A method for preparing a casein micelle-phenol-whey protein ternary complex

Abstract

This invention relates to the field of emulsion technology, specifically disclosing a method for preparing a casein micelle-phenol-whey protein ternary complex, comprising: S1, screening bovine milk with κ-casein genotype AA, and using it as raw material to prepare a casein micelle solution; S2, adding a phenolic compound to the casein micelle solution to form a casein micelle-phenol binary complex mixed solution; S3, dialyzing the mixed solution obtained in step S2; S4, adding whey protein; by combining a phenolic compound (such as EGCG) with casein micelles and whey protein to form a ternary complex, the degree to which proteins form dense clots in the stomach can be reduced, making the clot structure looser, thereby accelerating the digestion rate and reducing stomach discomfort.

Description

Technical Field

[0001] This invention relates to the field of emulsion technology, specifically to a method for preparing a casein micelle-phenol-whey protein ternary complex. Background Technology

[0002] Milk contains approximately 80% casein and 20% whey protein. Four types of casein (αS1-, αS2-, β-, κ-) interact to form casein micelles. These casein micelles in milk easily form curds in the stomach, while the phenolic compounds in tea readily bind to proteins or digestive enzymes, slowing down the rate of protein digestion and absorption. These phenomena can cause stomach discomfort in children and the elderly. Tea is rich in EGCG.

[0003] Among the three common κ-casein genotypes (AA, AB, and BB), BB and AB κ-casein have a higher degree of glycosylation, resulting in smaller casein micelle sizes. AA κ-casein micelles have larger sizes. β-lactoglobulin (β-LG) is the most common whey protein. During heating, casein micelles form thermal aggregates with whey protein through the outermost κ-casein. Therefore, we propose a method for preparing a casein micelle-phenol-whey protein ternary complex. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a casein micelle-phenol-whey protein ternary complex, so as to solve the problem of improving the digestibility of dairy products mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a casein micelle-phenol-whey protein ternary complex, comprising: Casein micelles; Phenolic compounds; whey protein; The casein micelles are derived from bovine milk with a κ-casein genotype of AA.

[0006] A method for preparing a casein micelle-phenol-whey protein ternary complex includes the following steps: S1. Screening bovine milk with κ-casein genotype AA and using it as raw material to prepare casein micelle solution; S2. Add phenolic compounds to the casein micelle solution, adjust the pH to neutral, and react at 50~70℃ to allow the phenolic compounds to bind with the casein micelles, forming a mixed solution of casein micelle-phenol binary complex. S3. Dialyze the mixed solution obtained in step S2 to remove unbound phenolic compounds and obtain a purified casein micelle-phenol binary complex solution. S4. Add whey protein to the purified casein micelle-phenol binary complex solution obtained in step S3, and perform instantaneous heat treatment at 130-140℃ for 5-15 seconds to obtain the casein micelle-phenol-whey protein ternary complex.

[0007] Preferably, in step S1, the genotype of κ-casein in milk is screened by high-resolution liquid chromatography-mass spectrometry.

[0008] Preferably, in step S1, the casein micelle solution is prepared by the following method: Skim milk with κ-casein genotype AA was adjusted to pH 4.6 to precipitate casein. The precipitate was collected by centrifugation and washed with pure water. The resulting casein precipitate was redispersed in phosphate buffer to obtain a casein micelle solution with a concentration of 8-12 mg / mL.

[0009] Preferably, the concentration of the casein micelle solution is 10 mg / mL.

[0010] Preferably, in step S2, the phenolic compound is epigallocatechin gallate (EGCG). The amount of epigallocatechin gallate added is such that the final concentration reaches 0.2-0.8 mg / mL.

[0011] Preferably, in step S3, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, the dialysis is performed at 4°C, and the dialysis time is 48 hours. During the dialysis process, the dialysis fluid is replaced every 6 hours.

[0012] Preferably, in step S4, the whey protein is β-lactoglobulin.

[0013] Preferably, the mass ratio of β-lactoglobulin to κ-casein is 0.5 to 2:1.

[0014] The present invention has at least the following beneficial effects: 1. This invention combines phenolic compounds (such as EGCG) with casein micelles and whey protein to form a ternary complex, which can reduce the degree to which proteins form dense clots in the stomach, making the clot structure looser, thereby accelerating the digestion rate and reducing stomach discomfort.

[0015] 2. The present invention first adds phenolic compounds (such as EGCG) to react with casein micelles, then performs dialysis purification, and finally introduces whey protein and performs instantaneous heat treatment. This sequence significantly improves the binding efficiency between the components, forming a ternary complex with a more stable structure and more uniform particle size.

[0016] 3. This invention uses milk with κ-casein genotype AA as raw material. Its micelles are larger and more loosely structured, which is more conducive to the binding of phenolic compounds and whey protein, and exhibits higher degree of hydrolysis and faster digestion kinetics during digestion. Attached Figure Description

[0017] Figure 1 This is the HPLC analysis chromatogram of milk containing AA-type κ-casein according to the present invention; Figure 2 This is a schematic diagram of the microstructure of the casein micelle-phenol-whey protein ternary complex of the present invention; Figure 3 Physicochemical data of the ternary complex obtained by adding β-lactoglobulin in this invention; Figure 4 Physicochemical data diagram of the ternary complex obtained by adding EGCG to the present invention; Figure 5 Transmission electron microscopy image of the casein micelle-phenol-whey protein ternary complex containing different κ-casein genotypes of the present invention; Figure 6 This is a scanning electron microscope image of the casein micelle-phenol-whey protein ternary complex containing different κ-casein genotypes of the present invention. Figure 7 The diagrams show the degree of digestion-hydrolysis and the kinetic model analysis of the ternary complex of this invention. Figure 8 This is a microscopic morphology observation image of the digestion-digestion products of the ternary complex of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0019] Please see Figure 1 The κ-casein genotype and glycosylation level were determined from Holstein milk from Modern Farm in Hefei.

[0020] Sample preparation: Holstein milk samples were collected from Modern Farm Farm in Hefei. Each fresh milk sample was collected in a sterile plastic bottle and centrifuged at 4°C and 2643 g for 30 min using a high-speed refrigerated centrifuge to remove impurities and defatting for later use.

[0021] The genotype of κ-casein in milk was analyzed by high-resolution liquid chromatography-mass spectrometry (HPLC). Chromatographic conditions included a C4 column (5 μm × 4.6 × 250 mm), mobile phase A of 0.05% trifluoroacetic acid aqueous solution, and mobile phase B of 0.05% trifluoroacetic acid acetonitrile solution, with gradient elution. The column temperature was 40°C, the injection volume was 6 μL, and the flow rate was 0.3 mL / min. The κ-casein genotype was determined by the peak time and position of different κ-casein variants in the HPLC chromatogram, and the degree of κ-casein glycosylation was calculated by the peak area of ​​different κ-casein variants in the HPLC chromatogram. Figure 1 Signal peaks of glycosylated κ-casein variants A / B and glycosylated variant A were observed at 12.7 min and 12.2 min, respectively, and a signal peak of κ-casein variant A was observed at 12.8 min. Therefore, the κ-casein genotype of this bovine milk was determined to be AA. Example 2

[0022] Please see Figure 2-4 This invention provides a technical solution: a method for preparing a casein micelle-phenol-whey protein ternary complex, comprising the following steps: S1. According to the method provided in Example 1, milk with κ-casein genotype AA is screened, and casein micelle solution is prepared using it as raw material. The preparation method of casein micelle solution is as follows: Equal volumes of milk with the same genotype were mixed, and the pH was adjusted to 4.6 to precipitate casein. 2.5 mL of 10% CH3COOH was added to 25 mL of skim milk, and the mixture was carefully mixed for 2 minutes until precipitation occurred. Then, 2.5 mL of 1 M CH3COONa was added, and the pH was confirmed to be 4.6. Stirring was stopped, and the mixture was allowed to stand for 5 minutes. The mixed milk was centrifuged at 1000 g at 4°C for 10 minutes. The casein precipitate was washed twice thoroughly with pure water, centrifuged at 1000 g at 4°C for 5 minutes, and then refrigerated at 4°C for later use.

[0023] The order of addition of phenolic compounds and whey protein was determined through competitive testing.

[0024] Experimental group 1: (EGCG added first) The optimal binding conditions for immobilizing phenolic compounds were: an addition amount of 6%, pH 7.0, and reaction in a water bath at 60°C for 1 h. The ratio of β-lactoglobulin (β-Lg) to κ-casein was successively adjusted to 0:1, 0.5:1, 1:1, 1.5:1, and 2:1 to achieve a casein concentration of 10 mg / ml. Three replicates were prepared for each sample.

[0025] Sample preparation: First, 200 mg of casein micelles were dissolved in 20 mL of PBS solution and stirred at room temperature at 1000 r / min to obtain a casein micelle solution. 4 mL of the above casein micelle solution was taken, and 2.4 mg of EGCG was added to each sample. The pH was adjusted to 7.0, and the mixture was reacted at 60℃ for 1 h to allow EGCG to fully react and bind with the casein micelles. After the reaction, unreacted phenolic compounds were removed using a dialysis bag (3500 Da), and the mixture was dialyzed at 4°C for 48 h, with water changed every 6 h. Different amounts of β-lactoglobulin were added to the dialyzed solution in proportions: 0 mg, 20 mg, 40 mg, 60 mg, and 80 mg, respectively. The solution was then placed in an oil bath at 137℃ and reacted for 5 s.

[0026] Experimental Group 2: (Whey protein added first) The optimal preparation conditions for immobilized casein micelles and β-lactoglobulin binary complexes were a β-lactoglobulin to κ-casein ratio of 2:1, a temperature of 137℃, and a pH of 7.0. The amount of phenolic substances added was adjusted sequentially to 0%, 2%, 4%, 6%, and 8% to achieve a casein concentration of 10 mg / ml. Three replicates were prepared for each sample.

[0027] Sample preparation: First, dissolve 200 mg of casein micelles in 20 mL of PBS solution and stir at room temperature at 1000 r / min to obtain a casein micelle solution. Take 4 mL of the above casein micelle solution, add 80 mg of β-lactoglobulin to each sample, adjust the pH to 7.0, and place in an oil bath at 137℃ for 5 s. Then, add different amounts of EGCG in proportions of 0, 0.8 mg, 1.6 mg, 2.4 mg, and 3.2 mg, and react in a water bath at 60℃ for 1 h.

[0028] The microstructure of the samples from experimental groups 1 and 2 was characterized using the following methods: Determination of κ-casein precipitation and whey protein attachment: HPLC was used to determine the precipitation and attachment of κ-casein and whey protein. Samples were added to 10 mL centrifuge tubes and centrifuged at 63000 × g (4°C) for 1 h to separate the whey and micelle phases. The relative content of β-lactoglobulin and κ-casein in the whey phase was determined by HPLC. 300 µL of whey and micelle samples were taken, and 900 µL of working solution and 24 µL of LDTE were added to each sample. The mixture was then centrifuged at 14000 r for 10 min at 4°C. After centrifugation, 1 mL of the mixture was transferred to a sample vial for HPLC analysis.

[0029] Determination of phenolic compound binding: The content of phenolic compounds involved in binding in the complex was determined using the Folin-Ciocalteu method. First, 0.5 mL of the sample solution (1 mg / mL) was mixed thoroughly with 2.5 mL of Folin-Ciocalteu reagent (0.2 N) and reacted in the dark for 5 min. Then, 2 mL of Na₂CO₃ (75 mg / mL) solution was added, mixed thoroughly, and reacted in the dark for 2 h. The absorbance was measured at 760 nm. The content of phenolic compounds bound to casein micelles was calculated by plotting different standard curves. Standard curves were constructed using different concentrations of EGCG.

[0030] The equation of the EGCG standard curve is: y = 0.008x + 0.1582 The amount of EGCG bound equals the total absorbance of the sample minus the absorbance of casein plus β-lactoglobulin.

[0031] x: EGCG binding amount y: Sample absorbance 10: Protein concentration is 10 mg / mL Particle size determination: Dissolve 1 mL of sample in 9 mL of deionized water, dilute the sample 10 times, and then determine the particle size. Shake the sample well before measurement. Nanoparticle size analyzer parameters: temperature 25°C, particle refractive index 1.450, particle absorptivity 0.8872, dispersant: water, dispersant refractive index 1.330. Glass sample cell with a 1 cm optical path and four-sided transparency. The same sample was measured three times, and the average value was taken.

[0032] Zeta potential measurement: Dissolve 1 ml of sample in 9 ml of pure water, dilute the sample 10 times, and then measure. Instrument parameters were set as follows: particle refractive index 1.450, particle absorptivity 0.001, dispersant water, dispersant refractive index 1.330. The measurement temperature was 25℃, and the Zeta potential of the same sample was measured three times.

[0033] Figure 2 This is a schematic diagram of the microstructure of the casein micelle-phenol-whey protein ternary complex. Figure 3It was found that when the EGCG concentration was 0 mg / mL, the casein micelle-β-LG complex had the largest particle size, approximately 330 nm. Heat treatment caused rapid denaturation of β-LG, disrupting its natural spherical structure and exposing a large number of hydrophobic amino acid residues. With increasing EGCG concentration, the casein micelle-β-LG complex gradually decreased in size. The hydrophobic benzene ring structure of EGCG preferentially binds to the exposed hydrophobic sites of the casein micelle-β-LG aggregates, occupying these sites and preventing further aggregate growth. When the EGCG concentration reached 0.8 mg / mL, the protein binding sites were saturated, and therefore the aggregate size no longer changed significantly. Figure 3 As the amount of EGCG added increases, when the EGCG concentration is 0 mg / mL, under neutral conditions, both casein micelles and β-lactoglobulin are negatively charged. However, heat treatment denatures β-LG, disrupting its charge distribution. EGCG, being a polyphenol, contains multiple phenolic hydroxyl groups. In a weakly alkaline environment (pH 7.0), these hydroxyl groups partially dissociate, making EGCG itself negatively charged. With increasing EGCG concentration, the EGCG binding sites on the complex surface are gradually occupied. When the EGCG concentration reaches 0.8 mg / mL, the binding sites on the complex surface are fully occupied, and a large number of negatively charged EGCG molecules are stably bound to the complex surface, significantly increasing the net negative charge density. Therefore, the zeta potential becomes further negative. This is consistent with the particle size results.

[0034] Figure 3 The physicochemical data of the ternary complex obtained by first adding β-lactoglobulin are shown in the figure. Figure 3 a represents the amount of phenolic compounds bound at different EGCG addition levels; Figure 4 b represents the amount of β-Lg adhesion at different EGCG addition levels; Figure 4 c represents the amount of κ-casein precipitated at different EGCG addition levels; Figure 4 d represents the particle size for different EGCG addition amounts; Figure 4 e represents the Zeta potential for different amounts of EGCG added.

[0035] Depend on Figure 3 As can be seen from this, with the increase of EGCG addition, EGCG molecules bind to the hydrophobic sites of denatured β-Lg or casein micelles through hydrophobic interactions, and simultaneously form hydrogen bonds with the amino and hydroxyl groups of the protein through phenolic hydroxyl groups. More EGCG molecules can occupy unused binding sites, thus the amount of EGCG binding gradually increases. When the amount of EGCG added is 0.6 mg / mL, the amount of EGCG is sufficient to occupy all the binding sites of the protein; both hydrophobic sites and hydrogen bond sites are saturated. Further increasing the amount of EGCG adds no more binding sites in the system, therefore the amount of EGCG binding no longer increases. Figure 3 b and Figure 3 As can be seen from the diagram, β-Lg is added first. Heating causes the natural structure of β-Lg to disintegrate and exposes a large number of hydrophobic groups. When enough β-Lg is added, the binding sites of casein micelles will be saturated. EGCG binds to the surface of the complex through hydrogen bonds and hydrophobic interactions. EGCG does not compete for the binding sites of β-Lg. Therefore, increasing the amount of EGCG added will not change the already bound β-Lg, and the amount of β-Lg attached remains stable.

[0036] Figure 4 Physicochemical data diagram of the ternary complex obtained by adding EGCG to the present invention; Figure 4 a represents the amount of phenolic compounds bound with different amounts of β-Lg added; Figure 4 b represents the amount of β-Lg adhesion at different β-Lg addition amounts; Figure 4 c represents the amount of κ-casein precipitated with different amounts of β-Lg added; Figure 4 d represents the particle size for different amounts of β-Lg added; Figure 4 e represents the Zeta potential for different amounts of β-Lg added.

[0037] Depend on Figure 4 As can be seen from d, with the increase of EGCG addition, the particle size of the complex did not differ significantly and remained at 75 nm. Figure 4 As can be seen from the diagram, adding EGCG first and then adjusting the amount of β-Lg sequentially did not result in a significant difference in the potential of the complex. Casein is a phosphoprotein rich in phosphate groups and carries a strong negative charge, remaining negatively charged at neutral pH. Figure 4 As can be seen from this, the amount of phenolic compounds bound does not change significantly with the increase of β-Lg content. First, EGCG is added. The benzene ring structure of EGCG is a strongly hydrophobic group, which will embed into the hydrophobic pocket of casein through a hydrophobic effect, thus achieving initial binding between the two. Figure 4 b and Figure 4 As shown in Figure c, with the increase of β-Lg concentration (0-10 mg / mL), the amount of κ-CN precipitated gradually increased; at 15-20 mg / mL, the precipitation rate tended to plateau. β-Lg binds to κ-CN on the surface of the casein micelle-EGCG complex through EGCG bridging or direct protein-protein interactions. With the increase of β-Lg concentration, the amount of β-Lg attached gradually increased; when the β-Lg concentration was 15-20 mg / mL, the attachment amount tended to stabilize.

[0038] In summary, the order of addition of the ternary complex was determined to be EGCG first, followed by β-Lg, with the addition amounts being casein:EGCG 1:6% and casein:β-Lg 1:1.5, respectively.

[0039] S2. Add phenolic compound (EGCG) to the casein micelle solution (the concentration of casein micelle solution is 10 mg / mL), adjust the pH to neutral, and react at 50~70℃ for 1 h to allow EGCG to fully react and combine with casein to form a mixed solution of casein micelle-phenol binary complex. S3. After the reaction is complete, the mixed solution obtained in step S2 is dialyzed to remove unreacted phenolic compounds using a dialysis bag (3500 Da). Dialysis is performed at 4°C for 48 h, with water changed every 6 h to remove unbound phenolic compounds, resulting in a purified casein micelle-phenol binary complex solution. S4. Add whey protein (β-Lg) to the purified casein micelle-phenol binary complex solution obtained in step S3, and perform instantaneous heat treatment at 130-140℃ for 5-15 s to obtain the casein micelle-phenol-whey protein ternary complex.

[0040] Example 3 Please see Figure 5 The protein morphology was observed using a transmission electron microscope (TEM). Figure 5 Transmission electron microscopy (TEM) images show casein micelles-phenol-whey protein ternary complexes containing different κ-casein genotypes. AA, AB, and BB represent three different κ-casein genotypes; AA-CM, AB-CM, and BB-CM represent three different κ-casein genotype casein micelles; and AA-CM-EGCG-β-Lg, AB-CM-EGCG-β-Lg, and BB-CM-EGCG-β-Lg represent three different κ-casein genotype casein micelles-phenol-whey protein ternary complexes. Protein morphology was observed at different magnifications (5000–100,000x) using a voltage of 100 kV. The microscopic morphology of casein micelles and ternary complexes containing different κ-casein genotypes was observed using TEM. As shown in the figure, the particle size of casein containing different κ-casein genotypes ranges from 120 nm to 200 nm. After adding EGCG and β-Lg to form a ternary complex, the particle size is significantly smaller than that of casein micelles, below 100 nm. This is because the addition of EGCG occupies the binding sites of β-Lg, resulting in less β-Lg binding and thus a smaller particle size after forming the ternary complex. Under the same conditions, the particle size of AA-type casein micelles and the ternary complex is larger than that of AB and BB types. Due to the influence of glycosylation degree and steric hindrance, the micelle size of AB and BB-type κ-casein is smaller, while the micelle size of AA-type κ-casein is larger, allowing for the binding of more EGCG. The hydroxyl groups introduced by phenolic compounds bind to casein micelles through hydrogen bonds and hydrophobic interactions, reducing intermolecular interactions and leading to a smaller particle size of the casein complex.

[0041] Example 4 Please see Figure 6 The morphology of the samples was observed and photographed using a Hitachi S-4800 scanning electron microscope. Figure 6 Scanning electron micrographs of casein micelles-phenol-whey protein ternary complexes containing different κ-casein genotypes are shown. AA, AB, and BB represent three different κ-casein genotypes; AA-CM, AB-CM, and BB-CM represent three different κ-casein genotype casein micelles; and AA-CM-EGCG-β-Lg, AB-CM-EGCG-β-Lg, and BB-CM-EGCG-β-Lg represent three different κ-casein genotype casein micelles-phenol-whey protein ternary complexes. The microscopic morphology of casein micelles and casein micelle-phenol-whey protein ternary complexes containing different κ-casein genotypes was observed using scanning electron microscopy. κ-casein micelles are large particles aggregated through hydrophobic interactions and calcium ion bridging, which can be clearly observed under an electron microscope. These aggregates are roughly spherical with a diameter of approximately 200-400 nm. Compared to casein micelles, the particle size of the ternary complex is significantly reduced, to approximately less than 100 nm, with a more uniform particle size distribution and a denser structure. This is because, with the addition of EGCG and β-Lg, multiple phenolic hydroxyl groups of EGCG can form hydrogen bonds and hydrophobic interactions with the hydrophobic regions, amino groups, and carboxyl groups of κ-casein. At the same time, the hydrophobic pockets of β-Lg also bind to the hydrophobic fragments of κ-casein, which breaks down the aggregated structure of the original micelles, causing the large micelles to dissociate. Furthermore, as a globular protein, β-Lg creates steric hindrance when it binds to casein micelles and EGCG. This steric hindrance further prevents particle aggregation, resulting in more dispersed and smaller composite particles, which is consistent with the particle size and transmission electron microscopy results.

[0042] Example 5 Please see Figure 7-8The digestion process of the ternary complex obtained in Example 2 was characterized. The in vitro digestion characteristics of the binary complex were determined according to the INFUGEST 2.0 protocol. 2 mL of sample solution was used for the in vitro digestion experiment; the added salt and protease were calculated based on the required casein concentration to be digested. A gas bath isothermal shaker was used to maintain the temperature at 37°C and simulate gastrointestinal stimulation. During the gastric digestion phase, an equal volume of simulated gastric juice containing pepsin was added for 0, 5, 15, 30, 60, and 120 min, and the results were named G0, G5, G15, G30, G60, and G120, respectively. The pH was adjusted to 8.0, and the reaction was stopped. After 120 min of gastric digestion, an equal volume of simulated intestinal juice containing trypsin was added to the sample for further intestinal digestion. After intestinal digestion for 0, 5, 15, 30, 60, and 120 min (designated as I0, I5, I15, I30, I60, and I120), the digestate was placed in a 95°C water bath for 5 min to stop the reaction. A time of 0 min indicates that the digestion reaction was stopped immediately after enzyme addition. For each digestion time point, a tube of the same mass of sample and digestion solution was prepared and the reaction was repeated three times.

[0043] Degree of protein hydrolysis determination: OPA reagent (prepare and use immediately): (1) Dissolve 7.620 g sodium tetraborate and 200 mg SDS in 150 ml deionized water. Other reagents must be added only after complete dissolution. (2) Dissolve 160 mg of o-phthalaldehyde in 4 mL of ethanol and add this solution to (1); (3) Add 176 mg DTT to (1), rinse with deionized water, and bring the volume to 200 mL.

[0044] Add 400 µL of deionized water as a blank sample to a test tube containing 3 mL of OPA reagent, vortex to mix, and react precisely for 2 min before measuring the absorbance at 340 nm. Then, add 400 µL of the digested sample solution to other test tubes containing 3 µL of OPA reagent, vortex to mix, and react precisely for 2 min before measuring the absorbance at 340 nm. Perform three parallel measurements and take the average value. Calculate the degree of protein hydrolysis:

[0045] In the above formula, and The concentrations of free amines in the samples hydrolyzed for x h and those not hydrolyzed for 0 h are respectively: the concentrations of free amines in the samples after hydrolysis in 6 M HCl at 110 °C for 24 h.

[0046] Figure 7a represents the change in the degree of hydrolysis of gastric digestion of casein micelles-phenol-whey protein ternary complexes containing different κ-casein genotypes; Figure 7 b represents the change in the degree of hydrolysis of casein micelles-phenol-whey protein ternary complexes containing different κ-casein genotypes during intestinal digestion; Figure 7 c represents a gastric digestion kinetic model containing a ternary complex of casein micelles, phenol, and whey protein with different κ-casein genotypes. Figure 7 d represents the intestinal digestion kinetics model of casein micelles-phenol-whey protein ternary complexes containing different κ-casein genotypes. AA, AB, and BB represent three different κ-casein genotypes; AA-CM, AB-CM, and BB-CM represent three different κ-casein genotype casein micelles; and AA-CM-EGCG-β-Lg, AB-CM-EGCG-β-Lg, and BB-CM-EGCG-β-Lg represent three different κ-casein genotype casein micelles-phenol-whey protein ternary complexes.

[0047] like Figure 7 a and Figure 7 As shown in b, the degree of hydrolysis of the ternary complexes significantly decreased during gastrointestinal digestion. Because EGCG can form hydrogen bonds with amino acid residues of proteases, disrupting the enzyme's catalytic conformation, and because EGCG binds to β-Lg, previously exposed hydrolysis sites are either embedded or conformationally altered, thus reducing the protease's recognition efficiency and protein digestibility. EGCG can bind to the hydrophobic pocket of β-Lg through hydrophobic interactions and simultaneously form hydrogen bonds with the phosphate groups of casein, further stabilizing the complex structure and reducing the hydrolysis sites accessible to the protease. Among the ternary complexes, the AA-type complex showed little change in degree of hydrolysis after 60 min of digestion, while the AB and BB-type complexes continued to increase in degree of hydrolysis. Due to different degrees of glycosylation, the ternary complex containing AA-type κ-casein had a larger particle size, resulting in a looser clot under acidic conditions in the stomach and a faster digestion rate.

[0048] Protein hydrolysis degree digestion kinetic model: The degree of hydrolysis of casein micelles and ternary complexes containing different κ-casein genotypes was nonlinearly fitted using a first-order kinetic model and a Weibull model. The maximum degree of hydrolysis and hydrolysis rate constant at different time points in gastrointestinal digestion were calculated based on the formulas of the first-order kinetic model and the Weibull model.

[0049] The formula for the first-order dynamic model is:

[0050] yt: Maximum degree of hydrolysis (%) at time t; A: Maximum degree of hydrolysis; k: hydrolysis rate constant; t: digestion time (min).

[0051] Weibull model formula:

[0052] yt: Maximum degree of hydrolysis (%) at time t; A: Maximum digestibility; k: digestion rate constant; xc: Delay time (if there is no significant delay in digestion, it can be set to 0); d: Shape parameter.

[0053] like Figure 7 c and Figure 7 As shown in Figure d, the degree of hydrolysis at different time points during gastrointestinal digestion was nonlinearly fitted using a first-order kinetic model and a Weibull model. Compared to casein micelles, the maximum degree of hydrolysis of the ternary complexes was lower. EGCG can form complexes with casein and β-Lg through hydrogen bonds and hydrophobic interactions. Simultaneously, β-Lg also interacts with casein. These interactions make the protein structure more compact, masking the action sites of digestive enzymes, thereby reducing the degree of hydrolysis and delaying digestion. The maximum degree of hydrolysis and the digestion rate constant of the AA-type ternary complex are greater than those of the AB and BB-type ternary complexes. The micellar structure of AA-type κ-casein is more loose, exposing more and a greater number of enzyme cleavage sites in the casein molecule, thus ultimately achieving the highest degree of hydrolysis.

[0054] like Figure 8 As shown, the microstructural characteristics of gastric digestate were observed using laser confocal microscopy. The excitation wavelength was 488 nm, and the emission wavelength was 498–532 nm. Flocculants were labeled with 0.1% rhodamine dye, allowed to stand in the dark for 20 min, and then imaged. In the figure, AA-CM-EGCG-β-Lg, AB-CM-EGCG-β-Lg, and BB-CM-EGCG-β-Lg represent three different κ-casein genotype casein micelle-phenol-whey protein ternary complexes.

[0055] At the start of digestion (0 min), after gastric acidification, all samples formed large and loose flocs with concentrated green fluorescence, indicating that the micelle structure was intact and highly aggregated. After 5 min of digestion with pepsin, large flocs formed, and the fluorescence distribution became more uniform, reflecting the initiation of gastrointestinal digestive enzymes (such as pepsin) and the rapid dissociation of the micelle structure. After 30 min of pepsin, the size of the flocs decreased, the particles became further refined, and the fluorescence intensity decreased, indicating that enzymatic digestion continued and the micelles were gradually hydrolyzed into smaller peptides. After 120 min, the flocs became even finer and smaller, the fluorescence decreased significantly, and there were very few residual particles, indicating that after long-term digestion, the protein structure was almost completely disintegrated and most of the protein was hydrolyzed.

[0056] Casein has an isoelectric point of approximately 4.6. During gastric digestion, when the pH drops to 3, far below its isoelectric point, the net positive charge of casein molecules increases significantly. At this point, the electrostatic repulsion between protein molecules decreases dramatically, disrupting the micellar dispersion previously maintained by negative charge. Molecules readily aggregate through hydrophobic interactions and van der Waals forces, forming flocs. EGCG can simultaneously bind to multiple protein molecules, forming a polyphenol-protein cross-linking network, accelerating floc formation. Although the positive charge of β-Lg repels the positive charge of casein, the hydrophobic water areas of the local structure and the bridging effect of EGCG overcome the electrostatic repulsion, promoting the co-aggregation of multiple components. Acidic conditions first induce protein aggregation to form flocs; subsequently, pepsin gradually degrades the protein molecules in the aggregates. Within the same digestion time, AA-type casein micelles exhibit a faster digestion rate, which may be related to the degree of glycosylation and steric hindrance of κ-casein. Previous studies have shown that glycosylated κ-casein has a slower hydrolysis rate than unglycosylated κ-casein. AA-type ternary complexes, due to their lower degree of glycosylation and larger particle size, form looser clots during gastric digestion, which facilitates the entry of digestive enzymes into the casein micelles, accelerates the enzymatic process, enhances protein digestion, and reduces the delaying effect of phenolic compounds on protein digestion.

[0057] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A casein micelle-phenol-whey protein ternary complex, characterized in that, include: Casein micelles; Phenolic compounds; whey protein; The casein micelles are derived from bovine milk with a κ-casein genotype of AA.

2. A method for preparing the casein micelle-phenol-whey protein ternary complex as described in claim 1, characterized in that, Includes the following steps: S1. Screening bovine milk with κ-casein genotype AA and using it as raw material to prepare casein micelle solution; S2. Add phenolic compounds to the casein micelle solution, adjust the pH to neutral, and react at 50~70℃ to allow the phenolic compounds to bind with the casein micelles, forming a mixed solution of casein micelle-phenol binary complex. S3. Dialyze the mixed solution obtained in step S2 to remove unbound phenolic compounds and obtain a purified casein micelle-phenol binary complex solution. S4. Add whey protein to the purified casein micelle-phenol binary complex solution obtained in step S3, and perform instantaneous heat treatment at 130-140℃ for 5-15 seconds to obtain the casein micelle-phenol-whey protein ternary complex.

3. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 2, characterized in that: In step S1, the genotype of κ-casein in milk is screened using high-resolution liquid chromatography-mass spectrometry.

4. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 3, characterized in that: In step S1, the casein micelle solution is prepared by the following method: Skim milk with κ-casein genotype AA was adjusted to pH 4.6 to precipitate casein. The precipitate was collected by centrifugation and washed with pure water. The resulting casein precipitate was redispersed in phosphate buffer to obtain a casein micelle solution with a concentration of 8-12 mg / mL.

5. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 4, characterized in that: The concentration of the casein micelle solution is 10 mg / mL.

6. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 2, characterized in that: In step S2, the phenolic compound is epigallocatechin gallate; The amount of epigallocatechin gallate added is such that the final concentration reaches 0.2-0.8 mg / mL.

7. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 2, characterized in that: In step S3, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da, at 4°C, for 48 hours. During the dialysis process, the external dialysis fluid is replaced every 6 hours.

8. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 2, characterized in that: In step S4, the whey protein is β-lactoglobulin.

9. The method for preparing the casein micelle-phenol-whey protein ternary complex according to claim 8, characterized in that: The mass ratio of β-lactoglobulin to κ-casein is 0.5~2:1.

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