Method for improving performance of concentrated milk protein and concentrated milk protein

Abstract

The invention belongs to the technical field of protein processing, and particularly relates to a method for improving the performance of concentrated milk protein and the concentrated milk protein. And immersing an ultrasonic probe into the concentrated milk protein solution, and carrying out probe ultrasonic treatment for 5-30 minutes under the power of 100-500W. By adopting the method provided by the invention, after the concentrated milk protein is subjected to probe ultrasonic treatment, the solubility of the concentrated milk protein is increased from 39.63% to 94.44%, which indicates that the probe ultrasonic treatment can destroy protein aggregates through the cavitation effect and improve the solubility and dispersion stability of the protein aggregates, thereby providing a theoretical basis for efficient application of the concentrated milk protein in the food industry.

Description

Technical Field

[0001] The invention belongs to the technical field of protein processing, and in particular relates to a method for improving the performance of concentrated milk protein and the concentrated milk protein. Background Art

[0002] Milk protein concentrates (MPCs), a novel dairy-based ingredient, are processed from milk. Produced through membrane filtration, evaporation, concentration, and drying, they are solid powders. The protein content of MPCs can range from 42% to 85% of total solids, thanks to innovations in membrane separation technology. Ultrafiltration and diafiltration selectively remove lactose and minerals from skim milk, followed by evaporation and concentration. MPCs offer a unique advantage in preserving the natural ratio of casein to whey protein (approximately 80:20) found in the milk source, without undergoing intense heat treatment or pH manipulation, thereby maximizing the preservation of the proteins' native conformation and biological activity. Compared to skim milk powder, MPCs offer high protein and low lactose content, while also combining the synergistic nutritional benefits of slowly digestible casein and rapidly released whey proteins to provide a sustained supply of amino acids. Due to its superior functional properties, such as thickening, emulsification, and gel-forming abilities, as well as its refreshing frankincense flavor, MPC has been widely used in infant formula, cheese fortifiers, fermented dairy products, and functional dairy beverages. Notably, its use in yogurt and cheese accounts for a particularly large proportion, which is closely related to its dual effects of improving texture and increasing protein content.

[0003] Although MPC has shown broad application prospects in the food industry, as storage time increases, the solubility of MPC in water decreases, and protein aggregation occurs, resulting in a solubility of MPC of only about 30%, which seriously restricts its practical application effectiveness. Studies have shown that the decrease in MPC solubility is due to the enhanced cross-linking between protein molecules during storage, including the synergistic effect of hydrogen bonds, disulfide bonds and hydrophobic interactions that lead to the formation of aggregates. This phenomenon is particularly significant in liquid systems such as functional beverages, often causing irreversible precipitation, which not only damages the sensory quality of the product, but also leads to the loss of protein functional properties. Therefore, exploring efficient and controllable MPC solubility modification technology has become a hot topic that the industry urgently needs to solve. Existing research has mostly focused on physical modification methods. Among them, static high-pressure treatment, temperature-time coordinated control and other technologies can effectively improve solubility, but they have the problems of cumbersome operation and long time. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for improving the performance of concentrated milk protein and the concentrated milk protein. The present invention can quickly and easily improve the solubility of concentrated milk protein by using a probe ultrasound method.

[0005] The specific technical solutions of the present invention are as follows: A first aspect of the present invention provides a method for improving the performance of concentrated milk protein, comprising the following steps: Immerse the ultrasonic probe in the concentrated milk protein solution and perform ultrasonic testing with the probe at a power of 100W to 500W for 5 minutes to 30 minutes; Improving the performance of concentrated milk protein refers to improving the solubility of concentrated milk protein.

[0006] The present invention uses probe ultrasound to increase the solubility of concentrated milk protein from 39.63% to 94.44% after ultrasound at a power of 100W to 500W for 5 minutes to 30 minutes. Compared with water bath ultrasound, probe ultrasound directly radiates a high-energy density sound field through the probe, which can achieve local and efficient treatment and effectively shorten the operation time. In addition, during the ultrasound process, only the power and time need to be controlled to achieve the improvement of the solubility of concentrated milk protein. The entire operation process is simple and convenient, which greatly improves work efficiency.

[0007] In another preferred embodiment, the specific process of the probe ultrasound is as follows: Immerse the ultrasonic probe in the concentrated milk protein solution, sonicate for 3s~5s, and then rest for 3s~5s in a cycle, and perform probe sonication for 5min~30min at a power of 100W~500W.

[0008] In another preferred embodiment, the specific process of the probe ultrasound is as follows: Immerse the ultrasonic probe in the concentrated milk protein solution, sonicate for 3s~5s, and then rest for 3s~5s in a cycle, and perform probe sonication for 5min~20min at a power of 100W~300W.

[0009] In another preferred embodiment, the temperature of the probe ultrasound is 23°C to 25°C.

[0010] In another preferred embodiment, the specific process of obtaining the concentrated milk protein solution is as follows: The concentrated milk protein powder is dissolved in water and stirred.

[0011] The second aspect of the present invention provides a concentrated milk protein prepared by the method.

[0012] In another preferred embodiment, the solubility of the concentrated milk protein in water is 90% to 94.44%.

[0013] In another preferred embodiment, the particle size of the concentrated milk protein is 190 μm to 200 μm.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention performs ultrasonic treatment on the concentrated milk protein solution, and performs probe ultrasonic treatment at a power of 100W to 500W for 5 minutes to 30 minutes, which can effectively improve the solubility of concentrated milk protein, up to 94.44%. The present invention also found through experiments that after 100W water bath ultrasonic treatment, the solubility of concentrated milk protein increased from 39.63% to 76.08%, while the probe ultrasonic treatment group reached 94.44% under the same conditions. In comparison, the probe ultrasonic treatment group showed a more significant increase in the solubility of concentrated milk protein under the same power of 100W and a duration of 30 minutes. This is mainly because probe ultrasonic treatment directly radiates a high-energy density sound field through the probe, which can achieve local and efficient treatment; while water bath ultrasonic treatment acts on the entire sample system with a uniform sound field. There are essential differences between the two in terms of thermodynamic effects, shear strength and processing uniformity. Therefore, the present invention effectively improves the solubility of concentrated milk protein by precisely controlling the probe ultrasonic parameters. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The protein standard curve.

[0016] Figure 2 This is a diagram showing the effect of ultrasonic treatment on the composition of MPC.

[0017] Figure 3 Figure 3 is the influence of ultrasonic treatment on the ultraviolet spectrum of MPC, A is the ultraviolet spectrum influence diagram of water bath ultrasound and probe ultrasound at 100 W, B is the ultraviolet spectrum influence diagram of water bath ultrasound and probe ultrasound at 300 W, and C is the ultraviolet spectrum influence diagram of water bath ultrasound and probe ultrasound at 500 W; in the figure, MPC represents milk protein concentrate, W-5M represents water bath ultrasound for 5 min, W-10M represents water bath ultrasound for 10 min, W-20M represents water bath ultrasound for 20 min, W-30M represents water bath ultrasound for 30 min, P-5M represents probe ultrasound for 5 min, P-10M represents probe ultrasound for 10 min, P-20M represents probe ultrasound for 20 min, and P-30M represents probe ultrasound for 30 min, the same below.

[0018] Figure 4 Figure 2 is the effect of ultrasonic treatment on the fluorescence spectrum of MPC; A is the fluorescence spectrum effect diagram of water bath ultrasound and probe ultrasound at 100W, B is the fluorescence spectrum effect diagram of water bath ultrasound and probe ultrasound at 300W, and C is the fluorescence spectrum effect diagram of water bath ultrasound and probe ultrasound at 500W.

[0019] Figure 5Figure 3 is the effect of ultrasonic treatment on the surface hydrophobicity of MPC; A is the surface hydrophobicity effect diagram of water bath ultrasound and probe ultrasound at 100W, B is the surface hydrophobicity effect diagram of water bath ultrasound and probe ultrasound at 300W, and C is the surface hydrophobicity effect diagram of water bath ultrasound and probe ultrasound at 500W.

[0020] Figure 6 Scanning electron micrographs of MPC under different ultrasonic treatment conditions; in the figure, 100W / P-5m means water bath ultrasonic treatment for 5 min at 100W power, and the rest are the same; 100W / W-5m means probe ultrasonic treatment for 5 min at 100W power, and the rest are the same.

[0021] Figure 7 Figure 2 is the effect of ultrasonic treatment on the turbidity of MPC. A is the turbidity effect diagram of water bath ultrasound and probe ultrasound at 100W, B is the turbidity effect diagram of water bath ultrasound and probe ultrasound at 300W, and C is the turbidity effect diagram of water bath ultrasound and probe ultrasound at 500W.

[0022] Figure 8 Figure 3 is the effect of ultrasonic treatment on the particle size of MPC. A is the particle size effect diagram of water bath ultrasound and probe ultrasound at 100W, B is the particle size effect diagram of water bath ultrasound and probe ultrasound at 300W, and C is the particle size effect diagram of water bath ultrasound and probe ultrasound at 500W.

[0023] Figure 9 Figure 3 is the effect of ultrasonic treatment on the particle size distribution of MPC. A is the particle size distribution effect diagram of water bath ultrasound and probe ultrasound at 100W, B is the particle size distribution effect diagram of water bath ultrasound and probe ultrasound at 300W, and C is the particle size distribution effect diagram of water bath ultrasound and probe ultrasound at 500W.

[0024] Figure 10 Figure 2 is the effect of ultrasonic treatment on the ζ-potential of MPC; A is the ζ-potential effect diagram of water bath ultrasound and probe ultrasound at 100W, B is the ζ-potential effect diagram of water bath ultrasound and probe ultrasound at 300W, and C is the ζ-potential effect diagram of water bath ultrasound and probe ultrasound at 500W. DETAILED DESCRIPTION

[0025] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0026] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0027] Ultrasonic waves (frequency > 20 kHz) are elastic mechanical waves whose mechanism of action stems from the localized high pressure (up to 100 MPa), transient high temperature (approximately 5000 K), and intense microfluidics induced by the cavitation effect. These physical effects can induce conformational rearrangements in protein molecules, disrupting hydrophobic regions and disulfide bond networks, promoting the dissociation of macromolecular aggregates into soluble subunits, thereby improving solubility. Notably, the regulation of protein functional properties by ultrasonic treatment is significantly parameter-dependent. Different ultrasonic treatment modes, such as probe-based and water-bath-based, differ fundamentally in their thermodynamic effects, shear strength, and treatment uniformity. Probe-based ultrasonic treatment directly radiates a high-energy-density acoustic field through a probe, achieving efficient localized treatment. Water-bath-based ultrasonic treatment, on the other hand, applies a uniform acoustic field to the entire sample system.

[0028] Based on this, the present invention uses MPC as the research object to systematically explore the effects of probe ultrasound and water bath ultrasound on its solubility and structural properties. By characterizing the particle size distribution, Zeta potential, surface charge, and changes in protein secondary structure, combined with dissolution kinetic analysis, the structure-activity relationship between the ultrasonic treatment parameters power, time, mode, and functional properties is clarified. At the same time, by comparing the energy transfer efficiency and thermal effect differences of the two ultrasonic modes, the differences in their mechanisms of action on proteins are revealed, thereby promoting the innovative application of highly soluble milk protein ingredients in the field of functional foods.

[0029] 1. Materials and Methods 1.1 Materials and Reagents All experimental reagents, including MPC powder, phosphate buffer powder, 8-anilino-1-naphthalenesulfonic acid (ANS), Coomassie Brilliant Blue R-250, Coomassie Brilliant Blue G-250, ethanol, and ultrapure water, were of analytical grade. Instruments are listed in Table 1.

[0030] Table 1 Main experimental instruments and equipment 1.2 Experimental methods 1.2.1 Sample processing 5 g of MPC powder was weighed, diluted to 100 mL with deionized water, stirred at 600 rpm in a magnetic stirrer for 4 h, and then placed in a refrigerator at 4°C overnight for hydration to obtain a concentrated milk protein solution.

[0031] 1.2.2 Ultrasonic treatment 1) Ultrasonic treatment of the probe 20 mL of concentrated milk protein solution was placed in an ultrasonic cell disruptor and sonicated with a working time of 3 s and a rest time of 3 s according to the following experimental protocol: Group 1: Adjust the power to 100W and process for 5 minutes, 10 minutes, 20 minutes, and 30 minutes respectively.

[0032] The second group: the power was adjusted to 300W and the treatments were performed for 5 minutes, 10 minutes, 20 minutes and 30 minutes respectively.

[0033] The third group: the power was adjusted to 500W and the treatments were conducted for 5 minutes, 10 minutes, 20 minutes and 30 minutes respectively.

[0034] Meanwhile, MPC powder without ultrasonic treatment was used as a control.

[0035] 2) Water bath ultrasonic treatment 20 mL of concentrated milk protein solution was placed in a 30°C water bath constant temperature oscillator and treated according to the following experimental protocol: Group 1: Adjust the power to 100W and process for 5 minutes, 10 minutes, 20 minutes, and 30 minutes respectively.

[0036] The second group: the power was adjusted to 300W and the treatments were performed for 5 minutes, 10 minutes, 20 minutes and 30 minutes respectively.

[0037] The third group: the power was adjusted to 500W and the treatments were conducted for 5 minutes, 10 minutes, 20 minutes and 30 minutes respectively.

[0038] Meanwhile, MPC powder without ultrasonic treatment was used as a control.

[0039] 1.2.3 SDS-PAGE electrophoresis The gel used a 12wt% separating gel and a 5wt% stacking gel. After ultrasonic treatment, the concentrated milk protein solution was centrifuged at 5000 rpm for 10 minutes. 8μL of the supernatant was mixed with 2μL of 5× SDS-PAGE Sample Loading Buffer for 10 seconds. The sample was then heated in a boiling water bath for 5 minutes and loaded. Gel electrophoresis was performed at 80V, then adjusted to a constant voltage of 120V after the sample entered the separating gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and destained with a destaining agent until the background was transparent and clear protein bands were visible.

[0040] 1.2.4 UV-visible spectrum measurement At room temperature, 3 mL of ultrasonically treated concentrated milk protein solution was added to a 1 cm quartz colorimetric cell using a 1000 μL microsyringe, and the UV-visible absorption spectrum was scanned in the range of 260 nm to 350 nm.

[0041] 1.2.5 Intrinsic fluorescence spectroscopy After ultrasonic treatment, the concentrated milk protein solution was diluted to a concentration of 0.2 mg / mL with PBS (0.01 mol / L, pH 7.0). Scan parameters were set as follows: voltage at 700 mV, excitation wavelength at 290 nm, slit at 5.0 nm, and emission wavelength between 300 and 460 nm.

[0042] 1.2.6 Surface hydrophobicity 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a fluorescent probe for the determination. Blank and ultrasonically treated concentrated milk protein solutions were diluted with phosphate buffer (0.1 mol / L, pH 7) to concentrations ranging from 0.05 g / L to 0.40 g / L. 2 mL of each diluted sample was then added to 10 μL of 8 mmol / L ANS (prepared in 0.01 mol / L, pH 7.0 phosphate buffer). Fluorescence intensity was immediately measured using a fluorescence spectrophotometer at an excitation wavelength of 390 nm (slit 2.5 nm) and an emission wavelength of 468 nm (slit 2.5 nm). A curve was plotted of fluorescence intensity versus protein concentration and extrapolated to a protein concentration of zero. The slope of the initial curve was used to determine the surface hydrophobicity index of the protein molecule.

[0043] 1.2.7 Scanning electron microscopy analysis The concentrated milk protein solution after ultrasonic treatment was fixed, dehydrated and dried, and the surface of the sample was sprayed with gold using a high vacuum plating machine for 60 seconds. The sample was then placed under a scanning electron microscope to observe the microstructure of the MPC.

[0044] 1.2.8 Determination of turbidity Use a microsyringe to transfer 200uL of the evenly stirred ultrasonically treated concentrated milk protein solution (1mg / mL) onto a 96-well plate, let it stand for 10 minutes, and then measure the absorbance of the above solution at a wavelength of 600nm on a microplate reader. Each sample was repeated 3 times.

[0045] 1.2.9 Particle size determination The concentrated milk protein solution after ultrasonic treatment was diluted to 1 mg / mL with phosphate buffer (0.01 mol / L, pH 7.0). The specifications of the particle size analyzer were: particle refractive index (1.460), equilibrium time 120 s, and dispersant refractive index (1.330).

[0046] 1.2.10 Determination of ζ-potential The concentrated milk protein solution after ultrasonic treatment was diluted to 1 mg / mL with phosphate buffer (0.01 mol / L, pH 7.0), and the ζ-potential of the protein solution was measured by a potentiometer at 25°C with the following parameters: temperature equilibrium for 2 minutes.

[0047] 1.2.11 Solubility determination 1) Drawing of the standard curve Take 7 test tubes and add 0mL, 0.01mL, 0.02mL, 0.03mL, 0.04mL, 0.05mL, and 0.06mL of standard protein (1mg / mL crystalline bovine serum albumin) solution, respectively. Add 0.15mol / LNaCl to 0.1mL, add 5mL of Coomassie Brilliant Blue reagent to each, shake well and mix. After 10 minutes, measure the absorbance of each tube at a wavelength of 595nm, using tube 0 as a blank. Plot a standard curve with the standard protein concentration (X) as the horizontal axis and the absorbance value (Y) as the vertical axis. The regression equation y=0.03x+0.6127 (R2=0.9994) is obtained. The standard curve is shown in the figure below. Figure 1 shown.

[0048] 2) Determination of protein content Prepare a 0.6 mg / mL solution of freeze-dried, ultrasonically treated concentrated milk protein solution (0.1 mL) in a test tube. Add 5 mL of Coomassie Brilliant Blue reagent to each tube, mix thoroughly, and let stand for 10 minutes. Measure the absorbance at 595 nm using a reagent blank as a control. Perform three replicates and measure the absorbance. Calculate the protein concentration (mg / mL) of the unknown sample by comparing the amount of standard protein on the standard curve. Compare the calculated protein concentration with the protein concentration of the unknown sample to determine the protein solubility.

[0049] 3) Data processing All experiments were repeated three times, and data were analyzed using SPSS Statistics 22.0 software. ANOVA was used for statistical analysis, with p < 0.05 considered significant. Origin software was used for graphic processing and atlas analysis.

[0050] 3. Results 3.1 Effect of ultrasonic treatment on the structure and properties of MPC 3.1.1 SPS-PAGE results SDS-PAGE is a common method for evaluating the molecular weight distribution of protein subunits. SDS-PAGE can be used to analyze the changes in the molecular weight of MPC subunits after treatment under different ultrasonic conditions. Figure 2As shown in the figure, there is no significant difference in protein bands under different ultrasonic power and time conditions, which indicates that ultrasonic treatment does not change the subunits of MPC.

[0051] 3.1.2 UV spectrum analysis results Ultraviolet spectroscopy can be used to analyze small molecule-protein interactions and reveal the conformational characteristics of proteins in solution. Ultraviolet absorption spectroscopy was used to examine the structural changes of MPC under different ultrasonic treatment conditions. The UV absorption characteristics are primarily determined by the side chain groups of aromatic amino acids such as tryptophan and tyrosine, with their exposure positively correlated with the absorption intensity. The characteristic peak at 278 nm originates from the n-π* electronic transitions of tryptophan (Trp) and tyrosine (Tyr). Figure 3 Shows the effect of ultrasonic treatment on the UV spectrum of MPC. After water bath ultrasonic treatment ( Figure 3 The intensity of the UV absorption peak increased continuously with the treatment time (0min~30min), and the 30min treatment group increased by 45% compared with the untreated group. Figure 3 The absorption intensity of C in the sample increased by 50.4% after 30 minutes. Comparison of the treatment conditions revealed that the UV absorption intensity was positively correlated with the ultrasonic power. The increase in absorption intensity may be due to the exposure of the internal aromatic groups caused by structural expansion. The characteristic absorption peak did not undergo a red shift (276nm~277nm) ( Figure 3 (A in Figure 1) indicates that sonication did not alter the protein microenvironment. The stability of the characteristic peaks confirms that the polarity of the microenvironment remains unchanged, a conclusion that is consistent with the SDS-PAGE results.

[0052] 3.1.3 Fluorescence spectrum analysis results Intrinsic fluorescence is an important optical property for characterizing protein structure. Fluorescence spectroscopy was used to analyze conformational changes in MPC under different sonication conditions. Fluorescence is derived from electronic transitions of tryptophan, tyrosine, and phenylalanine under excitation at 280 nm or 295 nm, and changes in its intensity reflect changes in protein conformation. Figure 4 The effect of ultrasonic treatment on the fluorescence spectrum of MPC is shown. The maximum fluorescence peak of the untreated group is at 366.5nm. After 30min of water bath ultrasonic treatment ( Figure 4 The fluorescence intensity of the probe ultrasonic treatment group ( Figure 4 (B) The intensity decreased by 81.9% after 30 minutes. Comparison of the various treatment conditions revealed a negative correlation between fluorescence intensity and ultrasound power. The maximum absorption peak shifted to the blue, indicating that ultrasound treatment altered the protein's tertiary structure. This phenomenon confirms that ultrasound can induce changes in protein tertiary structure.

[0053] 3.1.4 Surface hydrophobicity analysis results The changes in the surface hydrophobicity of proteins are closely related to the folding-extension dynamics of their molecular conformations. The surface hydrophobicity decreases when the molecules fold, while the hydrophobicity increases when the molecules extend. Figure 5 The system shows the influence of different ultrasonic treatment conditions on the hydrophobicity of MPC surface. The hydrophobicity index of the untreated MPC is 446.33. After water bath ultrasonic treatment ( Figure 5 The hydrophobicity index of the probe ultrasonic treatment group (A) increased to 739.43, an increase of 65.8% compared with the untreated group. Figure 5 The hydrophobicity index (F) increased to 663.06, a 48.6% increase. A comparison revealed that the H0 value was higher in the waterbath sonication group. The hydrophobicity index did not change significantly over time at both 300W and 500W ultrasonic powers. This suggests that sonication promotes protein conformational unfolding, exposing internal hydrophobic groups, indicating that different proteins respond differently to sonication.

[0054] 3.2 Effects of ultrasonic treatment on the physicochemical properties of MPC 3.2.1 Scanning electron microscopy analysis results Scanning electron microscopy was used to observe its microstructure. Micromorphology analysis can detect changes in protein structure. The morphological characteristics of MPC under different ultrasonic treatment conditions were observed by scanning electron microscopy. The surface of MPC in the untreated group was smooth and dense ( Figure 6 (blank in the image). After water bath and probe sonication, pores and fragmented structures appeared on the MPC surface. Morphological differences were evident between the 100W and 300W sonication groups, but no differences were observed between the 300W and 500W groups. At 300W, treatment times of 5 to 30 minutes did not cause any morphological changes. This suggests that there is a threshold power for the effect of sonication on MPC structure, indicating that different proteins respond similarly to sonication.

[0055] 3.2.2 Turbidity analysis results Turbidimetric analysis is used as a key indicator to assess protein aggregation status, and its quantitative characterization can be achieved by colorimetric methods. Figure 7 As shown in Figure 2, different ultrasonic treatment modes have different effects on the turbidity dynamics characteristics of the MPC suspension system. Figure 7 Figure B) During the treatment time of 0 min to 30 min, the turbidity value decreased from 0.57 to 0.39, indicating that this treatment helped maintain the stability of the system. This effect may be due to the uniform energy distribution characteristics of water bath sonication. Its gentle mechanical action promotes the formation of smaller particle size dispersion of proteins, thereby reducing turbidity. In contrast, probe sonication shows a different turbidity evolution pattern. In the power range of 100W to 300W ( Figure 7 The turbidity value continued to increase with the treatment time, with the highest increase reaching 53.48% (300W group). When the power was increased to 500W ( Figure 7(C in the figure), the turbidity values ​​tended to be stable, showing no significant time-dependent changes. This difference may be attributed to the high-intensity cavitation effect of probe ultrasound: the intense shear force and instantaneous extreme conditions cause the MPC structure to dissociate and reorganize, forming large aggregates, which enhance the light scattering effect and cause an increase in turbidity. This difference reflects the specific regulatory mechanism of different energy input methods on the protein structure-activity relationship.

[0056] 3.2.3 Particle size analysis results Particle size distribution is a common method to assess protein aggregation status, which reflects the aggregation process through changes in particle size. Figure 8 As shown in the figure, different ultrasonic treatment modes have different effects on the particle size of MPC solution. From the experimental results, it can be seen that after water bath ultrasonic treatment, the particle size of MPC solution is reduced from the initial 450.83μm to 136.1μm ( Figure 8 C in the figure), while that in the probe ultrasound treatment group was reduced to 194.47 μm ( Figure 8 C in the figure). This phenomenon may be related to the ultrasonic cavitation effect, which promotes the dissociation of protein aggregates into smaller subunit structures. Figure 9 As shown, the untreated sample exhibited a broad particle size distribution ranging from 100 μm to 1000 μm. However, after ultrasonic treatment, the proportion of particles in this range decreased, and the particle size distribution curve shifted to the right, indicating a more uniform distribution. This phenomenon suggests that the high-frequency mechanical vibrations generated during ultrasonic treatment caused collisional dissociation of large protein aggregates, ultimately forming a system of smaller particles. High-intensity ultrasonic experiments confirmed that the high shear forces generated by the cavitation effect could dissociate myosin aggregates.

[0057] 3.3 Effect of ultrasonic treatment on the functional properties of MPC 3.3.1 ζ-potential analysis results Zeta potential is a key indicator for characterizing the stability of colloidal dispersion systems and can effectively reflect the strength of electrostatic repulsion and interaction between protein molecules. The results of MPC solution zeta potential measurement are as follows: Figure 10 As shown in Figure 2, ultrasonic treatment at pH 7.0 changed the surface charge characteristics. The initial ζ-potential of the control group was -6.18 mV, which increased to -12.27 mV after 20 min of water bath ultrasonic treatment (100 W). Figure 10 The probe ultrasonic treatment group (100W) reached -11.03mV ( Figure 10 A in ). When the power is increased to 300W~500W ( Figure 10B and C in Figures 3 and 4 show no significant time-dependent changes in the ζ-potential. This phenomenon may be related to ultrasound-induced structural reorganization of the protein: the weakening of non-covalent bonds, such as hydrogen bonds and hydrophobic interactions, may enhance surface charge density, thereby improving solubility properties. Combined with these results, it is speculated that ultrasound treatment may restructure the protein's tertiary structure, weakening non-covalent binding forces such as hydrogen bond polarity between amino acid residues and hydrophobic interactions between non-polar residues, thereby enhancing surface charge density and optimizing protein-water interfacial compatibility. These findings collectively reveal the mechanism by which energy input modulates protein functional properties.

[0058] 3.3.2 Solubility analysis results Protein conformational evolution has a crucial influence on its functional properties. Solubility, a key parameter characterizing protein denaturation and aggregation behavior, is directly linked to the dynamic balance between molecular conformational rearrangements and interaction networks. As shown in Table 2, experimental data show that the solubility of MPC without sonication treatment is 40%, further confirming that the insufficient solubility performance of traditional MPC significantly limits its application in the food industry. Experimental data also reveal that solubility characteristics exhibit differentiated responses after sonication. The solubility of the waterbath sonication group (100 W, 30 minutes) increased from 39.63% to 76.08%, while the solubility of the probe sonication group reached 94.44% under the same conditions. In contrast, the solubility increase in the probe sonication group was even more significant under the same power (100 W) and duration (30 minutes). When the probe sonication power exceeded 300 W and the treatment time was prolonged, the solubility dropped to 67.71%. This phenomenon may be related to the local overheating effect caused by high-power sonication. The release of excessive heat energy may induce thermally induced aggregation of the protein, leading to a deterioration in solubility.

[0059] Table 2 Effect of ultrasonic treatment on the solubility of MPC Note: Significant differences (p<0.05) between different treatment times and powers are indicated by different lowercase and uppercase letters, respectively.

[0060] The present invention systematically studies the effects of probe ultrasound and waterbath ultrasound treatment on the physicochemical and functional properties of MPC. The study shows that different ultrasound treatment methods do not change the primary structure of MPC, but will cause its tertiary structure to unfold, resulting in a decrease in MPC fluorescence intensity and an increase in surface hydrophobicity. Scanning electron microscopy was used to observe the destruction of the apparent morphology of protein aggregates and the formation of pores on the MPC surface. In addition, ultrasound treatment significantly reduces the particle size of MPC and increases the absolute value of the Zeta potential of the solution system, indicating that the electrostatic repulsion of the MPC solution is enhanced and the dispersion stability is improved after ultrasound treatment. The solubility of MPC is significantly increased after ultrasound treatment. The effect of probe ultrasound treatment for 30 minutes is optimal under 100W conditions and is significantly better than waterbath ultrasound. However, 500W high-power probe ultrasound may cause secondary aggregation of proteins and a decrease in solubility. Probe ultrasound is suitable for rapid and efficient modification, and the present invention provides theoretical support for the application of MPC in the food industry.

[0061] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the performance of concentrated milk protein, characterized in that: The following steps are involved: Immerse the ultrasonic probe in the concentrated milk protein solution and perform ultrasonic testing with the probe at a power of 100W to 500W for 5 minutes to 30 minutes; Improving the performance of concentrated milk protein refers to improving the solubility of concentrated milk protein.

2. The method for improving the performance of concentrated milk protein according to claim 1, characterized in that: The specific process of the probe ultrasound is as follows: Immerse the ultrasonic probe in the concentrated milk protein solution, sonicate for 3s~5s, and then rest for 3s~5s in a cycle, and perform probe sonication for 5min~30min at a power of 100W~500W.

3. The method for improving the performance of concentrated milk protein according to claim 2, characterized in that: The specific process of the probe ultrasound is as follows: Immerse the ultrasonic probe in the concentrated milk protein solution, sonicate for 3s~5s, and then rest for 3s~5s in a cycle, and perform probe sonication for 5min~20min at a power of 100W~300W.

4. The method for improving the performance of concentrated milk protein according to claim 1, characterized in that: The temperature of the probe ultrasound is 20°C to 30°C.

5. The method for improving the performance of concentrated milk protein according to claim 1, characterized in that: The specific process of obtaining the concentrated milk protein solution is as follows: The concentrated milk protein powder is dissolved in water and stirred.

6. A concentrated milk protein prepared by the method according to any one of claims 1 to 5.

7. The concentrated milk protein according to claim 6, characterized in that The solubility of the concentrated milk protein in water is 90% to 94.44%.

8. The concentrated milk protein according to claim 6, characterized in that The particle size of the concentrated milk protein is 190 μm to 200 μm.

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