Preparation process and application of AA-CM-Fru modified emulsifier

Abstract

The invention provides a preparation process and application of an AA-CM-Fru modified emulsifier, and belongs to the technical field of emulsifiers, and the preparation process comprises the following steps: S1, pretreating cow milk to obtain skim cow milk, performing kappa-casein genotype analysis on the skim cow milk, screening cow milk containing AA genotype kappa-casein, and extracting casein micelles in the screened cow milk; s2, modifying casein micelles containing AA genotype kappa-casein; S21, preparing a phosphate buffer solution; s22, weighing casein micelles containing AA genotype kappa-casein extracted in the step S1, dissolving the casein micelles in a phosphate buffer solution, and stirring and mixing until the casein micelles are completely dissolved; according to the method, fructose is covalently grafted to the surface of AA genotype-containing kappa-casein micelles through Maillard reaction, the formed AA-CM-Fru modified emulsifier has stronger amphipathy and interfacial adsorption capacity, the emulsified medium-chain triglyceride emulsion is smaller in particle size and more uniform in distribution, and the emulsifying activity and the emulsion stability are obviously improved compared with those of a traditional casein emulsifier.

Description

Technical Field

[0001] This invention belongs to the field of emulsifier technology, specifically a preparation process and application of an AA-CM-Fru modified emulsifier. Background Technology

[0002] In the food, pharmaceutical, and cosmetic industries, emulsifiers are core materials for stabilizing oil-water mixtures, and their performance directly affects product quality and shelf life. Traditional emulsifiers, such as synthetic surfactants, can achieve basic emulsification functions, but they suffer from poor stability, easy oxidation, and insufficient biocompatibility, leading to easy emulsion separation and rancidity, which limits their application in high-end products. Protein emulsifiers have gradually become a research hotspot due to their natural origin and good biocompatibility. Among them, casein micelles (CM), as the main protein complex in bovine milk, have an amphiphilic structure, but their emulsifying properties are significantly affected by the κ-casein genotype. κ-casein is the only glycosylated subtype of CM, and its gene polymorphism (AA, AB, BB types) directly determines the particle size, electrostatic repulsion, and steric hindrance of CM. Studies have shown that AA-type κ-casein CMs have larger particle sizes and lower glycosylation levels, but higher emulsifying activity and stability indices. However, traditional emulsifier preparation processes do not fully utilize genotype differences and often use a single emulsifier, resulting in insufficient interfacial membrane mechanical strength. These membranes are prone to rupture under heating, ultraviolet light, or in the presence of metal ions, leading to poor emulsion stability. To improve emulsifier performance, existing technologies modify proteins through Maillard reactions to form protein-polysaccharide covalent complexes. While this technology can significantly improve emulsifying performance, it has the following key problems: the reaction conditions lack systematic optimization, resulting in low modification efficiency and unstable product performance. In addition, it does not screen and apply to specific genotypes, thus failing to fully utilize the regulatory role of genotypes on emulsifying performance.

[0003] In view of the above problems, this invention proposes a preparation process and application of AA-CM-Fru modified emulsifier. Through genotype screening, Maillard reaction modification and process parameter optimization, the synergistic improvement of emulsifying performance, nutrient retention and stability is achieved, providing a solution for the efficient utilization of functional lipids. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned problems in existing technologies by proposing a preparation process and application of AA-CM-Fru modified emulsifier.

[0005] The objective of this invention can be achieved through the following technical solutions: A preparation process and application of an AA-CM-Fru modified emulsifier, comprising the following steps: S1: Pre-treat the milk to obtain skim milk, perform κ-casein genotyping on the skim milk, screen for milk containing AA genotype κ-casein, and then extract casein micelles from the screened milk. S2: Modification of casein micelles containing AA genotype κ-casein, including the following steps: S21: Accurately weigh appropriate amounts of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or disodium hydrogen phosphate and sodium dihydrogen phosphate, dissolve them in ultrapure water or deionized water, and prepare a 0.01 M phosphate buffer solution with a pH of 7.0. S22: Weigh the casein micelles containing AA genotype κ-casein extracted in step S1 and dissolve them in phosphate buffer solution. Stir and mix until the casein micelles are completely dissolved. Adjust the volume to the target volume with PBS buffer to make the final casein micelle concentration accurate to 10 mg / mL. Adjust the pH to 7 to obtain a casein micelle solution containing AA genotype κ-casein (AA-CM). S23: Calculate the required mass of fructose (Fru) according to the ratio of proteoglycan 1:(1-3). Weigh the fructose and add it to a container containing AA-CM solution. Stir the mixture at 600 rpm for 15 minutes on a magnetic stirrer or vortex shaker until the fructose is completely dissolved and a homogeneous mixed solution is formed. The casein micelle-fructose mixture is obtained. Adjust the pH to 7. S24: Transfer the casein micelle-fructose mixture to a container suitable for a constant temperature rotary water bath, seal the container opening, and fix the seal. S25: Place the sealed and reinforced container containing the casein micelle-fructose mixture into a rotating water bath, ensuring that most of the container is submerged below the water bath surface to ensure good heat conduction. Set the temperature to (40-80)℃, the rotation speed to 500 r / min, and rotate at a constant temperature for (90-150) min to start the Maillard reaction. S26: After the Maillard reaction is complete, immediately remove the container containing the reaction sample from the constant temperature water bath; S27: Quickly immerse the container in a large beaker or water tank containing a sufficient amount of ice-water mixture, ensuring that the ice-water mixture completely surrounds the container, and continuously and gently shake or stir the outside of the container until the internal temperature of the sample drops to room temperature, to obtain the AA-CM-Fru modified emulsifier.

[0006] Preferably, in step S2, the liquid chromatography column is a Jupiter® 5 µm C4 300 Å (LC Column 250 × 2 mm); the column temperature is 40 °C; mobile phase A is a 0.05% aqueous solution of trifluoroacetic acid, and mobile phase B is a 0.05% solution of trifluoroacetic acid acetonitrile.

[0007] Preferably, the milk pretreatment in step S1 includes filtering the collected fresh milk to remove impurities such as sand and hair, rapidly cooling the sample temperature to 4°C, filtering the milk through a double-layer defatted gauze, centrifuging the milk to separate the fat layers and filter them out, and filtering the remaining fat again through a double-layer defatted gauze to obtain skimmed milk.

[0008] Preferably, the κ-casein genotype analysis in step S1 includes sampling the milk after S1 pretreatment, analyzing it by liquid chromatography to obtain milk containing AA genotype κ-casein, and then labeling it.

[0009] Preferably, the casein micelle extraction in step S1 includes mixing equal volumes of milk containing AA genotype κ-casein that was screened and labeled in step S1, adjusting the pH to 4.6, and extracting casein micelles containing AA genotype κ-casein by acid precipitation.

[0010] Preferably, the acid precipitation method for extracting casein micelles specifically includes the following steps: S31: Add 10% CH3COOH to milk containing AA genotype κ-casein at a volume ratio of 1:10, let stand for 5 minutes until precipitation occurs; S32: Add 1 M CH3COONa at a volume ratio of 1:10 to the milk containing precipitate from step S31, measure the pH value again to 4.6, and let stand for 5 min. S33: After centrifuging at 1000 g for 10 min at 4℃, the casein was washed twice in cold water and then centrifuged at 1000 g for 5 min at 4℃ to obtain casein micelles, which were then stored at 4℃.

[0011] Preferably, the AA-CM-Fru emulsion, prepared by emulsifying medium-chain triglycerides with a modified emulsifier, comprises the following components by weight: 200 parts of AA-CM-Fru modified emulsifier, 5 parts of medium-chain triglycerides, and 0.005 parts of β-carotene.

[0012] Preferably, the method for preparing the AA-CM-Fru emulsion specifically includes the following steps: S6: Weigh out β-carotene and dissolve it in medium-chain triglycerides. Stir and mix for 10 min at 500 r / min to obtain the oil phase. S7: Take AA-CM-Fru modified emulsifier as the aqueous phase, adjust the pH to 7, and add an appropriate amount of oil phase to the aqueous phase to make its mass fraction 2.5%; S8: Use a high-speed shearing machine to shear at high speed for 3 minutes at a speed of 12000 r / min to obtain a crude emulsion. Then use an ultra-high pressure homogenizer to perform secondary high-pressure homogenization, with the first stage at 85 MPa and the second stage at 10 MPa, for 3 cycles to obtain the AA-CM-Fru emulsion.

[0013] Compared with existing technologies, the medium-frequency heat treatment process for this support roller has the following advantages: 1. The present invention provides a preparation process and application of an AA-CM-Fru modified emulsifier. By covalently grafting fructose onto the surface of AA-genotype κ-casein micelles through Maillard reaction, the resulting AA-CM-Fru modified emulsifier has stronger amphiphilicity and interfacial adsorption capacity. The emulsified medium-chain triglyceride emulsion has smaller particle size and more uniform distribution, and its emulsifying activity and emulsion stability are significantly improved compared with traditional casein emulsifiers.

[0014] 2. The present invention provides a preparation process and application of an AA-CM-Fru modified emulsifier. By using κ-casein genotype analysis technology, milk containing AA genotype κ-casein is selected as raw material. The casein micelles containing AA genotype κ-casein have better hydrophobicity and surface activity than other genotypes. The extracted casein micelles are used as a modification substrate, ensuring the emulsification potential from the source. This increases the content of active ingredients in the final modified emulsifier by more than 20%, significantly improving the consistency and functionality of the product.

[0015] 3. The present invention provides a preparation process and application of AA-CM-Fru modified emulsifier. It uses milk by-products as raw materials and extracts casein micelles efficiently by acid precipitation, reducing resource waste. The modification process does not require organic solvents or chemical cross-linking agents, which is in line with the concept of green chemistry. Fructose, as a natural sugar source, replaces synthetic emulsifiers, reducing production costs and improving the natural properties of the product.

[0016] 4. The present invention provides a preparation process and application of AA-CM-Fru modified emulsifier. Through orthogonal experiments, the Maillard reaction conditions were systematically optimized to determine the optimal preparation parameters, which significantly improved the covalent binding efficiency of fructose and casein micelles. Variance analysis further verified the key influence of Maillard reaction conditions on the modification effect, providing reliable process parameters for large-scale production.

[0017] In summary, this invention provides a preparation process and application of AA-CM-Fru modified emulsifier. Through genotype screening, Maillard reaction modification, and process parameter optimization, it achieves a synergistic improvement in emulsifying performance, nutrient retention, and stability, providing a solution for the efficient utilization of functional lipids and possessing significant economic and social benefits. Attached Figure Description

[0018] Figure 1 This is an HPLC-MS chromatogram of AA genotype κ-casein in this invention; Figure 2 This is a comparison diagram of the emulsifying activities of AA-CM and AA-CM-Fru in this invention; Figure 3 This is a comparison chart of the emulsification stability of AA-CM and AA-CM-Fru in this invention; Figure 4 This is a microscopic image of AA-CM under a transmission electron microscope in this invention; Figure 5 This is a microscopic image of AA-CM-Fru in this invention under a transmission electron microscope; Figure 6 This is a low-field nuclear magnetic resonance imaging image of the AA-CM emulsion in this invention; Figure 7 This is a low-field nuclear magnetic resonance imaging image of the AA-CM-Fru emulsion in this invention; Figure 8 This is a microscopic image of the AA-CM emulsion in this invention under a transmission electron microscope; Figure 9 This is a microscopic image of the AA-CM-Fru emulsion in this invention under a transmission electron microscope; Figure 10 This is a microscopic image of the AA-CM emulsion in this invention under a laser confocal microscope; Figure 11 This is a microscopic image of the AA-CM-Fru emulsion in this invention under a laser confocal microscope. Detailed Implementation

[0019] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments.

[0020] Example 1: A preparation process for an AA-CM-Fru modified emulsifier includes the following steps: S1: Pre-treatment of the milk involved filtering the collected fresh milk to remove impurities such as sand and hair. The sample temperature was rapidly cooled to 4°C and maintained at this temperature before being transported to the laboratory for later use. The collected milk was then filtered through double-layered defatted gauze and centrifuged at 2643×g for 15 min at 25°C to remove the supernatant fat. The remaining fat was filtered again through double-layered defatted gauze to obtain skimmed milk, which was stored at -20°C. For genotyping using chromatography, samples were taken and analyzed using HPLC-MS, combining high-performance liquid chromatography and mass spectrometry. This allowed the components separated by HPLC to be directly analyzed by MS. HPLC-MS was used to detect and compare the κ-casein genotype of the milk samples. The results are as follows: Figure 1 As shown in the figure. Analysis revealed signal peaks for glycosylated κ-casein variants A / B and glycosylated variant A at 12.7 min and 12.2 min, respectively, and a signal peak for κ-casein variant A at 12.8 min. Therefore, it was determined that... Figure 1 For AA-type κ-casein, milk containing AA-type κ-casein was screened and labeled, and then mixed in equal volumes. The pH was adjusted to 4.6, and casein micelles containing AA-type κ-casein were extracted by acid precipitation. 10% CH3COOH was added to the milk containing AA-type κ-casein at a volume ratio of 1:10. After standing for 5 min until precipitation occurred, 1 M CH3COONa was added to the milk containing the precipitate at a volume ratio of 1:10 to flocculate the casein micelles. The pH value was measured again and controlled at 4.6. After standing for 5 min, the centrifuge tube containing the precipitated and flocculated milk was centrifuged at 1000 g for 10 min at 4℃. The casein micelles were then washed twice in cold water and centrifuged at 1000 g for 5 min at 4℃. This process was repeated twice to complete the casein micelle washing process, and the casein micelles were obtained and stored at 4℃. S2: Modification of casein micelles containing AA genotype κ-casein, including the following steps: S21: Accurately weigh appropriate amounts of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or disodium hydrogen phosphate and sodium dihydrogen phosphate, dissolve them in ultrapure water or deionized water, and prepare a certain volume of 0.01 M phosphate buffer solution with a pH of 7.0. S22: Weigh the casein micelles containing AA genotype κ-casein extracted in step S1 and dissolve them in phosphate buffer solution. Stir and mix until the casein micelles are completely dissolved and clear without precipitation. Adjust the volume to the target volume with PBS buffer to make the final casein micelle concentration accurate to 10 mg / mL. Adjust the pH to 7 with 6M HCl and 6M NaOH to obtain the casein micelle solution containing AA genotype κ-casein (AA-CM). S23: Calculate the required mass of fructose (Fru) according to the ratio of casein micelle polysaccharide 1:(1-3). Weigh the fructose and add it to a beaker containing AA-CM solution. Stir the mixture at 600 rpm for 15 minutes on a magnetic stirrer or vortex shaker until the fructose is completely dissolved and a homogeneous mixed solution is formed. The casein micelle-fructose mixture is obtained. Adjust the pH to 7 with 6M HCl and 6M NaOH. S24: Transfer the casein micelle-fructose mixture into an Erlenmeyer flask, insert a magnetic rotor, seal the container opening with tin foil, and secure the seal with a rubber band. S25: Place the sealed and reinforced container containing the casein micelle-fructose mixture into a water bath. Secure the water bath fixing ring to the mouth of the conical flask and fix the conical flask to ensure that most of the container is submerged below the water bath surface to ensure good heat conduction. Set the temperature to (40-80)℃, the rotation speed to 500 r / min, and oscillate at a constant temperature for (90-150) min to start the Maillard reaction. S26: After the Maillard reaction is complete, immediately remove the conical flask containing the reaction sample from the constant temperature water bath; S27: Quickly immerse the container in a large beaker containing a sufficient amount of ice-water mixture, ensuring that the ice-water mixture completely surrounds the container, and continuously and gently shake or stir the outside of the container until the internal temperature of the sample drops to room temperature, thus obtaining the Maillard product, namely the AA-CM-Fru modified emulsifier.

[0021] Table 1. Orthogonal experimental factor levels for the preparation conditions of casein micelles-fructose modified emulsifiers Table 2. Optimization results of orthogonal experiments on the preparation conditions of casein micelles-fructose modified emulsifiers Table 3. Results of orthogonal experimental variance analysis of preparation conditions for casein micelle-fructose modified emulsifiers As shown in Table 1, based on single-factor experiments, orthogonal optimization was performed on three factors affecting AA-CM-Fru (heating temperature, proteoglycan ratio, and heating time): heating temperature 40-80℃, proteoglycan ratio 1:(1-3), and heating time 90-150 min. Grafting degree was used as the evaluation criterion, and the results are shown in Table 2. The order of influence of the three factors on the grafting degree of AA-CM-Fru is heating temperature > proteoglycan ratio > heating time, as shown in Table 3. Analysis of variance shows that heating temperature and proteoglycan ratio have significant effects on the modified emulsifier, while heating time has no significant effect. Therefore, the optimal preparation conditions for AA-CM-Fru are selected as heating temperature 80℃, proteoglycan ratio 1:2, and heating time 90 min. Considering food safety issues, pH 7 was chosen.

[0022] Grafting degree determination method: The grafting degree of casein micelle-fructose modified emulsifier was determined by the OPA method. 80 mg of o-phthalaldehyde was accurately weighed into a 100 mL beaker, and 2 mL of methanol solution was added to dissolve it completely. Then, 5.0 mL of 20% SDS and 50 mL of 0.1 mol / L borax solution were added. Finally, 200 μL of β-mercaptoethanol was added, mixed well, and the solution was brought to a final volume of 100 mL with distilled water. The OPA solution was prepared fresh and stored away from light.

[0023] For the determination, accurately weigh 30 mg of casein micelle-fructose modified emulsifier lyophilized product, dissolve it in 10 mL of distilled water in a warm water bath to a concentration of 3.0 g / L, take 200 μL of sample solution, add 4 mL of OPA solution, and use distilled water as a control instead of the sample solution; after mixing evenly, place it in a constant temperature water bath at 35℃ and react in the dark for 2 min, then use a UV spectrophotometer to measure the absorbance at 340 nm, and calculate the grafting degree according to (Formula 1): (Formula 1) In Formula 1, DG: grafting degree, %, A0: absorbance of AA-CM solution, At: absorbance of AA-CM-Fru modified product.

[0024] Example 2: Based on Example 1, A preparation process for an AA-CM-Fru modified emulsifier includes the following steps: S1: Pre-treatment of the milk involved filtering the collected fresh milk to remove impurities such as sand and hair. The sample temperature was rapidly cooled to 4°C and maintained at this temperature before being transported to the laboratory for later use. The collected milk was then filtered through double-layered defatted gauze and centrifuged at 2643×g for 15 min at 25°C to remove the supernatant fat. The remaining fat was filtered again through double-layered defatted gauze to obtain skimmed milk, which was stored at -20°C. For genotyping using chromatography, samples were taken and analyzed using HPLC-MS, combining high-performance liquid chromatography and mass spectrometry. This allowed the components separated by HPLC to be directly analyzed by MS. HPLC-MS was used to detect and compare the κ-casein genotype of the milk samples. The results are as follows: Figure 1 As shown in the figure. Analysis revealed signal peaks for glycosylated κ-casein variants A / B and glycosylated variant A at 12.7 min and 12.2 min, respectively, and a signal peak for κ-casein variant A at 12.8 min. Therefore, it was determined that... Figure 1For AA-type κ-casein, milk containing AA-type κ-casein was screened and labeled, and then mixed in equal volumes. The pH was adjusted to 4.6, and casein micelles containing AA-type κ-casein were extracted by acid precipitation. 10% CH3COOH was added to the milk containing AA-type κ-casein at a volume ratio of 1:10. After standing for 5 min until precipitation occurred, 1 M CH3COONa was added to the milk containing the precipitate at a volume ratio of 1:10 to flocculate the casein micelles. The pH value was measured again and controlled at 4.6. After standing for 5 min, the centrifuge tube containing the precipitated and flocculated milk was centrifuged at 1000 g for 10 min at 4℃. The casein micelles were then washed twice in cold water and centrifuged at 1000 g for 5 min at 4℃. This process was repeated twice to complete the casein micelle washing process, and the casein micelles were obtained and stored at 4℃. S2: Modification of casein micelles containing AA genotype κ-casein, including the following steps: S21: Accurately weigh appropriate amounts of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or disodium hydrogen phosphate and sodium dihydrogen phosphate, dissolve them in ultrapure water or deionized water, and prepare a certain volume of 0.01 M phosphate buffer solution with a pH of 7.0. S22: Weigh the casein micelles containing AA genotype κ-casein extracted in step S1 and dissolve them in phosphate buffer solution. Stir and mix until the casein micelles are completely dissolved and clear without precipitation. Adjust the volume to the target volume with PBS buffer to make the final casein micelle concentration accurate to 10 mg / mL. Adjust the pH to 7 with 6M HCl and 6M NaOH to obtain the casein micelle solution containing AA genotype κ-casein (AA-CM). S23: Calculate the required mass of fructose (Fru) according to the ratio of casein micelle polysaccharide 1:2. Weigh the fructose and add it to a beaker containing AA-CM solution. Stir the mixture at 600 rpm for 15 minutes on a magnetic stirrer or vortex shaker until the fructose is completely dissolved and a homogeneous mixed solution is formed. The casein micelle-fructose mixture is obtained. Adjust the pH to 7 with 6M HCl and 6M NaOH. S24: Transfer the casein micelle-fructose mixture into an Erlenmeyer flask, insert a magnetic rotor, seal the container opening with tin foil, and secure the seal with a rubber band. S25: Place the sealed and reinforced container containing the casein micelle-fructose mixture into a water bath. Secure the water bath fixing ring to the mouth of the conical flask and fix the conical flask to ensure that most of the container is submerged below the water bath surface to ensure good heat conduction. Set the temperature to 80℃, the rotation speed to 500 r / min, and oscillate at a constant temperature for 90 min to start the Maillard reaction. S26: After the Maillard reaction is complete, immediately remove the conical flask containing the reaction sample from the constant temperature water bath; S27: Quickly immerse the container in a large beaker containing a sufficient amount of ice-water mixture, ensuring that the ice-water mixture completely surrounds the container, and continuously and gently shake or stir the outside of the container until the internal temperature of the sample drops to room temperature, thus obtaining the Maillard product, namely the AA-CM-Fru modified emulsifier.

[0025] Example 3: A preparation process for an AA-CM-Fru modified emulsifier includes the following steps: The milk was pretreated by filtering the collected fresh milk to remove impurities such as sand and hair. The sample temperature was rapidly cooled to 4℃ and maintained at this temperature before being transported to the laboratory for later use. The collected milk was then filtered through double-layered defatted gauze and centrifuged at 2643×g for 15 min at 25℃ to remove the supernatant fat. The remaining fat was filtered again through double-layered defatted gauze to obtain skimmed milk, which was stored at -20℃. For genotyping, samples were taken and analyzed using HPLC-MS, combining high-performance liquid chromatography and mass spectrometry, allowing the HPLC-separated components to be directly analyzed by MS. HPLC-MS was used to detect and compare the κ-casein genotypes of the milk samples. The results are as follows: Figure 1 As shown in the figure. Analysis revealed signal peaks for glycosylated κ-casein variants A / B and glycosylated variant A at 12.7 min and 12.2 min, respectively, and a signal peak for κ-casein variant A at 12.8 min. Therefore, it was determined that... Figure 1 For AA-type κ-casein, milk containing AA-type κ-casein was screened and labeled, and then mixed in equal volumes. The pH was adjusted to 4.6, and casein micelles containing AA-type κ-casein were extracted by acid precipitation. 10% CH3COOH was added to the milk containing AA-type κ-casein at a volume ratio of 1:10. After standing for 5 min until precipitation occurred, 1M CH3COONa was added to the milk containing the precipitate at a volume ratio of 1:10 to flocculate the casein micelles. The pH value was measured again and controlled at 4.6. After standing for 5 min, the centrifuge tube containing the precipitated and flocculated milk was centrifuged at 1000 g for 10 min at 4℃. The casein micelles were then washed twice in cold water and centrifuged at 1000 g for 5 min at 4℃. This process was repeated twice to complete the casein micelle washing process, and the casein micelles were obtained and stored at 4℃.

[0026] Accurately weigh appropriate amounts of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or disodium hydrogen phosphate and sodium dihydrogen phosphate, dissolve them in ultrapure water or deionized water, and prepare a certain volume of 0.01 M phosphate buffer solution with a pH of 7.0. Weigh the extracted casein micelles containing AA genotype κ-casein and dissolve them in phosphate buffer solution. Stir and mix until the casein micelles are completely dissolved and clear without precipitation. Adjust the volume to the target volume with PBS buffer to make the final casein micelle concentration accurate to 10 mg / mL. Adjust the pH to 7 with 6M HCl and 6M NaOH to obtain the casein micelle solution containing AA genotype κ-casein (AA-CM).

[0027] like Figure 2 and Figure 3 As shown, EAI represents emulsifying activity and ESI represents emulsifying stability. The modified casein micelles containing AA genotype κ-casein exhibit significantly increased emulsifying activity and stability. This enhanced emulsifying performance is mainly attributed to the interfacial binding of the covalent sugar complex, which promotes the assembly of sugar molecules into a highly viscoelastic film around the oil droplets. Figure 4 and Figure 5 As shown, the particle size of casein micelles containing AA genotype κ-casein is significantly increased after modification. This is because the binding of sugar molecules to proteins may change the surface charge or conformation of the micelles, leading to partial aggregation.

[0028] Example 4: An application of an AA-CM-Fru modified emulsifier is to emulsify medium-chain triglycerides with the modified emulsifier to form an AA-CM-Fru emulsion, which includes the following components by weight: 200 parts of AA-CM-Fru modified emulsifier (10 mg / mL solution), 5 parts of medium-chain triglycerides, and 0.005 parts of β-carotene.

[0029] Weigh β-carotene and place it in a beaker. Add medium-chain triglycerides to the beaker and place a magnetic rotor into the beaker. Stir the mixture magnetically for 10 min at 500 r / min to obtain the oil phase. Take AA-CM-Fru modified emulsifier as the aqueous phase and adjust the pH to 7. Add an appropriate amount of the oil phase to the aqueous phase to make its mass fraction 2.5%. Use a high-speed shearing machine to shear at high speed for 3 min at 12000 r / min to obtain a crude emulsion. Then use an ultra-high pressure homogenizer for secondary high-pressure homogenization, first stage 85 MPa, second stage 10 MPa, cycle 3 times to obtain AA-CM-Fru emulsion.

[0030] Example 5: Weigh out 200 parts of casein micelle solution (AA-CM) containing AA genotype κ-casein, 5 parts of medium chain triglycerides, and 0.005 parts of β-carotene.

[0031] The weighed β-carotene was placed in a beaker, and medium-chain triglycerides were added to the beaker. A magnetic rotor was then placed in the beaker and magnetically stirred for 10 min at a speed of 500 r / min to obtain the oil phase. Unmodified casein micelle solution (AA-CM) containing AA genotype κ-casein was used as the aqueous phase, and the pH was adjusted to 7. An appropriate amount of the oil phase was added to the aqueous phase to make its mass fraction 2.5%. The crude emulsion was obtained by high-speed shearing for 3 min at a speed of 12000 r / min. Then, a second high-pressure homogenization was performed using an ultra-high pressure homogenizer, with the first stage at 85 MPa and the second stage at 10 MPa, for a total of 3 cycles to obtain the AA-CM emulsion.

[0032] like Figure 6 and Figure 7 As shown, from a distribution perspective, lighter colors indicate smaller particle sizes and more uniform emulsion distribution. Generally, the larger the red area, the more abundant the hydrogen protons, indicating a higher water content. Higher proton concentrations result in brighter NMR images, reflecting higher water content in the sample. The gradual lightening of the red color indicates a decrease in the proton density of the emulsion to varying degrees, ultimately forming emulsion structures of different densities. Compared to the AA-CM emulsion, the modified AA-CM-Fru emulsion has a lighter color distribution, smaller particle size, and more uniform distribution.

[0033] like Figure 8 and Figure 9 As shown, the modified AA-CM-Fru emulsion has a smaller droplet size compared to the AA-CM emulsion. This is because the Maillard reaction modification introduces more hydrophilic groups into the casein micelles, enhancing the surface activity of casein and making it easier to adsorb onto the oil-water interface, forming smaller and more uniform emulsion droplets. On the other hand, the casein after the Maillard reaction may reduce interfacial tension through more efficient interfacial adsorption, promoting the dispersion and refinement of droplets.

[0034] like Figure 10 and Figure 11As shown, confocal laser microscopy (CLSM) can observe the embedding of emulsions by emitting lasers of different wavelengths. Green represents proteins stained by laser irradiation, red represents oils stained by laser irradiation, and yellow represents the superposition of two lasers. From the perspective of emulsion embedding, the modified AA-CM-Fru emulsion has smaller droplet sizes, more uniform distribution, and better embedding effect compared to the AA-CM emulsion. This is partly due to the improved emulsifying properties of casein micelles after Maillard reaction modification, allowing the emulsifier to quickly and uniformly cover the oil droplet surface during emulsification, forming a dense and uniformly thick interfacial film. The hydrophilic portion of the sugar chains extends outward to form a spatial barrier, inhibiting droplet aggregation after collision and maintaining particle size uniformity. Furthermore, the Maillard reaction modifies casein micelles, forming a sugar-protein covalent complex, significantly improving its adsorption capacity and interfacial film stability at the oil-water interface, thus resulting in better embedding.

[0035] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A preparation process for an AA-CM-Fru modified emulsifier, characterized in that, Includes the following steps: S1: Pre-treat the milk to obtain skim milk, perform κ-casein genotyping on the skim milk, screen for milk containing AA genotype κ-casein, and then extract casein micelles from the screened milk. S2: Modification of casein micelles containing AA genotype κ-casein, including the following steps: S21: Accurately weigh appropriate amounts of potassium dihydrogen phosphate and dipotassium hydrogen phosphate or disodium hydrogen phosphate and sodium dihydrogen phosphate, dissolve them in ultrapure water or deionized water, and prepare a 0.01 M phosphate buffer solution with a pH of 7.

0. S22: Weigh the casein micelles containing AA genotype κ-casein extracted in step S1 and dissolve them in phosphate buffer solution. Stir and mix until the casein micelles are completely dissolved. Adjust the volume to the target volume with PBS buffer to make the final casein micelle concentration accurate to 10 mg / mL. Adjust the pH to 7 to obtain a casein micelle solution containing AA genotype κ-casein (AA-CM). S23: Calculate the required mass of fructose (Fru) according to the ratio of proteoglycan 1:(1-3). Weigh the fructose and add it to a container containing AA-CM solution. Stir the mixture at 600 rpm for 15 minutes on a magnetic stirrer or vortex shaker until the fructose is completely dissolved and a homogeneous mixed solution is formed. The casein micelle-fructose mixture is obtained. Adjust the pH to 7. S24: Transfer the casein micelle-fructose mixture to a container suitable for a constant temperature rotary water bath, seal the container opening, and fix the seal. S25: Place the sealed and reinforced container containing the casein micelle-fructose mixture into a rotating water bath, ensuring that most of the container is submerged below the water bath surface to ensure good heat conduction. Set the temperature to (40-80)℃, the rotation speed to 500 r / min, and rotate at a constant temperature for (90-150) min to start the Maillard reaction. S26: After the Maillard reaction is complete, immediately remove the container containing the reaction sample from the constant temperature water bath; S27: Quickly immerse the container in a large beaker or water tank containing a sufficient amount of ice-water mixture, ensuring that the ice-water mixture completely surrounds the container, and continuously and gently shake or stir the outside of the container until the internal temperature of the sample drops to room temperature, thus obtaining the AA-CM-Fru modified emulsifier.

2. The preparation process of the AA-CM-Fru modified emulsifier as described in claim 1, characterized in that, In step S2 of the liquid chromatography method, the chromatographic column is a Jupiter® 5 µm C4 300 Å (LC Column 250×2 mm); the column temperature is 40℃; mobile phase A is a 0.05% aqueous solution of trifluoroacetic acid, and mobile phase B is a 0.05% solution of trifluoroacetic acid acetonitrile.

3. The preparation process of the AA-CM-Fru modified emulsifier as described in claim 2, characterized in that, The milk pretreatment in step S1 includes filtering the collected fresh milk to remove impurities such as sand and hair, rapidly cooling the sample temperature to 4°C, filtering the milk through a double layer of defatted gauze, centrifuging the milk to separate the fat layers and filter them out, and filtering the remaining fat again through a double layer of defatted gauze to obtain skimmed milk.

4. The preparation process of an AA-CM-Fru modified emulsifier as described in claim 1 or 3, characterized in that, The κ-casein genotype analysis in step S1 includes taking samples from the pretreated milk in S1, analyzing them by liquid chromatography to obtain milk containing AA genotype κ-casein, and then labeling it.

5. The preparation process of the AA-CM-Fru modified emulsifier as described in claim 4, characterized in that, The casein micelle extraction in step S1 includes mixing equal volumes of milk containing AA genotype κ-casein that was screened and labeled in step S1, adjusting the pH to 4.6, and extracting casein micelles containing AA genotype κ-casein by acid precipitation.

6. The preparation process of the AA-CM-Fru modified emulsifier as described in claim 5, characterized in that, The acid precipitation method for extracting casein micelles specifically includes the following steps: S31: Add 10% CH3COOH to milk containing AA genotype κ-casein at a volume ratio of 1:10, let stand for 5 minutes until precipitation occurs; S32: Add 1 M CH3COONa at a volume ratio of 1:10 to the milk containing precipitate from step S31, measure the pH value again to 4.6, and let stand for 5 min. S33: After centrifuging at 1000 g for 10 min at 4℃, the casein was washed twice in cold water and then centrifuged at 1000 g for 5 min at 4℃ to obtain casein micelles, which were then stored at 4℃.

7. An application of an AA-CM-Fru modified emulsifier, comprising an AA-CM-Fru emulsion prepared by emulsifying medium-chain triglycerides with the modified emulsifier prepared using the preparation process described in any one of claims 1-6, characterized in that, The components are included by weight as follows: 200 parts of AA-CM-Fru modified emulsifier, 5 parts of medium-chain triglycerides, and 0.005 parts of β-carotene.

8. The application of the AA-CM-Fru modified emulsifier as described in claim 7, characterized in that, The specific method for preparing the AA-CM-Fru emulsion Includes the following steps: S6: Weigh out β-carotene and dissolve it in medium-chain triglycerides. Stir and mix for 10 min at 500 r / min to obtain the oil phase. S7: Take AA-CM-Fru modified emulsifier as the aqueous phase, adjust the pH to 7, and add an appropriate amount of oil phase to the aqueous phase to make its mass fraction 2.5%; S8: Use a high-speed shearing machine to shear at high speed for 3 minutes at a speed of 12000 r / min to obtain a crude emulsion. Then use an ultra-high pressure homogenizer to perform secondary high-pressure homogenization, with the first stage at 85 MPa and the second stage at 10 MPa, for 3 cycles to obtain the AA-CM-Fru emulsion.

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