Micronized whey protein as well as preparation method and application thereof

Abstract

The invention discloses micronized whey protein as well as a preparation method and application thereof. In order to improve the stability of whey protein in ultra-high temperature (UHT) processing, a hot shearing-high pressure homogenization synergistic technology is combined with carbohydrate addition to prepare micronized whey protein (MWP), and the thermal stability of the micronized whey protein is systematically evaluated. The result shows that the denaturation degree of the MWP after micronization processing is increased. The MWP prepared under the conditions of neutral pH (6.6) and high protein concentration (greater than or equal to 9%) has excellent thermal stability, and still keeps good fluidity after UHT treatment. The invention provides an effective modification approach and theoretical support for developing a high-protein whey product suitable for a UHT process.

Description

Technical Field

[0001] This invention relates to the field of food processing, and more specifically, to micronized whey protein, its preparation method, and its application. Background Technology

[0002] Whey protein is an important protein in dairy products. It is rich in amino acids and bioactive peptides, has excellent nutritional value and functional characteristics, and is easily absorbed by the human body. It is an important component in the processing of high-protein products. However, whey protein is prone to denaturation, precipitation and gelation under high temperature and high concentration conditions, which limits its application in high-protein beverages, sports foods and special medical foods. Studies have found [1,2] that whey protein solutions will denature and aggregate when heated above 85°C, and at protein concentrations exceeding 10%, irreversible heat-induced gels are often formed. Due to its poor thermal stability, when beverages containing high concentrations of whey protein undergo strict heat treatment (such as boiling or ultra-high temperature instantaneous sterilization), precipitation, aggregation, flocculation or gelation will occur [3]. Therefore, improving the heat resistance of whey protein remains a serious challenge.

[0003] In recent years, improving the thermal stability of whey protein through physical, chemical or biological means has become a research hotspot. Among them, micronization process has attracted much attention due to its advantages such as high modification efficiency and no need for chemical additives. Micronization of whey protein has been proven to produce micro-aggregates with thermal stability [4]. Micronization is usually achieved by thermal aggregation or acid precipitation, combined with high shear or high pressure conditions [5]. However, Dilek Sağlam et al. [6] used a combination of two-step emulsification and thermally induced gelation to prepare whey protein microparticles. Pre-denatured whey protein microparticles can prevent thermal aggregation during secondary heating to a certain extent. Whey protein microparticles can be regarded as a combination of natural protein and controlled-size soluble and insoluble protein aggregates. The degree of denaturation and the degree of aggregation between protein molecules of whey protein vary depending on the processing method and process parameters during the preparation of micronized protein. Havea P et al. [7] published a patent on thermally stable micronized whey protein as early as 2012. Esra Çakır-Fuller [8] used the micronized whey protein prepared in the patent to produce a protein emulsion with a protein content as high as 11% wt / wt, and no visible aggregation or gelation was observed after high-temperature cooking. The micronized protein emulsion showed high thermal stability. However, most micronized proteins prepared are prone to particle swelling during secondary heating, which has a great impact on the thermal stability of whey protein. Dilek Sağlam [6] improved the swelling rate of particles by adding gum arabic as a stabilizer, and Jeroen Boeve et al. [9] increased the hardness of particles by adding aldehyde compounds. Currently, micronized whey protein has been applied in high-protein foods, but there are still few micronized proteins that can withstand UHT processing. The processing technology for micronized whey protein is still being explored. Summary of the Invention

[0004] The purpose of this invention is to provide micronized whey protein, its preparation method, and its application.

[0005] This invention utilizes thermal shearing and microfluidization techniques to prepare micronized whey protein. To improve particle hardness, after particle formation, it is mixed with carbohydrates to obtain micronized protein with heat-resistant properties, solving the problem that high-concentration whey protein solutions are difficult to treat with UHT or flocculate and precipitate during heating. By investigating the processing thermal stability, surface properties, microstructure, degree of denaturation, and structural changes of micronized protein, the molecular mechanism by which micronization improves the thermal stability of whey protein is systematically elucidated, providing a theoretical basis for the preparation of whey protein with excellent heat resistance.

[0006] To achieve the objective of this invention, in a first aspect, this invention provides a method for preparing micronized whey protein, comprising the following steps: (1) Prepare a whey protein solution with a concentration of 7%-11% (preferably 9%-11%) and adjust the pH value to 4.6-6.6 (preferably pH 6.0 to 6.6). (2) Add oil to the whey protein solution and perform thermal shearing treatment; (3) Mix the whey protein solution that has been subjected to thermal shearing with carbohydrates, adjust the pH of the system to neutral, and perform high-pressure homogenization or high-pressure microfluidic treatment. (4) Finally, spray drying is performed to obtain micronized whey protein.

[0007] Furthermore, (2) the added oil accounts for 0.1% to 1.0% of the total system volume; Preferably, the oil is a vegetable oil, which is selected from at least one of other commonly used oils such as sunflower seed oil, soybean oil, peanut oil, rapeseed oil, and flaxseed oil.

[0008] Further, (2) the method of hot shearing treatment includes: adding oil to whey protein solution, mixing the mixed solution at 8000 r / min for 5 min; then, using water as a medium, using a conventional heater to heat the mixed solution, selecting a temperature of 75℃-100℃ for heating (e.g., 85℃), holding for 10-60 min, and continuously shearing at 1500 r / min during the holding process.

[0009] Furthermore, (3) the carbohydrate is selected from at least one of lactose, sucrose, inulin, mannose, maltose, etc.

[0010] Preferably, the amount of lactose added is 5% to 20% of the total protein content.

[0011] Furthermore, (3) the pressure for high-pressure homogenization is 20 to 80 MPa, and the homogenization is performed 1 to 3 times.

[0012] Furthermore, (4) the spray drying method includes: the spray dryer is set with an inlet air temperature of 175°C, an outlet air temperature of 80°C, and a feed rate of 18.5 r / min.

[0013] Secondly, the present invention provides micronized whey protein prepared by the method described above. The micronized whey protein retains good flowability even after UHT treatment.

[0014] Thirdly, the present invention provides the application of the micronized whey protein in the beverage and food industries.

[0015] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention successfully prepared micronized whey protein (MWP) with different structural properties and thermal stability by using a combined hot shear-high pressure homogenization technique and adding carbohydrates (lactose). The thermal stability of MWP under 120℃ / 10 min and UHT (137℃ / 5 s) treatment was systematically evaluated by controlling the protein concentration (7%, 9%, 11%) and pH value (4.6, 5.6, 6.6) during processing. Its microstructure, surface properties, and changes in secondary and tertiary structures were also systematically characterized. The results show that micronization significantly alters the structural and functional properties of whey protein. MWP prepared under neutral pH (6.6) and high protein concentration (≥9%) conditions exhibited excellent thermal stability and maintained good flowability after UHT treatment without gelation. Structural analysis revealed alterations in the secondary structure (α-helix, β-sheet) of whey protein particles (MWPs), with a significant increase in surface hydrophobicity and free thiol content (P < 0.05) and a decrease in solubility. This indicates that controlled structural unfolding and orderly aggregation of the protein under controlled conditions is a key mechanism for its improved thermal stability. Further microstructural observation revealed significant differences in the structure of MWP particles formed under different pH conditions: a compact granular gel structure was formed under low pH conditions, while a complete, independent hollow spherical structure was formed under neutral conditions. This is consistent with the granular or fine chain-like structural types formed during the particle formation process of whey protein.

[0016] This invention systematically elucidates how the ordered aggregation and structural remodeling of whey proteins can be achieved by controlling processing conditions, thereby significantly improving their UHT processing stability. This result provides important theoretical basis for the development of high-performance, heat-resistant whey proteins, and can be further applied to high-temperature processed food systems such as high-protein beverages and special medical foods in the future. Attached Figure Description

[0017] Figure 1 This is a macroscopic view of the micronized protein before and after heat treatment in a preferred embodiment of the present invention.

[0018] Figure 2 The figures show the centrifugation sedimentation rate test results of micronized whey protein before and after heat treatment in a preferred embodiment of the present invention. Lowercase letters in the figure indicate significant comparisons between different samples under the same conditions (P ≤ 0.05).

[0019] Figure 3 The viscosity test results of micronized whey protein before heat treatment are shown in a preferred embodiment of the present invention.

[0020] Figure 4 The viscosity test results of micronized whey protein after heat treatment in a preferred embodiment of the present invention are shown.

[0021] Figure 5The electrophoretic test results of micronized whey protein in a preferred embodiment of the present invention are shown.

[0022] Figure 6 The image shows the scanning electron microscope observation results of micronized whey protein in a preferred embodiment of the present invention.

[0023] Figure 7 The figures show the solubility test results of micronized whey protein in a preferred embodiment of the present invention. Lowercase letters in the figures indicate significant comparisons between different samples under the same conditions (P ≤ 0.05).

[0024] Figure 8 The figures show the surface hydrophobicity test results of micronized whey protein in a preferred embodiment of the present invention. Lowercase letters in the figures indicate significant comparisons between different samples under the same conditions (P ≤ 0.05).

[0025] Figure 9 The figures show the results of free thiol group testing of micronized whey protein in a preferred embodiment of the present invention. Lowercase letters in the figures indicate significance comparisons between different samples under the same conditions (P ≤ 0.05).

[0026] Figure 10 The results of circular dichroism spectroscopy of micronized whey protein in a preferred embodiment of the present invention are shown.

[0027] Figure 11 The fluorescence spectral test results of micronized whey protein in a preferred embodiment of the present invention are shown. Detailed Implementation

[0028] To improve the stability of whey protein during ultra-high temperature (UHT) processing, this invention employs a combined thermal shear-high pressure homogenization technique with carbohydrate addition to prepare micronized whey protein (MWP). Its thermal stability was systematically evaluated and compared with commercially available WPC550. By controlling the protein concentration (7%, 9%, 11%) and pH value (4.6, 5.6, 6.6) during micronization, the effects of different processing conditions on the structural properties, denaturation degree, and thermal processing stability of MWP were systematically investigated, including the macroscopic state, centrifugal sedimentation rate, particle size distribution, and rheological behavior after treatment at 120 ℃ / 10 min and UHT (137 ℃ / 5 s). The results showed that the degree of denaturation of MWP increased after micronization. MWP prepared under neutral pH (6.6) and high protein concentration (≥9%) conditions exhibited excellent thermal stability and maintained good flowability after UHT treatment without gelation. Structural characterization revealed that micronization significantly altered the secondary and tertiary structures of the whey protein protein (MWP), resulting in decreased solubility and a marked increase in surface hydrophobicity and free thiol content (P < 0.05). This indicates that controlled protein unfolding and orderly aggregation are key mechanisms for improving its thermal stability. This invention provides an effective modification approach and theoretical support for developing high-protein whey products suitable for UHT processes.

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] Example 1: Preparation and thermal stability evaluation of micronized whey protein based on thermal shearing and high-pressure homogenization 1. Materials and Methods 1.1 Materials and Reagents Whey protein isolate (WPI90) and micronized whey protein (WPC550) were purchased from Fonterra. Sodium hydroxide (NaOH); glycine; ethylenediaminetetraacetic acid (EDTA); tris(hydroxymethyl)aminomethane (Tris); 8-aniline-1-naphthalenesulfonic acid (ANS); phosphate-buffered saline (PBS); 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB); glacial acetic acid; ethanol; and all organic solvents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0031] 1.2 Instruments and Equipment The APV-2000 high-pressure microfluidic homogenizer was purchased from APV GmbH, Germany; the YC-UL spray dryer was purchased from Shanghai Yacheng Instrument Equipment Co., Ltd.; the 3k15 high-speed centrifuge was purchased from Sigma GmbH, Germany; the full-wavelength microplate reader was purchased from SPARK GmbH; the S3500 laser particle size analyzer was purchased from Microtrac GmbH; the ultra-high temperature (UHT) sterilizer was purchased from Power Corporation, Japan; the WFY-28 fluorescence spectrophotometer was purchased from Tianjin Top Instrument Co., Ltd.; the S-5000 scanning electron microscope (SEM) was purchased from Hitachi Ltd.; the J-810 circular dichroism spectrometer was purchased from JASCO Corporation, Japan; and the Ni-PROTEAN... ® The electrophoresis apparatus was purchased from Bio-Rad, USA; Ease ® The FC gel imaging system was purchased from Alpha Corporation, USA; the MCR302e rotational rheometer was purchased from Anton Paar Corporation.

[0032] 1.3 Methods 1.3.1 Preparation of whey protein particles Whey protein isolate powder was used to prepare protein solutions with concentrations of 7%, 9%, and 11%, respectively. The pH of the dispersions was adjusted to 4.6, 5.6, and 6.6 using 2M HCl. An appropriate amount of sunflower seed oil was added to the solution, making the oil content 0.5% of the total volume. The mixture was then stirred using an emulsifier at 8000 r / min for 5 min until completely homogeneous. The treated protein mixture was then heat-treated at 85 °C using a conventional heater with water as the medium, and held at 85 °C for 20 min, with continuous shearing at 1500 r / min during the holding period. After heating, the whey protein and lactose were quickly and thoroughly mixed, with lactose accounting for 15% of the total protein content. The pH of the system was adjusted to neutral using 2M NaOH. The mixture was then homogenized once under high pressure at 50 MPa. After shearing, it was directly spray-dried. The spray dryer was set with an inlet air temperature of 175 °C, an outlet air temperature of 80 °C, and a feed rate of 18.5 r / min.

[0033] 1.3.2 Thermal processing stability test of micronized whey protein A 10% micronized whey protein solution was prepared and subjected to heat treatment in an oil bath at 120℃ for 10 min and in a UHT bath at 137℃ for 5 s. The macroscopic state, centrifugal sedimentation rate, particle size, apparent viscosity and other indicators of the micronized protein before and after heat treatment were observed to analyze the thermal processing stability of the micronized protein.

[0034] 1.3.2.1 Observation of the thermal stability of micronized whey protein The prepared micronized whey protein was subjected to an oil bath at 120°C for 10 min and then to a UHT bath at 137°C for 5 s. After the heat treatment, the macroscopic state of the protein solution was observed and photographed.

[0035] 1.3.2.2 Test of the precipitation rate of micronized whey protein by centrifugation The samples before and after heat treatment were centrifuged at 10,000 r for 10 min. After centrifugation, the supernatant was removed and the weight of the precipitate was recorded. The centrifugation precipitation rate was then calculated.

[0036] Centrifugal sedimentation rate = y0 / y1 × 100% In the formula: y0: weight of the precipitate after centrifugation (g); y1: weight of the material before centrifugation (g) 1.3.2.3 Particle size testing of micronized whey protein The particle size of micronized whey protein solutions before and after heat treatment was determined using a laser particle size analyzer. The average particle size was used to characterize the particle size of whey protein before and after heating. The parameters were set as follows: particle absorption index of 0.001, particle refractive index of 1.460, and refractive index of dispersant of 1.330.

[0037] 1.3.2.4 Apparent viscosity test of micronized whey protein The rheological properties of whey protein solutions were determined using a rheometer. The samples, after ultra-high temperature treatment, were kept at a constant temperature of 25°C with shear rates ranging from 0.1 to 100 s⁻¹. -1 Between loading and balancing, the sample is loaded onto a parallel plate geometry with a diameter of 40 mm and balanced for 1 min. Then, the rotor is lowered to a specified height of 0.099 mm and a steady-state scan is performed in rotation mode to measure the viscosity of the sample before and after heating.

[0038] 1.3.3 Dodecyl polypropylene gel electrophoresis test Dilute the protein concentration to 1 mg / mL. Mix the protein solution with the electrophoresis buffer at a volume ratio of 4:1. Set the voltage to 110 V, the separating gel concentration to 12%, the stacking gel concentration to 4%, the current to 15 mA and 30 mA, and the sample loading volume to 5 μL. Test the reducing and non-reducing electrophoresis of the samples respectively. After the test, stain with 0.1% Coomassie brilliant blue staining solution for 0.5 h, and then destain until clear with destaining solution (100 mL glacial acetic acid, 100 mL ethanol, and 300 mL water).

[0039] 1.3.4 Solubility test of micronized whey protein Whey protein was prepared into a 2% protein solution. The protein concentration was expressed as the absorbance value A at 280 nm using a UV spectrophotometer. The solution was centrifuged at 10,000 r / min for 15 min at 4 °C. The supernatant was collected, and the protein concentration of the supernatant was expressed as the absorbance value A280 at 280 nm using a UV spectrophotometer.

[0040] 1.3.5 Microscopic morphology observation of micronized whey protein The microstructure of micronized whey protein was tested using scanning electron microscopy (SEM). The prepared micronized whey protein was taken and evenly dispersed on weighing paper. Conductive tape was attached to the sample stage, and then the powder sample was evenly attached to the conductive tape. The microstructure of the whey protein was recorded using SEM.

[0041] 1.3.6 Surface hydrophobicity test of micronized whey protein The surface hydrophobicity of native and particulate proteins was determined using an ANS probe. A 5% whey protein solution and an 8 mM ANS solution were prepared using (0.01 M, pH 7) phosphate buffer. 20 μL of the ANS solution was added to 4 mL of the prepared protein solution, immediately shaken, and fluorescence intensity was measured after 15 minutes at room temperature. Excitation and emission wavelengths were set to 390 nm and 470 nm, respectively. Surface hydrophobicity (H0) is expressed as the initial slope of the fluorescence intensity versus protein concentration (mg / mL) graph.

[0042] 1.3.7 Free thiol group assay of micronized whey protein 1000 μL of sample suspension 20 (mg / mL) was mixed with 1000 μL of buffer (10.4 g Tris, 1.2 g EDTA and 6.9 g glycine dissolved in deionized water to a final volume of 1 L, adjusted to pH 8) and 50 μL of 4 (mg / mL) DTNB (5,5'-dithiobis(2-nitrobenzoic acid)). The mixture was then incubated at room temperature for 15 min, followed by centrifugation at 10000 rpm for 10 min at 4 °C. The absorbance was read at 412 nm using a UV-Vis spectrophotometer.

[0043] C SH =73.53×(A / C S ) In the formula: C SH Represents the thiol content (μmol / g); A is the absorbance at 412 nm; C S This is the sample concentration (mg / mL).

[0044] 1.3.8 Structural testing of micronized whey protein 1.3.8.1 Circular dichroism test A protein sample with a concentration of 0.3 mg / mL was placed in a quartz cell with a diameter of 1 mm. Deionized water was used as a blank control. The scanning rate was set to 10 nm / min, the scanning wavelength range was 190-260 nm, and the data point interval was 0.5 nm. The changes in the secondary structure of the protein were tested at room temperature using a circular dichroism chromatograph.

[0045] 1.3.8.2 Intrinsic Fluorescence Spectroscopy Test Fluorescence emission spectra of tryptophan (Trp) and tyrosine (Tyr) residues were recorded using a fluorescence spectrophotometer. Protein solutions with a concentration of 0.2 mg / mL were prepared using deionized water. The intrinsic fluorescence emission spectra of the sample solutions were recorded at 600 V with a scan rate of 1200 nm / min. Emission spectra were collected from 290 nm to 450 nm with an excitation wavelength of 280 nm. Both spectra had a slit width of 5 nm. The obtained spectra were corrected by subtracting the spectra from those of the deionized water solution. All fluorescence measurements were performed at room temperature.

[0046] 1.4 Data Analysis Experimental data are the average of three measurements. Data were analyzed using SPSS 27; P < 0.05 indicated statistical significance. Data were plotted using Origin 2024.

[0047] 2. Results and Analysis 2.1 Results of thermal processing stability test of micronized whey protein 2.1.1 Macroscopic results of thermal processing stability of micronized whey protein Figure 1The appearance of protein dispersions of control group (WPI90 and WPC550) and modified MWP under different pH conditions at a concentration of 10% is shown in the figure. As shown in the figure, in the unheated state, control group WPI90 is a clear and transparent light yellow liquid, while all MWP samples are milky white, indicating that whey protein has been denatured during the modification process, forming soluble aggregates, resulting in enhanced light scattering and increased turbidity. After a second heat treatment at 120℃, the thermal stability of each sample is significantly different. Control group WPI90 and MWP prepared under pH 4.6 and pH 5.6 conditions all showed obvious aggregation or precipitation; while MWP prepared at pH 6.6 still maintained fluidity and was accompanied by the formation of visible large particles, showing particle swelling behavior similar to WPC550. This swelling phenomenon is consistent with the research conclusion of Nieke Westerik

[10] , indicating that the protein aggregation mode is more controllable under this pH condition and no dense precipitation is formed. Further UHT treatment of the samples revealed that only commercially available WPC550 and MWP prepared at 9% and 11% concentrations under neutral pH conditions could pass through UHT smoothly without clogging or severe solidification. This result indicates that MWP with enhanced thermal stability can be produced under neutral conditions.

[0048] 2.1.2 Results of centrifugation precipitation rate test for micronized whey protein Centrifugation sedimentation rate is a key indicator for assessing the size and number of aggregates in whey protein dispersions, and can effectively reflect the solubility and stability of proteins. For example... Figure 2 As shown, this study systematically compared the changes in centrifugal sedimentation rates of MWP samples prepared under different pH conditions (4.6, 5.6, 6.6) and concentrations (7%, 9%, 11%) before heating and after different heat treatments. Before heat treatment, all protein dispersions maintained good flowability (…). Figure 1 Among them, the unmodified WPI90 had the lowest centrifugal precipitation rate, while the centrifugal precipitation rate of each MWP sample increased after micronization. This phenomenon indicates that micronization can cause partial unfolding and denaturation of protein structure, enhance intermolecular hydrophobic interactions, and promote the formation of larger and less stable aggregates, thus making them easier to precipitate during centrifugation. MWP particles swell during heating

[10] and the formation of aggregates are inevitable. After heat treatment at 120℃, the overall centrifugal precipitation rate of the protein solution increased (P < 0.05). The control group WPI90 and the MWP prepared at pH 4.6 and 5.6 aggregated in large quantities, forming solid gels ( Figure 1The centrifugal sedimentation rate increased to 10 times that of the unheated sample. However, WPC550 and MWP prepared at pH 6.6 retained flowability, with a centrifugal sedimentation rate 5 times higher than the unheated sample. Compared to MWP produced under neutral pH conditions, MWP produced at pH 4.6 and 5.6 exhibited stronger heat-induced gelation ability. UHT treatment further confirmed the excellent thermal processing properties of WPC550, and demonstrated that a combination of heat and high pressure at pH 6.6 and protein concentrations of 9% and 11% could produce MWP with good stability. This indicates that by adjusting the micronization processing conditions, the aggregation behavior and functional properties of whey proteins can be significantly affected to meet the needs of different foods.

[0049] 2.1.3 Results of Micronized Whey Protein Particle Diameter Test Table 1 shows the changes in powder particle diameter of MWP samples prepared at pH 4.6, 5.6, 6.6, and concentrations of 7%, 9%, and 11% before heating and after different thermal processing. After thermal denaturation and microfluidization, the average particle size of the untreated samples was between 1 and 10 μm. Among them, MWP produced at pH 6.6 generally had a larger particle diameter. Spiegel T

[14] proposed that the denaturation rate of β-lactoglobulin would be delayed as the pH value decreased in the pH range of 6.7 to 4.5. Therefore, the MWP aggregates produced at pH 4.6 and pH 5.6 both showed a small particle size (close to 5 μm), which can be attributed to the low reactivity of thiol groups and the decrease in the net charge of the protein in this pH range

[15] . After a second heating at 120 °C, the control group WPI90 and the MWP prepared at pH 4.6 and 5.6 existed in the form of aggregates, and their particle size could not be determined. The heat-induced gelation properties of micronized whey protein under low pH conditions may be related to its structural changes and colloidal stability under micronization processing conditions

[16] . MWP produced at pH 6.6 showed significantly improved thermal stability. After a second heating treatment at 120°C, although the protein dispersion did not exhibit the common gelation, the overall particle size increased (P < 0.05). After UHT processing, MWP produced at pH 6.6 and protein concentrations of 9% and 11% successfully maintained good fluidity, with particle sizes remaining between 20 μm and 25 μm. This phenomenon suggests that micronization processing under neutral conditions may guide protein molecules to form larger and more ordered aggregates, rather than disordered aggregate networks. This allows the system to maintain stability and fluidity during the second heating process.

[0050] Table 1. Particle diameters of micronized proteins after unheated treatment at 120°C and UHT treatment.

[0051] Note: Lowercase letters in the table indicate significance comparisons among all samples under different conditions (P ≤ 0.05).

[0052] 2.1.4 Results of viscosity test for micronized whey protein particles Figure 3 and Figure 4 The viscosity changes of MWP samples prepared at pH 4.6, 5.6, 6.6, and concentrations of 7%, 9%, and 11% before heating and after different heat treatments are shown. The viscosity of the control group WPI90, WPC550, and MWP samples before heat treatment (…) Figure 3 There was no significant difference. The protein particle dispersions exhibited non-Newtonian pseudoplastic behavior after heating, and shear-thinning behavior with increasing shear rate

[11] . At shear rates close to 40 s⁻¹, -1 It was subsequently observed that the viscosity of all samples was below 10 Pa·s. After heat treatment at 120℃ for 10 min, solid gels formed due to the formation of the control group WPI90 and the MWP samples prepared at pH 4.6 and 5.6. Figure 1 In this study, viscosity measurements could not be performed on the WPC550 dispersion. The WPC550 dispersion still exhibited good flowability and the lowest apparent viscosity after heating, which validated Esra Çakır-Fuller's [8] findings on the thermal stability of WPC550. Compared to the viscosity of the unheated sample, the viscosity of the WPC dispersion obtained at pH 6.6 with protein concentrations of 9% and 11% increased by 3 times, while the viscosity of the WPC dispersion obtained at pH 6.6 with a protein concentration of 7% increased by 10 times. The increase in viscosity due to heat treatment can be explained by intermolecular interactions of protein molecules and the subsequent formation of aggregates

[12] . Thermal denaturation of whey protein above 70°C exposes hydrophobic amino acid residues in the protein molecules, enhancing intermolecular interactions and leading to aggregation

[13] . Due to this aggregation, the viscosity increases with heating. After UHT processing, only WPC550 and the MWP dispersions obtained at pH 6.6 and protein concentrations of 9% and 11% exhibited fluid properties, and the viscosity of the MWP samples was twice that of WPC550. Exploring the formation of heat-stable whey protein particles is a research hotspot. The balance between shear-controlled aggregate growth and shear-induced aggregate rupture determines the properties of aggregates during micronization. Dissanayake M[4] modified whey protein by combining heat and high pressure. Compared with natural whey protein, this process resulted in MWP with higher thermal stability. This study verified this conclusion and further showed that MWP produced by heat and high pressure has better thermal stability under neutral conditions with a protein concentration greater than 9%.

[0053] 2.2 Electrophoresis results of micronized whey protein To investigate the effects of pH and protein concentration on disulfide bond formation during micronization modification, this study performed SDS-PAGE analysis under both reducing and non-reducing conditions. Figure 5 Whey protein exhibited three main bands at approximately 18.3, 13.8, and 66.6 kDa, corresponding to β-lactoglobulin (β-Lg), α-lactalbumin (α-La), and bovine serum albumin (BSA), respectively. In the unmicronized WPI90 sample (C1), clear β-Lg, α-La, and BSA monomer bands were visible under non-reducing conditions. After modification, the β-Lg and α-La monomers in micronized whey protein (MWP) were significantly reduced, and the BSA monomer band disappeared, a trend consistent with the protein banding observed in WPC550. This indicates that whey protein undergoes significant denaturation through a combination of heat and microfluidization. The denaturation and aggregation behaviors of the proteins showed significant differences under different micronization conditions. When the concentration increased from pH 4.6 to 6.6, the β-Lg and α-La monomers in MWP decreased significantly, indicating that as the concentration increased, the formation of disulfide bonds was enhanced, the denaturation and aggregation degree increased, affecting their migration rate and visibility in electrophoresis. Dissanayake M

[15] found that due to micronization, multiple protein molecules aggregated to form dimers, polymers and even larger insoluble aggregates. Therefore, the monomers disappeared on the electrophoretic gel and appeared at the 245 kDa position. However, in this experiment, no large molecular aggregates were observed at the top, based on the solubility test results ( Figure 7 It is speculated that the aggregates formed by micronization are insoluble aggregates and cannot migrate in the gel and are blocked. Under reducing conditions, after heating and treatment with β-mercaptoethanol, more separation bands appeared in the electrophoretic pattern. Compared with the unreduced conditions, the presence of β-mercaptoethanol led to the release of β-Lg and α-La monomers, and the bands were enhanced. At the same time, BSA monomer bands also appeared, indicating that the aggregates formed by heating in MWP mainly originated from the cross-linking of disulfide bonds between protein molecules

[18] .

[0054] 2.3 Scanning Electron Microscope like Figure 6The figure shows the microstructure of MWP powders obtained under different pH values ​​and protein concentrations. As can be seen from the figure, WPI90 has a smooth surface structure and relatively large particle volume, which is consistent with the previous research results of Agarwal S

[19] . WPC550 and other MWP particles all show surface wrinkles and collapsed microstructures, which is due to the fact that whey protein is a heat-sensitive protein, and the rapid evaporation of water during spray drying leads to the combined effect of internal and external pressure differences. Whey protein can form different particle structures through relative changes in physicochemical conditions. The particle structures observed in the MWP samples are somewhat similar to the gel structure types formed by changing pH value and ionic strength. The main protein in whey protein is β-lactoglobulin, and the gelation properties of β-lactoglobulin are very complex

[20] . Depending on the effect of pH value, two types of gel structures can usually be formed, granular or fine chain. A third structure, called a mixed structure, can also be observed under certain conditions, which contains both granular and fine chain characteristics

[20] . The MWP obtained at pH 4.6 and 5.6 formed dense particulate gels with broken particulate structures. Due to the proximity of the isoelectric point of whey protein (pH 5.0) to this pH condition, the electrostatic repulsion between protein molecules was significantly weakened. They were more likely to bind together and form tight aggregates. These aggregates were then broken up during high-pressure homogenization. The particulate gels were thought to contain proteins with relatively low denaturation and formed mainly through hydrophobic interactions

[21] . They consisted of relatively uniform primary aggregates that formed through phase separation and then bonded together to form a gel. The MWP powder obtained at pH 6.6 had a complete and independent particulate structure. At this pH condition, the electrostatic repulsion increased, and the proteins remained in a relatively stable and ordered state as individual molecules or aggregates in the solution. After spray drying, they formed larger and more complete hollow spherical structures. Based on these observations, it can be explained that lower pH values ​​tend to form tight packing structures and produce smaller particle sizes.

[0055] 2.4 Results of micronized whey protein solubility test Figure 7The changes in solubility of MWP samples prepared by adjusting pH and protein concentration were summarized. Solubility is usually one of the key indicators of protein functional properties and is affected by protein-water and protein-protein interactions

[15] . Whey protein has high solubility over a wide pH range, and conditions such as pH, ionic environment and temperature are crucial for controlling the solubility of whey protein

[22] . The results in the figure show that the solubility of MWP was significantly reduced by heating and shearing compared to the control WPI90 (P < 0.05). The isoelectric point (pI) of whey protein is about pH 5.0. Therefore, at pH 4.6 and 5.6, due to the proximity to the isoelectric point, the electrostatic repulsion between molecules is reduced, and some whey protein molecules may partially aggregate or change their existing conformation, leading to aggregation and subsequent partial loss of solubility

[23] . Secondly, due to the combined effect of heating and shearing on whey protein, whey protein also loses its ability to regulate hydration. Although lower than the control WPI90, the obtained MWP was similar to that of WPC550 in overall solubility (P > 0.05), indicating that heat-induced protein aggregation usually reduces the solubility of whey proteins. As previously shown by Dissanayake et al. [4], this loss of solubility is directly attributable to the combination of high-pressure shear and changes in processing conditions.

[0056] 2.5 Results of Hydrophobicity Test on the Surface of Micronized Whey Protein Most of the surface functional properties of proteins are affected by surface hydrophobicity. In this study, the surface hydrophobicity was obtained by using an ANS probe, which can bind to the hydrophobic regions on the protein surface, thereby significantly increasing its fluorescence emission intensity

[24] . The changes in MWP surface hydrophobicity at pH values ​​of 4.6, 5.6, and 6.6 and protein concentrations of 7%, 9%, and 11% are shown in the figure. Figure 8As shown. The surface hydrophobicity of MWPs after thermal shearing treatment was significantly increased (P < 0.05). This is the same trend as commercially available WPC550, indicating that the unfolding of the native conformation of whey protein exposes hydrophobic amino acids, resulting in a significant change in the ability of ANS to enter the hydrophobic sites of protein molecules

[24] . The lowest surface hydrophobicity was observed in MWPs obtained at pH 4.6 and the same concentration, because at pH 4.6, which is close to the isoelectric point of whey protein (approximately pH 5), the charge density is the lowest, the conformation is the most compact, resulting in most of the hydrophobic regions being buried inside. In contrast, the surface hydrophobicity of MWPs obtained at pH 6.6 was generally higher, and the loss of surface hydrophobicity after micronization followed the order of pH 4.6 > pH 5.6 > pH 6.6. This is basically consistent with the study of Zahra Allahdad

[25] . These findings confirm that changing the charge of proteins can further prevent the interaction between hydrophobic proteins and the formation of aggregates.

[0057] 2.6 Results of Free Thiol Group Test for Micronized Whey Protein Natural whey protein has a globular structure, and thermal shearing can induce protein unfolding, exposing hidden thiol groups in the molecule. These exposed free thiol groups (-SH) may participate in the formation of intermolecular disulfide bonds through exchange reactions or oxidation reactions with disulfide bonds (-SS-)

[13] . Therefore, the free thiol content can be used to further evaluate the effects of pH and protein concentration on the denaturation, unfolding, and aggregation of WPI90. Figure 9 As shown, the free thiol content of MWP showed a significant increase compared to the control group (untreated WPI90). This increase indicates that thermal shear modification leads to the formation of more disulfide bonds inside whey protein. At pH 6.6 and the same protein concentration, the prepared MWP had a higher free thiol content (P < 0.05). This is because as the pH increases from the isoelectric point of whey protein, electrostatic repulsion intensifies, leading to the complete unfolding of the WPI90 structure and the exposure of more thiol groups. With the increase of protein concentration during the modification process, the free thiol content of MWP generally showed an increasing trend, but at pH 4.6, it showed the opposite trend. This can be explained by the relative induced crowding effect. At pH 4.6, which is close to the isoelectric point of whey protein, the aggregation reaction is accelerated. Compared with low concentration modification, higher concentration leads to incomplete unfolding of protein, thereby reducing the exposure of free thiol groups

[26] . Secondly, the oxidation of thiol groups caused by denaturation or the exchange reaction with disulfide bonds is a key factor in the formation of macromolecular polymers

[13] . Wang et al.

[27] also believed that the reduction of free thiol groups in PWP may be due to the oxidation of thiol groups or their conversion into disulfide bonds.

[0058] 2.7 Results of micronized whey protein structure testing 2.7.1 Circular dichroism CD spectroscopy is used to characterize changes in the secondary structure of micronized proteins after their formation. Figure 10 The far-ultraviolet CD spectra of MWP obtained at different pH values ​​and protein concentrations are shown. The far-ultraviolet CD spectra of MWP have a positive peak curve in the range of 190 to 200 nm and an extremely positive value at 195 nm, which indicates the presence of β-sheet structure in the secondary structure of whey protein

[25] . A large negative peak curve is shown at 208 and 222 nm, and the large overlap of the curve with a single elliptical arc centered at 215 nm means that α-helices and β-sheet structures appear simultaneously. And the trend is roughly constant at all pH values ​​and protein concentrations. The intensity of this broad peak is significantly weakened after micronization, indicating that the overall ordered secondary structure of the protein is changed, and its conformation may become more loose or disordered

[29] . Although the peaks of MWP obtained at different pH values ​​and protein concentrations are similar, micronization has the greatest impact on the secondary structure at pH 6.6, with both α-helices and β-sheets being significantly reduced. The commercially available WPC550 only exhibits a significant negative peak at 198 to 208, lacking the corresponding minimum values ​​for α-helical and β-sheet structures, which may be due to the use of other elements of the secondary structure for some α-helical and β-sheet structures

[30] .

[0059] 2.7.2 Intrinsic Fluorescence Spectrum Aromatic amino acids (phenylalanine, tyrosine, and tryptophan) can produce intrinsic fluorescence at appropriate excitation wavelengths, and environmental modifications of tryptophan residues can be determined by irreversible changes in the structure of whey proteins

[31] . The fluorescence intensities of control samples WPI90, WPC550, and MWP are as follows: Figure 11As shown. The MWP sample prepared by thermal shearing at a concentration of 7% and pH 5.6 had a higher maximum fluorescence intensity than the untreated WPI90. Thermal treatment caused more protein molecules to unfold, exposing more hydrophobic groups in the protein core, which led to an increase in fluorescence intensity. Studies have shown

[32] that the fluorescence intensity of micronized proteins gradually increases with increasing concentration, but in this study, it was found that the fluorescence intensity of commercially available WPC550 and other MWP samples was lower than that of WPI90. This may be because in the natural state, tryptophan residues are mostly located inside the hydrophobic core of the protein molecule, in a relatively nonpolar microenvironment, which is conducive to its strong fluorescence. However, the micronization process will destroy the natural conformation of the protein, leading to molecular unfolding and aggregation. This exposes some of the originally embedded tryptophan residues to the hydrophilic environment. This reduces the fluorescence yield of tryptophan, thus leading to a decrease in overall fluorescence intensity. Secondly, the micronization process promotes the formation of insoluble or soluble aggregates between protein molecules through hydrophobic interactions, disulfide bonds, etc. These aggregates may embed tryptophan residues within them, greatly hindering the effective excitation of tryptophan by excitation light

[33] .

[0060] This invention successfully prepared micronized whey protein (MWP) with different structural properties and thermal stability by using a combined hot shear-high pressure homogenization technique and adding carbohydrates (lactose). The thermal stability of MWP under 120℃ / 10 min and UHT (137℃ / 5 s) treatment was systematically evaluated by controlling the protein concentration (7%, 9%, 11%) and pH value (4.6, 5.6, 6.6) during processing. Its microstructure, surface properties, and changes in secondary and tertiary structures were also systematically characterized. The results show that micronization significantly alters the structural and functional properties of whey protein. MWP prepared under neutral pH (6.6) and high protein concentration (≥9%) conditions exhibited excellent thermal stability and maintained good flowability after UHT treatment without gelation. Structural analysis revealed alterations in the secondary structure (α-helix, β-sheet) of whey protein particles (MWPs), with a significant increase in surface hydrophobicity and free thiol content (P < 0.05) and a decrease in solubility. This indicates that controlled structural unfolding and orderly aggregation of the protein under controlled conditions is a key mechanism for its improved thermal stability. Further microstructural observation revealed significant differences in the structure of MWP particles formed under different pH conditions: a compact granular gel structure was formed under low pH conditions, while a complete, independent hollow spherical structure was formed under neutral conditions. This is consistent with the granular or fine chain-like structural types formed during the particle formation process of whey protein.

[0061] This invention systematically elucidates how the ordered aggregation and structural remodeling of whey proteins can be achieved by controlling processing conditions, thereby significantly improving their UHT processing stability. This result provides important theoretical basis for the development of high-performance, heat-resistant whey proteins, and can be further applied to high-temperature processed food systems such as high-protein beverages and special medical foods in the future.

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

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Claims

1. A method for preparing micronized whey protein, characterized in that, Includes the following steps: (1) Prepare a whey protein solution with a concentration of 7%-11% and adjust the pH value to 4.6-6.6; (2) Add oil to the whey protein solution and perform thermal shearing treatment; (3) Mix the whey protein solution that has been subjected to thermal shearing with carbohydrates, adjust the pH of the system to neutral, and perform high-pressure homogenization or high-pressure microfluidic treatment. (4) Finally, spray drying is performed to obtain micronized whey protein.

2. The method according to claim 1, characterized in that, (1) The concentration of the whey protein solution prepared in the formula is 9%-11%; Preferably, the pH value is adjusted to 6.0 to 6.

6.

3. The method according to claim 1, characterized in that, (2) The added oil accounts for 0.1% to 1.0% of the total system volume; Preferably, the oil is a vegetable oil, which is selected from at least one of sunflower seed oil, soybean oil, peanut oil, rapeseed oil, and flaxseed oil.

4. The method according to claim 1, characterized in that, (2) The hot shearing treatment method includes: adding oil to the whey protein solution, mixing the mixed solution at 8000 r / min for 5 min; then, using water as a medium, using a conventional heater to heat the mixed solution, selecting a temperature of 75℃-100℃ for heating, holding for 10-60 min, and continuously shearing at 1500 r / min during the holding process.

5. The method according to claim 1, characterized in that, (3) The carbohydrates mentioned are selected from at least one of lactose, sucrose, inulin, mannose and maltose.

6. The method according to claim 5, characterized in that, The amount of lactose added is 5% to 20% of the total protein content.

7. The method according to claim 1, characterized in that, (3) The pressure for high-pressure homogenization is 20 to 80 MPa, and the homogenization is performed 1 to 3 times.

8. The method according to any one of claims 1-7, characterized in that, (4) The spray drying method includes: the spray dryer is set with an inlet air temperature of 175℃, an outlet air temperature of 80℃, and a feed rate of 18.5 r / min.

9. Micronized whey protein prepared by the method according to any one of claims 1-8.

10. The application of the micronized whey protein of claim 9 in the beverage and food industries.

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