextran was added to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to dextran was 1:5. The reaction was carried out by heating at 90 °C for 4 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 hours. Then it was filtered, evaporated, and crystallized in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Glycosylation-modified casein activation: Add the glycosylation-modified casein prepared in step S3 to 0.1 M Hepes buffer (pH 8.0), and add the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4. The molar ratio of glycosylation-modified casein to N-hydroxysuccinimide ester is 1:1.5. Incubate at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: Add the modified casein solution to 0.5 M hydroxylamine phosphate buffer and hydrolyze at pH 8.0 for 2 h; S7. Casein purification: Purify the modified casein solution by dialysis or gel filtration; S8. Lyophilization: Lyophilize the purified casein to obtain N-Boc-phenylalanine-glycosylation-modified casein; S9. Removal of Boc group: Treat 50 mg of N-Boc-phenylalanine-glycosylation-modified casein obtained in S8 with 1 mL of anhydrous trifluoroacetic acid for 30 min. Rotate and evaporate to remove anhydrous trifluoroacetic acid. Disperse the residue in Tris-HCl buffer and slowly adjust the pH to 8 - 9 with a lower concentration of sodium hydroxide solution. After dialysis, lyophilize to obtain a glycosylation-modified casein-phenylalanine conjugate. Comparative Example 3 The difference between this comparative example and Example 1 is that casein is not glycosylation-modified, as follows: S1. Casein solution preparation: Dissolve 10 g of casein powder in 100 mL of 0.01 M phosphate buffer at pH 7.0 and stir magnetically for 30 min to obtain a casein solution; S2. N-hydroxysuccinimide ester preparation: Add N-Boc-phenylalanine, N-hydroxysuccinimide, and dicyclohexylcarbodiimide in a molar ratio of 1:1.5:1.5 to dimethylformamide and react at 4 °C for 16 h. Then filter, evaporate, and crystallize in 2-propanol to obtain N-hydroxysuccinimide ester; S3. Casein activation: Add the casein solution prepared in step S1 to 0.1 M Hepes buffer (pH 8.0), and add the N-hydroxysuccinimide ester solution (10 wt%) prepared in S2. The molar ratio of casein to N-hydroxysuccinimide ester is 1:1.5. Incubate at room temperature for 2 h to obtain a modified casein solution; S4. Ester bond hydrolysis: Add the modified casein solution to 0.5 M hydroxylamine phosphate buffer and hydrolyze at pH 8.0 for 2 h; S5. Casein purification: Purify the modified casein solution by dialysis or gel filtration; S6. Lyophilization: Lyophilize the purified casein to obtain N-Boc-phenylalanine-casein; Removal of the Boc group: 50 mg of N-Boc-phenylalanine-casein obtained from S6 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a lower concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the casein-phenylalanine conjugate. Comparative Example 4 The difference between this comparative example and Example 1 is that lysine was used instead of phenylalanine, which is specifically as follows: S1. Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and magnetically stirred for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then, the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain the acid-base modified casein solution; S3. Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to N-acetylglucosamine was 1:5. It was heated and reacted at 90 °C for 4 h to obtain the glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: N-Boc-lysine, N-hydroxysuccinimide, and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 h, then filtered, evaporated, and crystallized in 2-propanol to obtain the N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 was added to 0.1 M Hepes buffer (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 was added. The molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester was 1:1.5, and incubated at room temperature for 2 h to obtain the modified casein solution; S6. Ester bond hydrolysis: The modified casein solution was added to 0.5 M hydroxylamine phosphate buffer and hydrolyzed at pH 8.0 for 2 h; S7. Purification of casein: The modified casein solution was purified by dialysis or gel filtration; S8. Freeze-drying: The purified casein was freeze-dried to obtain N-Boc-lysine-glycosylated modified casein; Removal of Boc group: 50 mg of N-Boc-lysine-glycosylated modified casein obtained from S8 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a lower concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-lysine conjugate. Comparative Example 5 The difference between this comparative example and Example 1 is that glutamic acid was used instead of phenylalanine, as follows: S1. Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and stirred magnetically for 30 min to obtain a casein solution; S2. Acid-base modification of casein: The pH of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; S3. Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to N-acetylglucosamine was 1:5. It was heated and reacted at 90 °C for 4 h to obtain glycosylated modified casein; S4. Preparation of N-hydroxysuccinimide ester: N-Boc-glutamic acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 h, then filtered and evaporated, and crystallized in 2-propanol to obtain N-hydroxysuccinimide ester; S5. Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 was added to 0.1 M Hepes buffer (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 was added. The molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester was 1:1.5, and incubated at room temperature for 2 h to obtain a modified casein solution; S6. Ester bond hydrolysis: The modified casein solution was added to 0.5 M hydroxylamine phosphate buffer and hydrolyzed at pH 8.0 for 2 h; S7. Purification of casein: The modified casein solution was purified by dialysis or gel filtration; S8. Freeze-drying: The purified casein was freeze-dried to obtain N-Boc-glutamic acid-glycosylated modified casein; Removal of Boc group: 50 mg of N-Boc-glutamic acid-glycosylated modified casein obtained from S8 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a low concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-glutamic acid conjugate. Comparative Example 6 The difference between this comparative example and Example 1 lies in that the mass ratio of acid-base modified casein to N-acetylglucosamine is (1:6), which is specifically as follows: Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and stirred magnetically for 30 min to obtain a casein solution; Acid-base modification of casein: The pH of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain an acid-base modified casein solution; Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution, and the mass ratio of acid-base modified casein to N-acetylglucosamine was 1:6. It was heated and reacted at 90 °C for 4 h to obtain glycosylated modified casein; Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 h, then filtered and evaporated, and crystallized in 2-propanol to obtain N-hydroxysuccinimide ester; Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 was added to 0.1 M Hepes buffer (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 was added. The molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester was 1:1.5, and it was incubated at room temperature for 2 h to obtain a modified casein solution; Hydrolysis of ester bond: The modified casein solution was added to 0.5 M hydroxylamine phosphate buffer and hydrolyzed at pH 8.0 for 2 h; Purification of casein: The modified casein solution was purified by dialysis or gel filtration; Freeze-drying: The purified casein was freeze-dried to obtain N-Boc-phenylalanine-glycosylated modified casein; Removal of Boc group: 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained in S8 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a lower concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-phenylalanine conjugate. Comparative Example 7 The difference between this comparative example and Example 1 is that the heating reaction time of the acid-base modified casein solution and N-acetylglucosamine is 5 h, which is specifically as follows: Preparation of casein solution: 10 g of casein powder was dissolved in 100 mL of 0.01 M phosphate buffer at pH 7.0 and magnetically stirred for 30 min to obtain a casein solution; Acid-base modification of casein: The pH value of the casein solution was adjusted to 12.0 with 2.0 M sodium hydroxide solution, and magnetic stirring was continued for 30 min. Then the pH of the solution was adjusted to 7.0 with hydrochloric acid solution and diluted to 2 wt% with pure water, and stirred to obtain the acid-base modified casein solution; Preparation of glycosylated modified casein: N-acetylglucosamine was added to the acid-base modified casein solution. The mass ratio of the acid-base modified casein to N-acetylglucosamine was 1:6, and the reaction was heated at 90 °C for 5 h to obtain the glycosylated modified casein; Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide were added to dimethylformamide at a molar ratio of 1:1.5:1.5 and reacted at 4 °C for 16 hours, then filtered and evaporated, and crystallized in 2-propanol to obtain the N-hydroxysuccinimide ester; Activation of glycosylated modified casein: The glycosylated modified casein prepared in step S3 was added to 0.1 M Hepes buffer (pH 8.0), and the N-hydroxysuccinimide ester solution (10 wt%) prepared in S4 was added. The molar ratio of the glycosylated modified casein to the N-hydroxysuccinimide ester was 1:1.5, and incubated at room temperature for 2 h to obtain the modified casein solution; Hydrolysis of ester bond: The modified casein solution was added to 0.5 M hydroxylamine phosphate buffer and hydrolyzed at pH 8.0 for 2 h; Purification of casein: The modified casein solution was purified by dialysis or gel filtration; Freeze-drying: The purified casein was freeze-dried to obtain N-Boc-phenylalanine-glycosylated modified casein; Removal of Boc group: 50 mg of N-Boc-phenylalanine-glycosylated modified casein obtained from S8 was treated with 1 mL of anhydrous trifluoroacetic acid for 30 min. The anhydrous trifluoroacetic acid was removed by rotary evaporation. The residue was dispersed in Tris-HCl buffer, and the pH was slowly adjusted to 8 - 9 with a lower concentration of sodium hydroxide solution. After dialysis, it was freeze-dried to obtain the glycosylated modified casein-phenylalanine conjugate. Performance measurement: 1. Preparation of Pickering emulsion The prepared conjugate was uniformly dispersed in ultrapure water to be fully dissolved as the aqueous phase (3%). Lycopene was dissolved in tomato seed oil as the oil phase. They were mixed evenly at a ratio of aqueous phase:oil phase = 4:1, and homogenized with a high-speed homogenizer at 20000 rpm for 3 min to obtain Pickering emulsion. 2. Average particle size of Pickering emulsion Droplets of different Pickering emulsion samples were taken, diluted by the same multiple with ultrapure water, and a certain amount was taken into a sample bottle to measure its particle size. Particle size is an important indicator to characterize the stability of the emulsion. Generally, the smaller the particle size, the higher the stability of the emulsion system. As Figure 1 shown, with the increase of N-acetylglucosamine, the particle size of the emulsion decreased to a certain extent. When the ratio of casein to N-acetylglucosamine was 1:5 (Example 1), the particle size was the smallest. When the ratio of N-acetylglucosamine continued to increase (Comparative Example 6), the particle size began to increase. In addition, when the heating time was 2 - 4 h, the particle size decreased with the increase of heating time, and there was no significant difference in particle size when it increased from 4 h to 5 h (Comparative Example 7). The results of the particle size of the Pickering emulsion prepared in Example 1 and Comparative Examples 1 - 5 are as Figure 2 shown. Compared with Comparative Examples 1 - 3, the particle size in Example 1 decreased significantly. Although the particle sizes in Comparative Examples 4 and 5 were lower than those in Comparative Examples 1 - 3, they were still larger than that in Example 1, indicating that lysine and glutamic acid can also reduce the particle size of the emulsion, but the effect is lower than that of phenylalanine. 3. Determination of the encapsulation rate of lycopene in Pickering emulsion Take 0.5 mL of the sample Pickering emulsion, extract lycopene with an organic phase solution, collect the supernatant, and calculate the content of free lycopene according to the formula using the calibration curve of lycopene, and calculate the encapsulation rate of lycopene: Encapsulation rate (%) = C1 / C0 × 100% Where: C1 is the mass concentration (μg / mL) of lycopene in the newly prepared emulsion; C0 is the mass concentration (μg / mL) of the added lycopene. As Figure 3As shown, different carriers have significantly different encapsulation rates of lycopene in Pickering emulsions. Among them, the Pickering emulsion of lycopene encapsulated by the glycosylated modified casein-phenylalanine conjugate (Example 1) has the highest encapsulation rate, reaching 73.19%; the Pickering emulsions stabilized by conjugates prepared with lysine (Comparative Example 4) or glutamic acid (Comparative Example 5) instead of phenylalanine have lower encapsulation rates, which are 67.08% and 64.03% respectively; however, the Pickering emulsions stabilized without adding monosaccharides (Comparative Example 3) or amino acids (Comparative Example 1) have significantly lower encapsulation rates than Example 1, only 41.85% and 59.18%. This is mainly because the structure of the conjugate used for encapsulation has changed, losing the interaction between sugar, protein and amino acid, and the stability of the interfacial film has decreased. In addition, the increase in the encapsulation rate with the addition of monosaccharides and amino acids may be due to the increased solubility of the conjugate and the thicker wall layer resulting in better stabilization of lycopene in the oil phase. 3. Determination of the creaming index of Pickering emulsions The creaming index is an index characterizing the stability of emulsions. The smaller the creaming index, the higher the emulsification stability of the emulsion. The calculation formula for the creaming index (CI) is: where Hc is the height of the creaming layer and Ht is the total height. The results of the creaming index are as Figure 4 shown. The glycosylated modified casein-amino acid conjugates (Example 1, Control Examples 4 - 5) can form a mixed multi-molecular layer structure near the droplets, thereby reducing the degree of droplet aggregation and achieving the purpose of improving emulsification. The emulsion prepared with non-glycosylated casein (Control Example 3) has the lowest stability, and the covalent complexes of sugar and protein (Control Examples 1 and 2) have significantly improved emulsification stability. This is because casein stabilizes the emulsion through interfacial adsorption, steric hindrance and electrostatic repulsion, while monosaccharides enhance the mechanical strength and stability of the interfacial film through hydrogen bonding. The synergistic effect of the two significantly improves the interfacial stability, anti-aggregation and anti-external stress ability of Pickering emulsions. After adding amino acids, the three interact with each other, and the sugar and amino acids further enhance the stability of the interfacial film through intermolecular interactions. 4. Determination of the in vitro antioxidant activity of Pickering emulsions Lycopene itself has strong antioxidant activity. Encapsulating it in a covalent complex and homogenizing it into an emulsion may affect its antioxidant activity. The antioxidant activity of the emulsion was evaluated by the DPPH and ABTS radical scavenging rates. The higher the radical scavenging rate, the stronger the antioxidant ability. (1) Determination of the DPPH radical scavenging rate Prepare a 1.75×10-4mol / L DPPH solution with ethanol. Take 2 ml of the sample emulsion diluted to a certain concentration, dissolve the analyte with the DPPH solution, mix well, and let stand for 30 min. Measure its absorbance at 517 nm. Where: Ai is the absorbance of 2 mL of DPPH solution and 2 mL of sample solution; Aj is the absorbance of 2 mL of sample extract and 2 ml of ethanol; Ac is the absorbance of 2 mL of DPPH solution and 2 mL of ethanol. (2) Determination of ABTS radical scavenging rate Take 1 ml of the sample solution diluted to a certain concentration, add 3 ml of ABTS+ solution (7 mM ABTS solution and 2.45 mM K2S2O2 are mixed in equal proportions and reacted overnight (12 h - 16 h) to prepare the ABTS·+ stock solution. Adjust the absorbance value of the ABTS·+ reaction solution at 734 nm to 0.70 ± 0.02;), shake for 30 s, react in the dark at room temperature for 60 min, and then measure the absorbance value at 734 nm wavelength. ABTS· + Scavenging rate (%) = (A control - A test ) * 100 / A contro Where: Acontrol- is the absorbance value of the control tube; Atest is the absorbance value of the analyte sample. As Figure 5 shown, the DPPH radical scavenging rate of the lycopene Pickering emulsion stabilized by casein without glycosylation modification (Comparative Example 3) is the lowest. After modification with polysaccharide (Control Example 2) or monosaccharide (Control Example 1), the DPPH radical scavenging rate increases, but is significantly lower than that of Example 1. The ABTS radical scavenging rate is consistent with DPPH. The reason for this result may be that the Pickering emulsion prepared from the glycosylated modified casein-phenylalanine conjugate has the highest encapsulation rate, the stability of the encapsulated lycopene is improved, the loss caused by the decomposition of lycopene is reduced, and its antioxidant property is enhanced. 6. Effect of pH on Pickering emulsion Take equal amounts of different emulsions and adjust the pH of the emulsions to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and measure the particle size of the emulsions. Since casein has pH responsiveness, it affects the solubility, aggregation behavior of casein complexes at different pH values, and their interactions with other molecules. As Figure 6As shown, with the increase of pH value, the particle size of Pickering emulsion gradually decreases and the stability improves. Under acidic conditions (pH < 4.6), the glycosylated modified casein-phenylalanine conjugate may aggregate and its solubility decreases, resulting in a decline in its ability as an emulsifier. In the range of pH 6.0 - 8.0, the protein has good solubility and can effectively adsorb at the oil-water interface to form a stable Pickering emulsion. By adjusting the pH or adding stabilizers, the performance of the emulsion can be optimized to meet different application requirements. As described above, it is only the preferred embodiment of the present invention, and there is no any form of restriction on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into an equivalent embodiment with equivalent changes without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a glycosylated modified casein-phenylalanine conjugate, characterized in that: The following steps are involved: S1. Preparation of glycosylated modified casein: N-acetylglucosamine is added to the acid-base modified casein solution and then heated to obtain glycosylated modified casein; S2. Preparation of N-hydroxysuccinimide ester: N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide are added to dimethylformamide for reaction, and then filtered, evaporated and crystallized to obtain N-hydroxysuccinimide ester; S3. Activation of glycosylated modified casein: adding the glycosylated modified casein prepared in step S1 to a buffer, adding the N-hydroxysuccinimide ester solution prepared in step S2, and incubating at room temperature to obtain a modified casein solution; S4. Casein purification: The modified casein solution prepared in step S3 is subjected to esterification, purification, and freeze-drying to obtain N-Boc-phenylalanine-glycosylated modified casein; S5. Removal of Boc group: Treat the N-Boc-phenylalanine-glycosylated modified casein obtained in S4 with anhydrous trifluoroacetic acid, remove the anhydrous trifluoroacetic acid, disperse the residue in Tris-HCl buffer, adjust the pH to 8-9 with sodium hydroxide solution, dialyze and freeze-dry to obtain a glycosylated modified casein-phenylalanine conjugate.

2. The method for preparing the glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: The preparation method of the acid-base modified casein solution comprises the following steps: S11. Preparation of casein solution: casein powder was dissolved in phosphate buffer to obtain a casein solution with a concentration of (10-15) wt%; S12. Acid-base modification of casein: adjust the pH value of the casein solution to 9.0-12.0 with sodium hydroxide solution, then adjust the pH value of the solution to 7.0 with hydrochloric acid solution, and dilute to obtain an acid-base modified casein solution with a concentration of (2-5) wt%.

3. The method for preparing a glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: In step S1, the mass ratio of acid-base modified casein and N-acetylglucosamine is (2-1):(1-5); the heating reaction temperature is 70-90° C. and the time is 2-4 hours.

4. The method for preparing the glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: In step S2, the molar ratio of N-Boc-phenylalanine, N-hydroxysuccinimide and dicyclohexylcarbodiimide is 1:(1.1-1.5):(1.1-1.5), the reaction time is 10-20 hours, and the temperature is 0-10°C.

5. The method for preparing a glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: In step S3, the glycosylated modified casein is added to a Hepes buffer or a sodium borate buffer with a pH of 8.0-9.0, and the concentration of the N-hydroxysuccinimide ester solution is (8-12) wt%.

6. The method for preparing a glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: In step S3, the molar ratio of glycosylated modified casein to N-hydroxysuccinimide ester is 1:(1.2-2.0), and the incubation time at room temperature is 2-3 hours.

7. The method for preparing the glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: The step S4 casein purification comprises the following steps: S41. adding the modified casein solution obtained in S3 to a phosphate buffer containing hydroxylamine and hydrolyzing it under alkaline conditions; S42. A purified casein solution is obtained by dialysis or gel filtration; S43. Freeze-dry the purified casein solution to obtain N-Boc-phenylalanine-glycosylated modified casein.

8. The method for preparing a glycosylated modified casein-phenylalanine conjugate according to claim 7, characterized in that: In the step S41, the concentration of hydroxylamine in the phosphate buffer is 0.4-0.6 M; the alkaline condition is pH (8.0-9.0), and the hydrolysis is carried out for 2-3 hours.

9. The method for preparing a glycosylated modified casein-phenylalanine conjugate according to claim 1, characterized in that: In step S5, the mass volume ratio of N-Boc-phenylalanine-glycosylated modified casein to anhydrous trifluoroacetic acid is 100:(1-2), and the treatment time is 30-50 min.

10. Use of the glycosylated modified casein-phenylalanine conjugate prepared according to the preparation method according to any one of claims 1 to 9 in preparing a high-load and high-stability Pickering emulsion.

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