A biological macromolecule stabilizer, its preparation method and application

Abstract

The application relates to a biological macromolecule stabilizer, a preparation method and application thereof, and particularly relates to a crystal nucleus covalently combined with a biological monomer macromolecule by using a polyphenol substance to form a complex, wherein the complex can induce the biological macromolecule to form a supramolecule as an inducer to improve the stability of the biological macromolecule.

Description

Technical Field

[0001] This invention relates to the field of emulsion preparation, specifically to a biomacromolecule stabilizer, its preparation method and application, and more specifically to improving the emulsification stability of biomacromolecules by altering the active regions on the surface of crystal nuclei. Background Technology

[0002] Changes in protein structure lead to alterations in functional properties. For whey protein, under conventional treatment conditions (e.g., heat treatment for 10 minutes), conventional polymers are formed through the interaction of covalent and non-covalent bonds; these structures are typically disordered and random. However, under unconventional treatment conditions, such as low pH, 90℃, and heat treatment for more than 10 hours, protein molecules unfold, and the secondary structures—β-sheets—are stacked through non-covalent bonds such as hydrogen bonds, van der Waals forces, hydrophobic interactions, and π-π bonds to form ordered supramolecular structures. These structures typically exist in the form of nanofibers, exhibiting a compact and ordered structure. Homogenization slightly improves emulsification performance, generally by about 20%, demonstrating the structural advantages of supramolecular structures in emulsification. Structurally, compared with natural whey protein and its conventional polymer structure, supramolecular structure has a significant improvement in surface hydrophobicity and charge quantity, which should be more suitable for the structural requirements of interface stability. However, this protein assembly structure has excessively strong molecular rigidity and poor flexibility, with molecular flexibility only half that of conventional whey protein polymers. It cannot meet the adsorption requirements of interfacial molecular denaturation and unfolding, thus limiting the improvement of its emulsifying performance.

[0003] Polyphenols are important plant secondary metabolites, widely distributed in vegetables, fruits, tea, and coffee. Common polyphenols include catechins, gallic acid, curcumin, quercetin, and chlorogenic acid. Polyphenols possess strong anti-inflammatory, antibacterial, and antioxidant properties. Polyphenols can interact with proteins. Taking gallic acid (GA) as an example, GA interacts with proteins in two main ways: one is through non-covalent bonding with aromatic amino acids or carboxyl groups under acidic conditions; the other is through covalent bonding with amino groups under alkaline conditions to form quinones. Due to the influence of polyphenols' own structure, food matrix, and food processing, their bioavailability is relatively poor, thus affecting their health benefits in vivo. In recent years, many studies have reported on improving the bioavailability of polyphenols through interactions between food polyphenols and proteins. In addition, existing technologies for protein-polyphenol interactions can also improve protein properties, with applications in enhancing solubility, gel strength, thermal stability, and emulsification. Although the binding of GA to proteins improves the bioavailability of GA, the emulsifying properties of the proteins are still unsatisfactory.

[0004] In their previous research, the inventors addressed the issue of the rigid supramolecular structure of whey protein formed by spontaneous assembly, which hindered the improvement of emulsification performance. This improvement primarily involved a multiple-cycle induction method to obtain a loosely structured supramolecular polymer. Compared to spontaneous assembly, this approach improves molecular flexibility and enhances emulsification performance. However, this method of improving molecular flexibility through multiple cycles has limited effect on emulsification performance. It requires repeated, prolonged heating treatments, with each cycle slightly altering the stacking structure on the crystal nucleus surface. Multiple cycles are needed to obtain a supramolecular structure with a certain degree of looseness. This method cannot effectively control the surface activity of the crystal structure; the looseness of the supramolecular structure depends solely on the number of cycles, which is uncertain. For example, some processes require heating at 90°C for 10 hours, repeated 6 times, which is energy-intensive, time-consuming, and difficult to implement. Furthermore, the improvement in emulsification performance is limited.

[0005] In summary, how to effectively regulate the supramolecular structure to improve the emulsifying properties of whey protein is a problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This invention aims to provide a protein structure that is more suitable for interfacial adsorption through a novel protein assembly mode, thereby further improving the stability of biomacromolecules such as proteins, and providing corresponding products.

[0007] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0008] A first aspect of the present invention is to provide a biomacromolecule stabilizer, wherein the biomacromolecule stabilizer is a supramolecular nucleus-polyphenol complex, characterized in that the stabilizer is prepared by the following method:

[0009] 1) Prepare solutions of biological macromolecule monomers;

[0010] 2) The solution prepared in step 1) is homogenized at high speed to prepare a supramolecular solution with a crystal nucleus structure;

[0011] 3) Add polyphenols to the supramolecular solution formed in step 2) to obtain a supramolecular nucleus-polyphenol complex solution.

[0012] In one specific embodiment, the biomolecule is a protein; preferably whey protein isolate.

[0013] In another specific embodiment, the polyphenol is gallic acid.

[0014] A second aspect of the present invention is to provide the use of the biomacromolecule stabilizer described in the first aspect in improving the stability of biomacromolecules.

[0015] In one specific embodiment, the biomacromolecule stabilizer is added as an inducer to the monomer solution of the biomacromolecule; preferably, the biomacromolecule is a protein, more preferably whey protein isolate.

[0016] In another specific embodiment, a solution of crystal nucleus-polyphenol complex and a solution of biological macromolecule monomer are thoroughly mixed, the pH value is adjusted, the mixture is heated in a high-temperature water bath and stirred, and then cooled to room temperature; preferably, the biological macromolecule is a protein, more preferably whey protein isolate.

[0017] In another specific embodiment, the solution cooled to room temperature is concentrated and freeze-dried to obtain biomacromolecules with improved stability.

[0018] In one specific implementation, when the biomolecule is whey protein isolate, the following steps are performed:

[0019] (1) Preparation of milk monosomal protein solution

[0020] Dissolve whey protein isolate in deionized water, mix thoroughly, adjust the pH value, centrifuge at high speed under low temperature, take the supernatant, and dilute with deionized water of the same pH value to obtain a whey protein isolate solution of 1-4 g / 100 mL.

[0021] (2) Preparation of whey protein supramolecular fragments with crystal nucleus structure

[0022] The partial whey protein monosodium solution obtained in step (1) was heated and stirred in a high-temperature water bath, then cooled to room temperature, and homogenized at high speed to prepare a whey protein supramolecular fragment solution with a crystal nucleus structure.

[0023] (3) Preparation of the crystal nucleus-GA complex solution

[0024] According to the protein mass contained in the supramolecular fragment solution (2), add gallic acid (GA), mix thoroughly, and obtain a whey protein supramolecular crystal nucleus-GA complex solution bound by non-covalent bonds;

[0025] (4) Crystal nucleus-GA induces the formation of whey protein supramolecular solution

[0026] Prepare a mixed solution consisting of the crystal nucleus-GA complex solution (3) and the emulsion monosodium protein solution from step (1), mix thoroughly, adjust the pH value, heat and stir in a high-temperature water bath, and cool to room temperature;

[0027] (5) The whey protein supramolecular solution obtained in step (4) is concentrated and freeze-dried to obtain whey protein isolate with high emulsification stability.

[0028] Preferably, in step (1), the pH value is adjusted to 1.5-2.5; the low temperature conditions are 1-4℃, the centrifugation speed is 16000-20000g, and the centrifugation time is 15-30min.

[0029] Preferably, the high-temperature water bath in step (2) is 80-95°C, the water bath heating and stirring time is 8-20h, and the high-speed homogenization conditions are 18000-20000rpm high-speed homogenization for 1-2min.

[0030] Preferably, the method of adding GA in step (3) is as follows: GA is added to the supramolecular fragment solution at a mass concentration of 8-12 mg / g (GA mg / protein g), mixed thoroughly, and stirred in the dark at 20-25°C for 18-24 hours.

[0031] Preferably, in step (4), a mixed solution is prepared consisting of the crystal nucleus-GA complex solution (3) and the emulsion monosomal protein solution from step (1), with a volume ratio of 1:1 to 4; the mixture is thoroughly mixed, the pH is adjusted to 1.5 to 2.5, heated in a water bath at 80 to 95°C and stirred for 8 to 20 hours, and then cooled to room temperature.

[0032] Preferably, in step (3), GA is added as follows: GA is added to the supramolecular fragment solution at a mass concentration of 11 mg / g (GA mg / protein g), and the solution is magnetically stirred at 25°C in the dark for 24 hours.

[0033] Preferably, in step (4), a mixed solution is prepared consisting of the crystal nucleus-GA complex solution (3) and the lactose monosomal protein solution from step (1), with a volume ratio of 1:1, and the two are thoroughly mixed.

[0034] Preferably, the water bath temperature in steps (2) and (4) is 90°C; and the pH in steps (1) and (4) is 2.0.

[0035] Preferably, step (4) is repeated for induction; preferably, the number of inductions is 1.

[0036] A third aspect of the present invention is to provide the use of gallic acid (GA) in weakening the surface activity of crystal nuclei and improving the stability of biomacromolecules; preferably, the biomacromolecule is a protein, more preferably a whey protein isolate; the stability is preferably the emulsification stability of the protein.

[0037] In one specific embodiment, the GA is added to a whey protein supramolecular fragment solution having a crystal nucleus structure; preferably, the GA is added to the supramolecular fragment solution at a mass concentration of 8-12 mg / g (GA mg / protein g), mixed thoroughly, and stirred in the dark at 20-25°C for 18-24 h; more preferably, the GA is added to the supramolecular fragment solution at a mass concentration of 11 mg / g (GA mg / protein g).

[0038] The beneficial effects achieved by this invention include:

[0039] 1. This invention can effectively regulate the surface activity of crystal nuclei. An unexpected discovery during experiments revealed that among various polyphenols, polyphenols, especially GA, can most effectively and precisely control the degree of shielding, thereby moderately weakening crystal nuclei activity without affecting the stacking of biomolecules (preferably lactone monoproteins) on the modified crystal nuclei surface. This is difficult to achieve with other types of polyphenols. Weakening crystal nuclei surface activity through shielding is actually a microscopic and complex process, and precise control is quite difficult. However, a specific amount of GA can achieve precise control of crystal nuclei surface activity. In the experiment, selecting the amount of GA added was challenging, and existing technologies lacked guidance. A low GA addition ratio resulted in less binding to amino acid residues on the active region of the crystal nuclei surface, a low degree of shielding, and minimal impact on the stacking ability of lactone monoproteins on the crystal nuclei surface. The resulting supramolecular rigid structure was not significantly improved, which was detrimental to emulsification stability. Conversely, a high GA addition ratio resulted in too much GA binding to the crystal nuclei surface, excessively strong shielding of crystal nuclei activity, blocking the stacking of lactone monoproteins on the crystal nuclei surface, inhibiting supramolecular formation, and failing to improve emulsification stability.

[0040] 2. Under specific process and parameter conditions, this invention simultaneously weakens the surface activity of crystal nuclei to an appropriate degree using gallic acid, thereby altering the supramolecular structure of biological macromolecules (preferably whey protein) in terms of assembly method, making it easier to improve emulsification stability; it consumes less energy and time, and is easier to implement.

[0041] 3. Adding GA at a mass concentration of 8-12 mg / g (GA mg / protein g) to the supramolecular fragment solution achieved unexpected results. The shielding treatment effectively and precisely weakened the surface activity of the crystal nuclei, and more GA was bound. This significantly improved the emulsion stability and also gave the product stronger nutritional benefits. Attached Figure Description

[0042] Figure 1 Adsorption capacity at the emulsion interface for different supramolecular structures of whey proteins.

[0043] Figure 2 Surface hydrophobicity (A) and molecular flexibility (B) of whey protein supramolecular polymers induced by nucleus-GA and formed by existing methods.

[0044] Figure 3 Hydrogen bond strength (A) and π-π packing strength (B) within supramolecular structures induced by nucleus-GA and formed by existing methods.

[0045] Figure 4 Compared with spontaneous assembly, the nucleus-GA-induced release of peptides and hydrophobicity values ​​are compared with the nucleus-GA-induced assembly method.

[0046] Figure 5 Comparison of the ability of different polyphenols to induce the formation of supramolecular polymers after binding with crystal nuclei. Detailed Implementation

[0047] The specific implementation methods and technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to realize and apply the technology of the present invention. The present invention is illustrated through specific embodiments, but the present invention is not limited thereto.

[0048] Example 1: Preparation of whey protein isolate with high emulsification stability

[0049] 1. Preparation Example 1

[0050] Step 1: Preparation of Milk Monosodium Protein Solution

[0051] Dissolve 4.0g of whey protein isolate in deionized water, mix thoroughly, adjust the pH to 1.5, centrifuge at 16000g for 20min at 4℃, take the supernatant, and dilute with deionized water of the same pH to obtain a whey protein isolate solution of 2g / 100mL.

[0052] Step 2: Preparation of whey protein supramolecular fragments with crystal nucleus structure

[0053] A whey protein supramolecular fragment solution with a crystal nucleus structure was prepared by heating a 2g / 100mL whey monosodium protein solution in an 80℃ water bath, stirring for 20h, cooling to room temperature, and homogenizing at 20000rpm for 1min.

[0054] Step 3: Preparation of the nucleus-GA complex solution

[0055] GA was added to the supramolecular fragment solution at a mass concentration of 11 mg / g (GA mg / protein g), mixed thoroughly, and magnetically stirred at 25°C in the dark for 24 h to obtain a whey protein supramolecular crystal nucleus-GA complex solution.

[0056] Step 4: Crystal nucleus-GA induction to form whey protein supramolecular solution

[0057] Prepare a mixed solution consisting of crystal nucleus-GA and the monosodium protein solution from the emulsion in step one above, with a volume ratio of 1:2; mix thoroughly, adjust the pH to 1.5, heat in an 80°C water bath, stir for 20 hours, and cool to room temperature;

[0058] Step 5: Concentrate the obtained whey protein supramolecular solution and freeze-dry it to obtain whey protein isolate sample 1 with high emulsification stability.

[0059] 2. Preparation Example 2

[0060] Step 1: Preparation of Milk Monosodium Protein Solution

[0061] Dissolve 6.0g of whey protein isolate in deionized water, mix thoroughly, adjust the pH to 2.0, centrifuge at 20000g for 20min at 4℃, take the supernatant, and dilute with deionized water of the same pH to obtain a whey protein isolate solution of 3g / 100mL.

[0062] Step 2: Preparation of whey protein supramolecular fragments with crystal nucleus structure

[0063] A whey protein supramolecular fragment solution with a crystal nucleus structure was prepared by heating a 3g / 100mL whey monosodium protein solution in a 90℃ water bath, stirring for 10h, cooling to room temperature, and homogenizing at 20000rpm for 2min.

[0064] Step 3: Preparation of the nucleus-GA complex solution

[0065] GA was added to the supramolecular fragment solution at a mass concentration of 11 mg / g (GA mg / protein g), mixed thoroughly, and magnetically stirred at 25°C in the dark for 24 h to obtain a whey protein supramolecular crystal nucleus-GA complex solution.

[0066] Step 4: Crystal nucleus-GA induction to form whey protein supramolecular solution

[0067] Prepare a mixed solution consisting of crystal nucleus-GA and the monosodium protein solution from the emulsion in step one above, with a volume ratio of 1:1; mix thoroughly, adjust the pH to 2.0, heat in a 90°C water bath, stir for 10 hours, and cool to room temperature;

[0068] Step 5: Concentrate the obtained whey protein supramolecular solution and freeze-dry it to obtain whey protein isolate sample 2 with high emulsification stability.

[0069] 3. Preparation Example 3

[0070] Step 1: Preparation of Milk Monosodium Protein Solution

[0071] Dissolve 6.0g of whey protein isolate in deionized water, mix thoroughly, adjust the pH to 2.5, centrifuge at 18000g for 20min at 4℃, take the supernatant, and dilute with deionized water of the same pH to obtain a whey protein isolate solution of 3g / 100mL.

[0072] Step 2: Preparation of whey protein supramolecular fragments with crystal nucleus structure

[0073] A whey protein supramolecular fragment solution with a crystal nucleus structure was prepared by heating a 3g / 100mL whey monosodium protein solution in a 95℃ water bath, stirring for 8 hours, cooling to room temperature, and homogenizing at 20000rpm for 2 minutes.

[0074] Step 3: Preparation of the nucleus-GA complex solution

[0075] GA was added to the supramolecular fragment solution at a mass concentration of 11 mg / g (GA mg / protein g), mixed thoroughly, and magnetically stirred at 25°C in the dark for 24 h to obtain a whey protein supramolecular crystal nucleus-GA complex solution.

[0076] Step 4: Crystal nucleus-GA induction to form whey protein supramolecular solution

[0077] Prepare a mixed solution consisting of crystal nucleus-GA and the monosodium protein solution from step one above, with a volume ratio of 1:3; mix thoroughly, adjust the pH to 1.5, heat in a 95°C water bath, stir for 8 hours, and cool to room temperature;

[0078] Step 5: Concentrate the obtained whey protein supramolecular solution and freeze-dry it to obtain whey protein isolate sample 3 with high emulsification stability.

[0079] 4. Stability testing

[0080] Emulsion stability test

[0081] Sodium azide (0.02%, w / v) was added to different sample solutions with a protein concentration of 2.0 g / 100 mL to inhibit microbial growth. The sample solutions were mixed with soybean oil at a volume ratio of 10:7 and homogenized at 20,000 rpm for 2 min. The freshly prepared emulsion was transferred to graduated test tubes, ensuring that each sample volume was 20 mL. The height of the emulsion layer was recorded at different times.

[0082]

[0083] In the formula: A t —Emulsion height after 60 days of emulsion settling; A0 —Emulsion height when the emulsion has been settling for 0 days.

[0084] The results are shown in Table 1:

[0085] Table 1. Comparison of emulsifying stability of whey protein isolates obtained by different methods (60 days) %

[0086]

[0087] Note: Different lowercase letters indicate significant differences (P<0.05).

[0088] Experimental group nucleus-GA induction: carried out according to preparation examples 1, 2 and 3 of this application.

[0089] Control groups 1-5 are as follows:

[0090] Control 1: Conventional Polymer: The whey protein monosolid protein solution was prepared according to step one of Preparation Example 2. The solution was then heated in a 90°C water bath, stirred for 10 min, and cooled to obtain the conventional whey protein polymer. Control 1 included two groups of samples: those with and without GA. For the GA-added samples, the same mass concentration of GA as the experimental group was added before determining emulsion stability. The emulsion stability was determined according to the emulsion stability method.

[0091] Control 2: Monomeric Proteins: The monomeric protein solution for the emulsion was prepared according to step one of Preparation Example 2. Control 2 included two groups of samples: those with GA and those without GA. For the samples with GA, the same mass concentration of GA as the experimental group was added before determining the emulsion stability. The emulsion stability was determined according to the emulsion stability method.

[0092] Control 3: Spontaneous assembly of whey protein. A whey protein monosodium protein solution was prepared according to step one of Preparation Example 2. The protein solution was heated in a 90°C water bath and stirred for 10 hours, then cooled to obtain the spontaneously assembled whey protein supramolecular polymer. Control 3 included two groups of samples: those with and without GA. For the GA-added samples, the same mass concentration of GA as in the experimental group was added before determining emulsion stability. The emulsion stability was determined according to the emulsion stability method.

[0093] Control 4: Crystal nucleation-induced whey protein supramolecular fragment solution with crystal nuclei was prepared according to steps one and two of Preparation Example 2. The whey protein supramolecular fragment solution with crystal nuclei was thoroughly mixed with the whey protein monosodium solution prepared according to step one of Preparation Example 2 at a ratio of 1:1; the mixture was heated in a 90°C water bath with stirring for 10 hours, and then cooled to obtain the crystal nucleation-induced whey protein supramolecular polymer. Control 4 included two groups of samples: with and without GA. For the GA-added samples, the same mass concentration of GA as the experimental group was added before determining emulsion stability. The emulsion stability was determined according to the emulsion stability method.

[0094] Control 5 (6-cycle induction): A whey protein supramolecular fragment solution with a crystal nucleus structure was prepared according to steps one and two of Preparation Example 2. The whey protein supramolecular fragment solution with the crystal nucleus structure was thoroughly mixed with the whey protein monosodium solution prepared according to step one of Preparation Example 2 at a ratio of 1:1; heated in a 90°C water bath and stirred for 10 hours, then cooled to obtain a whey protein supramolecular polymer induced once. The whey protein supramolecular polymer induced once was thoroughly mixed with the whey protein monosodium solution prepared according to step one of Preparation Example 2 at a ratio of 1:1; heated in a 90°C water bath and stirred for 10 hours, then cooled to obtain a whey protein supramolecular polymer induced twice. The above steps were repeated 6 times to prepare a whey protein supramolecular polymer induced 6 times. Control 5 included two groups of samples: with and without GA. For the sample with GA, the same mass concentration of GA as the experimental group was added before determining emulsification stability. The emulsion stability was determined according to the emulsification stability method.

[0095] 1) The supramolecular whey protein sample 1 with high emulsifying performance obtained in Preparation Example 1 has an emulsifying stability that is 185.3% higher than that of conventional polymers (control 1+GA) and 109.3% higher than that of unmodified crystal nucleus-induced (control 4+GA);

[0096] 2) The supramolecular whey protein sample 2 with high emulsifying performance obtained in Preparation Example 2 has an emulsifying stability that is 205.5% higher than that of conventional polymers (control 1+GA) and 124.1% higher than that of unmodified crystal nucleus-induced (control 4+GA).

[0097] 3) The supramolecular whey protein sample 3 with high emulsifying performance obtained in Preparation Example 3 showed that its emulsifying stability was 198.6% higher than that of conventional polymers (control 1+GA) and 119.1% higher than that of unmodified crystal nucleus-induced (control 4+GA).

[0098] Example 2: Comparison of properties of whey protein isolate with high emulsification stability with whey protein isolate obtained by existing methods.

[0099] 1. Property Parameter Detection Method

[0100] 1) Determination of interfacial adsorption capacity

[0101] The prepared emulsion was centrifuged at 13,000 r / min for 15 min, and the aqueous phase at the bottom of the centrifuge tube was removed. Protein content was determined using the Bradford method, where C0 is the total protein content in the emulsion, and C... f Protein content in the aqueous phase layer at the bottom of the centrifuge tube.

[0102]

[0103] 2) Method for determining surface hydrophobicity

[0104] The ANS (8-phenylamino-1-naphthylsulfonic acid) fluorescent probe method was used, with modifications. A 3.0 g / 100 mL whey protein supramolecular solution was diluted with 0.01 mol / L phosphate buffer to obtain four concentrations: 0.025, 0.05, 0.1, and 0.2 mg / mL. 6 mL of the diluted whey protein solution was taken, and 20 μL of 8 mmol / L ANS solution was added. The mixture was incubated at room temperature in the dark for 15 min. The fluorescence intensity was then measured using a fluorescence spectrophotometer with an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and a slit width of 5 nm. A graph was plotted with fluorescence intensity on the ordinate and whey protein solution concentration on the abscissa, with the initial slope representing the sample surface hydrophobicity.

[0105] 3) Methods for determining molecular flexibility

[0106] Add 250 μL of 1 mg / mL trypsin (dissolved in 0.05 mol / L, pH 8.0 Tris-HCl buffer, enzyme activity 7852 U / g) to 4 mL of 1 mg / mL whey protein sample solution and incubate at 38 °C for 2 h. Then, add 4 mL of 5% trichloroacetic acid solution to terminate the enzymatic reaction and centrifuge at 3000 rpm for 5 min. The supernatant was then measured at 280 nm, and the absorbance was expressed as absorbance A0.

[0107] 4) Hydrogen bond strength determination method

[0108] The whey protein supramolecular solution sample was diluted to 0.1 mg / mL with ultrapure water (pH 2.0). 200 μL of the diluted protein sample was transferred to a quartz cuvette with a 0.2 cm optical path and scanned using a circular dichroism spectrometer. Specific parameter settings were as follows: scan range 190 nm–260 nm, scan rate 100 nm / min. All samples were scanned three times.

[0109] 5) Method for determining π-π stacking strength

[0110] Aromatic amino acid residues play a crucial role in supramolecular polymer formation due to their influence on side-chain interactions caused by π-π stacking. Information on conformational changes during supramolecular assembly can be indirectly reflected by measuring changes in the fluorescence intensity and maximum emission wavelength of tryptophan (Trp) at an excitation wavelength of 280 nm. The sample was diluted to 0.02 g / 100 mL with deionized water (pH 2.0). The excitation wavelength was 280 nm, the emission spectrum was 300-400 nm, and the excitation and emission slits were both 5 nm.

[0111] 6) Identification of the releasing peptide

[0112] Whey protein supramolecular samples were hydrolyzed using alkaline protease (AP, enzyme activity 7447 U / g). The pH of the whey protein supramolecular samples was adjusted to 8.0 using 6M NaOH. Alkaline protease solution was added at an enzyme-to-substrate ratio (E / S) of 1 / 30, and the mixture was incubated at 55°C for 2 hours with stirring. The hydrolysis reaction was terminated by heating the mixture in a 90°C water bath for 20 min. The hydrolysate was centrifuged at 10,000 × g for 15 min. The resulting supernatant was desalted using C18 Stage Tips and then detected using QExactive Plus. Raw data were analyzed using Proteome Discoverer software and the UniProt protein database. β-Lg was used as a template, and only hydrolyzed peptides associated with the β-Lg primary sequence were analyzed. The hydrophobicity value of the peptide fragments was calculated by summing the hydrophobicity values ​​of each amino acid (in kcal / mol).

[0113] 7) Determination of supramolecular polymers

[0114] The T(ThT) method was used to indirectly obtain the supramolecular polymer level of whey protein. 0.080 g of thiosulfate T (ThT) was dissolved in 0.01 mol / L phosphate buffer (0.2 mol / L NaCl, pH 7.0) and the volume was adjusted to 100 mL. Insoluble matter was removed by filtration through a 0.22 μm aqueous syringe filter to obtain the ThT stock solution. For the experiment, the ThT stock solution was diluted 50 times with the aforementioned phosphate buffer to prepare the ThT working solution. 50 μL of the test sample was mixed with 5 mL of the ThT working solution in a 10 mL brown centrifuge tube, vortexed for 1 min, and then measured colorimetrically using a fluorescence spectrophotometer. The measurement parameters were: excitation wavelength 460 nm, emission wavelength 490 nm, and slit width 5 nm.

[0115] Each detection method can also be replaced by other conventional methods in the field.

[0116] 2. Differences in interfacial adsorption characteristics and structure between nucleus-GA induced supramolecular polymer emulsions and those obtained by existing preparation methods

[0117] Experimental group nucleus-GA induction*: carried out according to preparation example 2 of this application.

[0118] Control groups: Control group 3 and control group 4 mentioned above.

[0119] Experimental results are as follows Figure 1-5 As shown:

[0120] Figure 1 The results show that, compared with the supramolecular polymer structure formed by spontaneous assembly, the structural features of the supramolecular polymer obtained by the present invention are more conducive to adsorption at the oil-water interface, and the adsorption amount at the emulsion interface increases by 98%.

[0121] Figure 2 The results show that, compared with spontaneously assembled supramolecular polymers, the supramolecular polymers obtained in this invention have a 47.38% improvement in surface hydrophobicity. Figure 2 A); Molecular flexibility was improved by 59.65% ( Figure 2 B).

[0122] Figure 3 The results indicate that the GA-modified crystal nuclei, due to the moderate weakening of nucleus activity, induce the formation of supramolecular forces (hydrogen bonds) within whey proteins. Figure 3 A) and π-π bonds ( Figure 3 The weakening of B) indicates that GA effectively shields the activity of the crystal nucleus.

[0123] Figure 4 The results show that, compared with the supramolecular polymers formed by spontaneous assembly, the released building block peptides in the supramolecular structure obtained by this invention have higher hydrophobicity, which effectively improves the hydrophobic properties and is beneficial to the improvement of emulsion stability.

[0124] Figure 5 The results show that among various polyphenols, only GA, after binding to the crystal nucleus, can still induce the formation of supramolecular polymers from single lactone proteins, exhibiting a higher formation capacity than spontaneous assembly methods. Therefore, only GA, and under appropriate dosage conditions, can most effectively and precisely control crystal nucleus activity without affecting the supramolecular formation capacity, thus still enabling the formation of supramolecular polymers with crystal nucleus structures. This is something that other types of polyphenols cannot achieve.

[0125] 3. Emulsification stability of supramolecular polymers formed after modification of whey protein crystal nuclei by different polyphenols

[0126] Experimental group: Prepared according to Preparation Example 2 of this application.

[0127] Control group: GA in preparation example 2 was replaced with chlorogenic acid, naringin, and quercetin, respectively, to obtain control group 6, control group 7, and control group 8.

[0128] The results are shown in Table 2:

[0129] Table 2. Emulsification stability (after 60 days) of supramolecular polymers formed by different types of polyphenols after modification of whey protein crystal nuclei.

[0130]

[0131] Note: Different lowercase letters indicate significant differences (P<0.05).

[0132] As shown in Table 2, among various polyphenolic compounds, GA exhibits the best effect.

[0133] 4. Emulsification stability of supramolecular polymers formed after modification of whey protein nuclei with different mass concentrations of GA

[0134] Experimental group: Prepared according to Preparation Example 2 of this application.

[0135] Control group: GA was added to the supramolecular fragment solution at mass concentrations of 8 mg / g, 9 mg / g, 10 mg / g, and 12 mg / g (GA mg / protein g) in step 3 of preparation example 2, respectively, as controls 9, 10, 11, and 12. Other steps were the same.

[0136] The experimental results are shown in Table 3.

[0137] Table 3. Emulsification stability (after 60 days) of supramolecular polymers formed after nucleus modification by different mass concentrations of GA.

[0138]

[0139] Note: Different lowercase letters indicate significant differences (P<0.05).

[0140] The results from Tables 2 and 3 show that the degree of weakening of the crystal nucleus surface activity is key to improving emulsion stability, and this invention can effectively regulate the crystal nucleus surface activity. The choice of polyphenols has a significant impact on the degree of shielding of crystal nucleus surface activity. Unexpectedly, it was found that among polyphenols, GA can most effectively and precisely control the degree of shielding. A low GA addition ratio results in fewer bindings to amino acid residues on the active region of the crystal nucleus surface, leading to a low degree of shielding and minimal impact on the stacking ability of whey protein on the crystal nucleus surface. This also results in little improvement in the formed supramolecular rigid structure, which is detrimental to improving emulsion stability. Conversely, a high GA addition ratio results in too much GA binding on the crystal nucleus surface, leading to excessive shielding of crystal nucleus activity, blocking the stacking of whey protein on the crystal nucleus surface, inhibiting supramolecular formation, and failing to improve emulsion stability. Determining a precise GA addition ratio can effectively regulate the crystal nucleus surface activity. The above description is only a preferred embodiment of this invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. A method for preparing whey protein isolate with high emulsification stability, characterized in that, The preparation steps are as follows: (1) Preparation of monosomal protein solution of milk Dissolve whey protein isolate in deionized water, mix thoroughly, adjust the pH to 1.5-2.5, centrifuge at high speed at 1-4℃ for 15-30 minutes, take the supernatant, and dilute with deionized water of the same pH to obtain a whey protein isolate solution of 1-4 g / 100 mL. (2) Preparation of whey protein supramolecular fragments with crystal nucleus structure The partial whey protein monosomal solution obtained in step (1) was heated and stirred in a high-temperature water bath at 80-95℃ for 8-20 hours, then cooled to room temperature, and homogenized at high speed for 18000-20000 rpm for 1-2 minutes to prepare a whey protein supramolecular fragment solution with a crystal nucleus structure. (3) Preparation of the crystal nucleus-GA complex solution According to the protein mass contained in the supramolecular fragment solution, add gallic acid (GA) at a mass concentration of 8-12 mg / g. Mix thoroughly and stir in the dark at 20-25°C for 18-24 hours to obtain a whey protein supramolecular crystal nucleus-GA complex solution bound by non-covalent bonds. (4) Crystal nucleus-GA induces the formation of whey protein supramolecular solution Prepare a mixed solution consisting of the crystal nucleus-GA complex solution and the emulsion monosodium protein solution from step (1) with a volume ratio of 1:1-4. Mix thoroughly, adjust the pH to 1.5-2.5, heat and stir in a water bath at 80-95℃ for 8-20 hours, and cool to room temperature. (5) The whey protein supramolecular solution obtained in step (4) is concentrated and freeze-dried to obtain whey protein isolate with high emulsification stability.

2. Use of gallic acid (GA) in improving the emulsification stability of whey protein isolate, wherein the whey protein isolate with high emulsification stability is prepared according to the method described in claim 1.

3. A method for preparing whey protein isolate with high emulsification stability, characterized in that, The preparation steps are as follows: Step 1: Preparation of Milk Monosodium Protein Solution Dissolve 6.0g of whey protein isolate in deionized water, mix thoroughly, adjust the pH to 2.0, centrifuge at 20000g for 20min at 4℃, take the supernatant, and dilute with deionized water of the same pH to obtain a whey protein isolate solution of 3g / 100mL. Step 2: Preparation of whey protein supramolecular fragments with crystal nucleus structure A whey protein supramolecular fragment solution with a crystal nucleus structure was prepared by heating and stirring a 3g / 100mL whey monosodium protein solution in a 90℃ water bath for 10h, cooling to room temperature, and homogenizing at 20000rpm for 2min. Step 3: Preparation of the nucleus-GA complex solution Based on GA mg / protein g, GA was added to the supramolecular fragment solution at a mass concentration of 11 mg / g, mixed thoroughly, and magnetically stirred at 25°C in the dark for 24 h to obtain a whey protein supramolecular crystal nucleus-GA complex solution. Step 4: Crystal nucleus-GA induction to form whey protein supramolecular solution Prepare a mixed solution consisting of the crystal nucleus-GA complex solution and the emulsion monosodium protein solution from step one above, with a volume ratio of 1:1; mix thoroughly, adjust the pH to 2.0, heat in a 90°C water bath with stirring for 10 hours, and then cool to room temperature; Step 5: Concentrate the obtained whey protein supramolecular solution and freeze-dry it to obtain whey protein isolate with high emulsification stability.

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