A method for preparing whey protein isolate-junco powder conjugate by ultrasonic-assisted wet heating

Abstract

The application discloses a method for preparing whey protein isolate-chicory powder conjugates by means of ultrasonic-assisted wet heat treatment, and the whey protein isolate-chicory powder conjugates are obtained by 80 DEG C wet heat treatment for 1-5 hours and ultrasonic treatment. The ultrasonic treatment conditions are optimized by means of a response surface method, so that the grafting degree value is maximized. In addition, the generation of the whey protein isolate-chicory powder conjugates is proved by grafting degree, browning degree, FT-IR, SDS-PAGE, CD and fluorescence spectrum. The emulsifying activity, emulsion stability, thermal stability and antioxidant activity of the whey protein isolate-chicory powder conjugates generated by Maillard reaction are improved. Compared with the conjugates prepared by means of wet heat treatment, the conjugates prepared by means of ultrasonic treatment exhibit better structural performance and functional performance.

Description

Technical Field

[0001] This invention relates to the field of food science and technology, specifically to a method for preparing whey protein isolate-inulin conjugates using an ultrasound-assisted wet heat method. Background Technology

[0002] Whey protein isolate (WPI), as a food ingredient, is rich in nutrients, easily digested and absorbed, and possesses various physiological activities. WPI is mainly composed of α-lactalbumin (α-LA), β-lactoglobulin (β-LG), bovine serum albumin (BSA), immunoglobulins, bioactive substances, and enzymes.

[0003] WPI (wax polyimide) is widely used in a range of food products, including dairy products, ice cream, desserts, sauces, infant formula, sausages, and beverages, exhibiting excellent emulsifying, shaping, and gelling properties. Furthermore, as a natural emulsifier, WPI is frequently used to stabilize emulsions, particularly oil / water emulsions. However, WPI typically exhibits poor solubility and functional properties near its isoelectric point (PI) or at higher temperatures, limiting its application in the food industry and affecting the physicochemical stability of transport systems. Therefore, improving the functional properties of WPI and expanding its applications in the food industry is of great significance.

[0004] The preparation of protein-polysaccharide covalent complexes via Maillard reactions to improve their functional properties, such as water solubility, emulsification, thermal stability, and antioxidant activity, has attracted considerable attention. It has been reported that whey protein (WPI) and polydextrose (PDX) conjugates exhibit improved water solubility, antioxidant activity, and emulsification properties. Microencapsulation of whey protein and xylooligosaccharide conjugates has demonstrated better encapsulation performance and improved bioavailability of lycopene. Furthermore, WPI conjugates show significantly improved pH stability and antioxidant capacity, while reducing lipolysis. Therefore, glycosylation is a promising protein modification method for improving the functional properties of proteins. However, traditional methods, such as dry and wet heating, are time-consuming and have low grafting efficiency, significantly limiting their commercial application. In addition, proteins are more prone to denaturation and polymerization at prolonged high temperatures.

[0005] Therefore, it is crucial to provide a method for preparing whey protein isolate-inulin conjugates using an ultrasound-assisted wet heat method to accelerate the grafting reaction between proteins and polysaccharides and control the reaction to develop in a favorable direction. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing whey protein isolate-inulin conjugates by ultrasound-assisted wet heat treatment. The thermal stability, foaming properties, emulsifying activity, emulsion stability, and antioxidant activity of the conjugates after ultrasound treatment are significantly improved.

[0007] The objective of this invention is achieved as follows:

[0008] A method for preparing whey protein isolate-inulin conjugates using an ultrasound-assisted hydrothermal method includes the following steps:

[0009] Step 1: Prepare whey protein isolate solution and inulin solution, ensuring that the concentrations of the whey protein isolate solution and inulin solution are equal. Then, mix equal volumes of the whey protein isolate solution and inulin solution to obtain the first mixture for later use.

[0010] Step 2: Adjust the pH of the first mixture from Step 1 to 7.0 using an acidic or alkaline solution to obtain the second mixture;

[0011] Step 3: Under the conditions of ultrasonic frequency of 20-25KHz and ultrasonic power of 150-450W, the second mixture in step 2 is ultrasonically treated for 30-150 minutes to obtain the third mixture.

[0012] Step 4: After hydrating the third mixture from Step 3 overnight at 4°C, heat it at 80°C for 1-5 hours to form conjugates with different grafting degrees. Then immediately place it in an ice bath to stop the reaction and perform dialysis to separate unreacted inulin to obtain whey protein isolate-inulin conjugate. Freeze-dry the whey protein isolate-inulin conjugate and store it at -18°C.

[0013] Step 5: The whey protein isolate-inulin conjugate obtained in Step 4 was subjected to structural and functional characterization tests.

[0014] The specific operation of step 1 is as follows: prepare whey protein isolate solution and inulin solution with milliq water, stir at room temperature for 2 hours to completely dissolve them, and obtain whey protein isolate solution with a concentration of 100 mg / mL and inulin solution with a concentration of 100 mg / mL.

[0015] The specific operation of step 2 is as follows: the pH value of the first mixture is adjusted to 7.0 using 0.1M HCl solution and 0.1M NaOH solution.

[0016] In step 4, the conjugate is dialyzed for 48 hours using a dialysis bag with a molecular weight of 12000.

[0017] The beneficial effects of this invention are as follows: The whey protein isolate-inulin conjugate is obtained by wet heating at 80℃ for 1-5 hours followed by ultrasonic treatment. The ultrasonic treatment conditions were optimized using response surface methodology to maximize the grafting degree. Furthermore, the formation of the whey protein isolate-inulin conjugate was confirmed by grafting degree, browning degree, FT-IR, SDS-PAGE, CD, and fluorescence spectroscopy. The whey protein isolate-inulin conjugate exhibiting Maillard reaction showed improved emulsifying activity, emulsifying stability, thermal stability, and antioxidant activity. Compared to the conjugate prepared by wet heating, the ultrasonically treated conjugate exhibited better structural and functional properties. Attached Figure Description

[0018] Figure 1 The results of a single-factor experiment show the effects of different factors on the GD of WPI-inulin conjugates: (A) the effect of ultrasonic time on GD, (B) the effect of ultrasonic power on GD, (C) the effect of pH on GD, and (D) the effect of the ratio of WPI to inulin on GD.

[0019] Figure 2 Contour plots showing the interaction of ultrasound time and ultrasound power (A), ultrasound time and pH (B), ultrasound time and WPI-inulin ratio (C), ultrasound power and pH (D), ultrasound power and WPI-inulin ratio (E), and pH and WPI-inulin ratio (F) on WPI-inulin conjugate GD.

[0020] Figure 3 The response surface plots show the interaction between ultrasound time and ultrasound power (A), ultrasound time and pH (B), ultrasound time and WPI-inulin ratio (C), ultrasound power and pH (D), ultrasound power and WPI-inulin ratio (E), and pH and WPI-inulin ratio (F) on WPI-inulin conjugate GD.

[0021] Figure 4 Electrophoresis results of WPI and WPI-inulin covalent grafts under different treatment conditions (1-5 h of wet heat treatment respectively); Emulsification properties EAI and ES (A), ABTS radical scavenging ability (B), and DPPH radical scavenging antioxidant ability (C) are shown respectively. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] A method for preparing whey protein isolate-inulin conjugates using an ultrasound-assisted hydrothermal method includes the following steps:

[0024] Step 1: Prepare whey protein isolate and inulin solution to make their concentrations equal. Use Milliq water to prepare the whey protein isolate and inulin solution, and stir at room temperature for 2 hours to completely dissolve them, obtaining a whey protein isolate solution with a concentration of 100 mg / mL and an inulin solution with a concentration of 100 mg / mL. Then, mix equal volumes of the whey protein isolate solution and the inulin solution to obtain the first mixture for later use.

[0025] Step 2: Adjust the pH of the first mixture from Step 1 to 7.0 using an acidic or alkaline solution to obtain the second mixture; adjust the pH of the first mixture to 7.0 using a 0.1M HCl solution and a 0.1M NaOH solution.

[0026] Step 3: Under the conditions of ultrasonic frequency of 20-25KHz and ultrasonic power of 150-450W, the second mixture in step 2 is ultrasonically treated for 30-150 minutes to obtain the third mixture.

[0027] Step 4: After hydrating the third mixture from Step 3 overnight at 4°C, heat it at 80°C for 1–5 hours to form conjugates with different grafting degrees. Then immediately place it in an ice bath to stop the reaction. Dialyze the conjugates using a dialysis bag with a molecular weight of 12,000 for 48 hours to separate unreacted inulin, and obtain whey protein isolate-inulin conjugate. Freeze-dry the whey protein isolate-inulin conjugate and store it at -18°C.

[0028] Step 5: The whey protein isolate-inulin conjugate obtained in Step 4 was subjected to structural and functional characterization. Materials:

[0029] The whey protein isolate was from Hilmar Ingredients International (Hilmar 9410, USA), and the inulin was from Beneo. GR, Belgium). OPA (purity ≥98%) was supplied by Shanghai Maclean Biochemical Co., Ltd., China. SDS-PAGE protein markers with molecular weights of 10–180 kDa were from Thermo Fisher Scientific (Waltham, MA, USA). 2,2′-N-bis(3-ethylbenzothiazol-6-sulfonic acid) (ABTS, purity 98%) and 2,2-diphenyl-1-pyridinehydrazine (DPPH, purity 98%) were purchased from Shanghai Yuanye Co., Ltd., China.

[0030] All reagents used in this invention are analytical grade reagents.

[0031] The ultrasound equipment used in step 3 is an ultrasonic cell disruptor (JY92-IIDN, China), and the Φ6 probe is used to process the mixed solution of whey protein isolate and inulin in pulse mode (on / off for 2 seconds).

[0032] To optimize the performance of the WPI-inulin conjugate, the levels of each factor were first determined through a series of single-factor, one-time (OFAT) experiments. Specifically, the grafting degree (GD) of the sample was used as the response, and the ultrasonic time (40, 60, 80, 100, 120 min), ultrasonic power (20%, 25%, 30%, 35%, 40%, with a maximum power of 900 W), pH value (7.0, 8.0, 9.0, 10.0, 11.0), and the ratio of WPI to inulin (3:1, 2:1, 1:1, 1:2, 1:3) were considered as independent influencing factors. Secondly, the reaction conditions were optimized using the Box-Behnken Design response surface methodology (RSM) to maximize the response (Table 1). Finally, the experimental results were analyzed using Design-Expert to obtain the optimal results.

[0033] The structural and functional properties of whey protein isolate-inulin conjugate were analyzed using the following methods.

[0034] Browning intensity: The appropriately diluted sample solution was measured at 290 nm (to detect the formation of Maillard reaction intermediates) and 420 nm (to detect the formation of brown polymers) using a microplate (MultiskanSkyHigh).

[0035] Grafting degree: The free amino content of WPI-inulin conjugate and WPI conjugate was determined using the OPA method, and the GD of the conjugate was analyzed. In short, 40 mg OPA was dissolved in 1 mL of ethanol, mixed with 25 mL of sodium tetraborate buffer (0.1 M, pH 9.5), 2.5 mL of 20% (w / v) SDS, and 100 μL of β-mercaptoethanol, and diluted to 50 mL with distilled water. 100 μL of the sample solution (10 mg / mL, dispersed in ultrapure water) was reacted with 4 mL of OPA reagent at 25 °C for 2 min, and the absorbance was recorded at 340 nm using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan).

[0036] The formula for calculating grafting degree is as follows:

[0037] GD=(A1-A2) / A1×100 (1)

[0038] In the formula, A1 and A2 represent the absorbance of whey protein isolate and WPI-inulin conjugate, respectively.

[0039] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): 40 μL of sample solution (10 mg / mL) was mixed with 10 μL of loading buffer and placed in a boiling water bath for 5 min to denature the proteins. SDS-PAGE electrophoresis used a separating gel (12%) and a concentrating gel (5%), with a loading volume of 5 μL. The voltages for the concentrating gel and separating gel were 80 V and 120 V, respectively. After running, the gels were stained with Coomassie Brilliant Blue R-250 for 2 h, followed by destaining with methanol-glacial acetic acid until the protein bands were clear. Images were obtained using a gel scanner (GelDocXR+, Bio-Rad, USA).

[0040] Size, polydispersity index (PDI), and zeta potential measurements: The average particle size and zeta potential of WPI and its conjugate were determined using a Zetasizer NanoSeries (NanoZS, Malvern Instruments, UK) according to the methods described above. Samples were diluted with ultrapure water to a protein concentration of 2 mg / mL.

[0041] Fourier Transform Infrared Spectroscopy (FT-IR): FT-IR spectral analysis of samples commonly uses the KBr pellet method (IS50, Suzhou, China). Simply put, freeze-dried powder is dispersed in KBr at a ratio of 1:100, then ground and pressed into thin pellets. The resolution and wavenumber range are 4 ohms and 4000-400 cm⁻¹, respectively.

[0042] Circular dichroism (CD) spectroscopy: Circular dichroism (J-1500, JASCO, Japan) is a key method for analyzing the secondary structure of whey protein isolates and glycosylated proteins in the wavelength range of 190–260 nm. WPI and WPI-inulin conjugate were dissolved in ultrapure water with a protein concentration of 0.1 mg / mL. The scan rate was 100 nm / min, the path length was 1 mm, and the temperature was 25 °C. The measured spectrum is the average of three measurements. The secondary structure composition was calculated using CD Pro software.

[0043] Intrinsic fluorescence spectroscopy: Intrinsic fluorescence intensity was measured using a fluorescence spectrophotometer (F-4600, Foss, Denmark). WPI and the conjugate were dispersed at 2 mg / mL in PBS buffer (pH 7.0, 0.01 M), with the PBS buffer serving as a blank. The excitation wavelength was 280 nm, and the emission wavelength was 300–500 nm. The excitation and emission slit widths were set to 5.0 nm.

[0044] Differential Scanning Calorimetry (DSC): The thermal stability of whey protein isolate and WPI-inulin conjugate was analyzed using DSC (DSC 204F1, NETZSCH, Germany). Approximately 3.0 mg of sample was placed in an aluminum crucible and sealed, with an empty crucible used as a control. The sample was then heated from 20 °C to 200 °C at a heating rate of 10 °C / min. The initial denaturation temperature, thermal denaturation temperature, and enthalpy change of WPI-inulin and WPI-inulin conjugate were obtained from the thermal profiles.

[0045] Emulsifying activity (ESI) and emulsion stability (ES): The EAI and ES of WPI and WPI-inulin conjugate were determined by turbidimetric method, and the changes were not significant. In short, soybean oil was mixed with the sample solution (2 mg / mL) at a ratio of 1:4 and homogenized for 3 minutes at 12000 rpm / min using an IKAULTRA-TURRAX high-speed homogenizer. 50 μL of the emulsion was extracted from the bottom at 0 min and 10 min, respectively, and dispersed in 5 mL of 0.1% (w / v) sodium dodecyl sulfate (SDS) solution. The absorbance of the diluted emulsion at 500 nm was recorded using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan). The EAI and ES indices were calculated according to Eq.(2) and Eq.(3).

[0046]

[0047] ES(min)=A0 / (A0-A t )×t (3)

[0048] In the formula, N represents the dilution factor (N = 101), ρ represents the protein concentration (g / mL), φ represents the optical path (φ = 0.01 m), θ represents the oil phase volume fraction (θ = 0.20), and A0 and A t The absorbance values ​​represent the absorbance of the sample at 0 min and t min (t = 10), respectively.

[0049] Antioxidant capacity includes ABTS free radical scavenging activity and DPPH free radical scavenging activity, as detailed below:

[0050] ABTS radical scavenging activity: The ABTS radical scavenging assay was slightly modified from the previously published report. A working ABTS solution was prepared by reacting 7 mM ABTS with 2.5 mM potassium persulfate at 25°C in the dark for 16 hours. The working solution was then diluted with deionized water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. Subsequently, 0.4 mL of sample solution (from 1 mg / mL to 5 mg / mL, dissolved in ultrapure water) was added to 3 mL of ABTS solution, and the reaction was allowed to proceed in the dark at room temperature for 30 minutes until completion. The absorbance of the sample was measured at 734 nm using a UV-Vis spectrophotometer. The ABTS radical scavenging activity of the sample was calculated as follows:

[0051] ABTS radical scavenge activity (%) = (A0-A1) / A0×100 (4)

[0052] In formula A1, A0 and A0 represent the absorbance of the sample and ultrapure water, respectively.

[0053] DPPH radical scavenging activity: The DPPH scavenging ability of the samples was slightly modified as described above. Soon, 0.5 mL of sample (from 1 mg / mL to 5 mg / mL, dissolved in ultrapure water) was incubated with 2.5 mL of DPPH (0.1 mM, dissolved in anhydrous ethanol) in the dark at 25°C for 30 minutes, with anhydrous ethanol as a control. The absorbance was then recorded at 517 nm using a UV-Vis spectrophotometer. The DPPH radical scavenging activity was calculated as follows:

[0054] DPPH radical scavenging activity(%)=1-((A s -A c ) / A B )×100 (5)

[0055] In the formula A s A represents the absorbance of the sample. c The absorbance of the control group is A. B The absorbance of the blank group is shown.

[0056] Statistical analysis: All experiments were repeated three times, and results are expressed as mean ± standard deviation (SD). Data were analyzed using Excel 2019 and IBM SPSS. Duncan's multiple range test was used to determine statistical significance, with a significance level of p < 0.05.

[0057] Results and Discussion:

[0058] The optimization of ultrasonic treatment reaction conditions includes single-factor experiments to determine the levels of each factor, model fitting, response surface analysis, and model optimization and validation.

[0059] Single-factor experiment to determine the levels of each factor: Figure 1 (AD) shows the changes in GD value with sonication time, sonication power, pH, and the WPI to inulin ratio. The GD value initially increased and then decreased with increasing sonication time, sonication power, pH, and the WPI to inulin ratio. According to the OFAT experiment, the maximum GD value for all WPI-inulin conjugates was obtained at sonication time = 80 min, sonication power = 25%, pH = 8, and a WPI to inulin ratio of 1:1. Therefore, in the RSM study, the reaction conditions were further optimized by selecting the points before and after the maximum GD value (Table 1).

[0060] Table 1. Variables and levels of the design

[0061]

[0062] Model Fitting: The experimental data were analyzed using Design-Expert software to establish a mathematical regression model. Table 3 shows the results of the analysis of variance for the model.

[0063] Table 3 Analysis of Variance

[0064]

[0065]

[0066] It can be seen that the model is highly significant (p<0.0001), while the model lacks a significant fit (p>0.05). The R-squared value of the model is 0.9571, indicating that the model has a good correlation. According to the F-values ​​of each factor, the significance of the influence of each factor is in the following order: ultrasonic power > WPI / inulin ratio > ultrasonic time > pH. It is worth mentioning that the quadratic polynomial regression equation between each factor and the grafting degree is as follows:

[0067] GD=+2.68-1.58A-1.78B+0.9524C-1.70D-1.42A+0.9774AC+1.28AD+0.1128BC+1.17BD+0.0125CD-1.62A 2 -2.31B 2 -1.21C 2 -5.12D 2 (6)

[0068] Response surface methodology: The interaction of sonication time, sonication power, pH, and the ratio of WPI to inulin on the grafting degree of WPI-inulin conjugates. Figure 2 and Figure 3 As shown. Figure 2As shown in (AF), the contour lines are all elliptical, indicating a significant interaction between the factors. Figure S3 clearly shows that the GD value of the conjugate first increases and then decreases with increasing sonication time or power, consistent with the results of the OFAT study. Ultrasound can accelerate the unfolding of protein structures, thereby exposing free amino groups and accelerating the Maillard reaction process within an appropriate range. Unfortunately, excessively long sonication times or excessively high power can lead to protein denaturation. Figure 3 (B, D, E) It can be seen that the grafting degree first increases and then decreases as the pH increases from 7 to 11. It is generally believed that increasing the pH value can effectively promote the Maillard reaction, especially under alkaline conditions. However, at pH 11, the GD value decreases sharply, which may be related to the racemic mixing and aggregation of proteins. From... Figure 3 As shown in (C, E, F), when the ratio of WPI to inulin changes from 3:1 to 1:3, the GD value first increases slowly and then decreases significantly. On the one hand, the accessibility of the reduced carbonyl group of the polysaccharide to the free amino group of the protein decreases with the reduction of inulin. On the other hand, excessive inulin may inhibit the reaction process through steric hindrance.

[0069] Model Optimization and Validation: The model and its multiple regression equation were optimized using Design-Expert software. The optimal preparation parameters for GD were determined to be: ultrasonic time 72.47 min, ultrasonic power 23.37%, pH 8.2, and a WPI to inulin ratio of 3:4. Under these conditions, the maximum grafting degree reached 27.38%. To verify the reliability of the model, three parallel experiments were conducted under the above optimal preparation conditions. The measured value of GD was 27.07 ± 0.94, close to the predicted value. Furthermore, the absorbance of the conjugate at 290 nm and 420 nm were 1.35 ± 0.01 and 1.22 ± 0.01, respectively. For convenience, the conjugate is named conjugate-c.

[0070] Grafting degree and browning intensity: GD value and browning intensity are commonly used to determine the degree of reaction between WPI and inulin. From Figure 1 As shown in (A) and (B), the glycemic index (GD) of the WPI-inulin conjugate increases with increasing reaction time. When the heating time reaches 5 h, the GD does not increase significantly, which may be due to protein denaturation caused by excessive heating. With prolonged reaction time, the absorbance of the conjugate at 290 nm and 420 nm increases significantly, indicating that a large number of Maillard reaction intermediates and melanoidins appear in the WPI-inulin conjugate with prolonged reaction time. Meanwhile, the grafting degree of conjugate-c is the highest compared to the wet heat method, while the browning intensity is only comparable to the sample treated with wet heat for 2 h. This indicates that ultrasound not only significantly shortens the Maillard reaction time but also yields a conjugate with a high grafting degree and low browning intensity.

[0071] SDS-PAGE: SDS-PAGE confirmed the formation of WPI-inulin conjugates. Electrophoretic results of WPI and WPI-inulin covalently grafted conjugates under different treatment conditions (1–5 h of moist heat treatment) are shown below. Figure 4 As shown, the two main bands of natural WPI are located near 13.9 and 16.0 kDa of the α-lactalbumin and β-lactoglobulin monomers, respectively. Additionally, a lighter band near 70.0 kDa corresponds to bovine serum albumin (BSA), consistent with previous studies. Compared to natural WPI, new bands were found in the top regions of lanes 2, 3, 4, 5, and 6 of the WPI-inulin conjugate, indicating the formation of higher molecular weight WPI-inulin complexes. With increasing reaction time, more new bands appeared in the top regions, while the bands near 13.9 and 16.0 kDa became lighter and slightly migrated upwards. These results indicate that the Maillard reaction of WPI with inulin generates high molecular weight covalent grafts, and the formation of more conjugates increases with prolonged heating time. However, when the reaction time reached 5 h, the top bands became lighter, possibly due to protein denaturation caused by prolonged heating, leading to a reduction in covalent grafting.

[0072] Size, PDI, and Zeta: Figure 1 (C) presents the particle size and PDI characterization of WPI and its conjugates. The particle size of natural WPI is approximately 527.20 ± 4.10 nm. Clearly, the average particle size of the conjugates is smaller than that of WPI and increases with increasing reaction time. The polydispersity index (PDI) of both WPI and the conjugates is less than 0.6, and the PDI of the conjugates is lower than that of WPI, indicating that the solution system is homogeneous. The average particle size of the ultrasonically treated sample is only 245.87 ± 3.35 nm, significantly lower than that of the wet heat-treated sample. This is because ultrasonic treatment generates a strong cavitation effect, thereby reducing the size of the WPI particles. The surface charge density of the samples is usually determined by the zeta potential value. Figure 1 As shown in (D), the zeta potential of natural WPI is -18.6 mV, while the zeta potential of the WPI-inulin conjugate does not change much, indicating that different treatments have little effect on the electrical properties of the complex.

[0073] FT-IR spectroscopy: FT-IR is used to detect and predict information and composition of protein secondary structure, and is an effective means of studying protein-polysaccharide interactions. The amide band is a typical and key absorption band for proteins, with 1700–1600 cm⁻¹, 1550–1450 cm⁻¹, and 1300–1200 cm⁻¹ representing amide I (C=O stretching), amide II (NH₄⁺ bending), and amide III (CN stretching and NH₄⁺ deformation), respectively. From... Figure 3(A) It can be seen that WPI exhibits absorption peaks at 1654 cm⁻¹ and 1541 cm⁻¹, consistent with previous findings. Compared to WPI, the positions of the amide I and amide II bands of the conjugate are slightly shifted, indicating that WPI is covalently linked to inulin. Furthermore, the absorption of the amide band in the whey protein isolate-inulin conjugate weakens after saccharification with inulin. Compared to whey protein isolate, the conjugate band intensity at 1100 cm⁻¹–1000 cm⁻¹ is significantly increased, indicating the formation of a new CN covalent bond via the Maillard reaction. This finding is consistent with previous studies on soy protein isolate (SPI) and glucose, and WPI and xylooligosaccharides. The band intensity of the conjugate treated with ultrasound is significantly stronger than that treated with wet heating, indicating that appropriate ultrasound accelerates the Maillard reaction. The overall signal intensity of the conjugate obtained by the ultrasound / pH shift combined method is much lower than that of other conjugates.

[0074] CD spectroscopy: CD spectroscopy is used to determine the secondary structure information of proteins in the wavelength range of 190–260 nm. For example... Figure 3 As shown in (B), the secondary structure of WPI changed significantly after glycosylation. Similarly, the peak height and position also changed, with an increase in the intensity of the negative absorption peak, likely due to the combination with heating, sonication, and inulin. Table 1 shows that the contents of α-helices, β-sheets, β-turns, and random coils in the natural whey protein isolate were approximately 22.4 ± 0.98%, 21.70 ± 2.34%, 20.43 ± 0.64%, and 35.47 ± 2.20%, respectively. Compared to WPI, the Maillard reaction resulted in a decrease in the contents of α-helices and β-turns, and an increase in the content of β-sheets. The combination of the carbonyl group of inulin with the ε-amino group of WPI is one of the main reasons for the decrease in α-helices. Furthermore, the content of random coils increased slightly, indicating that the structure of the coupling compound became more loosely structured and disordered due to prolonged heating. It is worth noting that the contents of α-helix, β-sheet, β-turn, and random coil in the ultrasonically treated conjugate were approximately 19.30±1.10%, 36.10±1.01%, 10.80±1.05%, and 33.83±1.05%, respectively. Compared with wet heat treatment, the structure of conjugate c is closer to that of natural WPI, which means that ultrasound is a more promising method for obtaining covalent proteoglycan grafts.

[0075] Fluorescence spectroscopy: Intrinsic fluorescence spectroscopy is commonly used to study changes in protein structure and the sensitivity of fluorophores to their microenvironment. Tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe) are generally considered to be the sources of fluorescence. Therefore, they are often used as fluorescent probes to study the interactions between proteins and polysaccharides. Figure 3As shown in (C), the fluorescence intensity of the conjugate decreased compared to whey protein isolate alone. The fluorescence intensity gradually decreased with increasing reaction time, indicating that glycosylation occurred between WPI and inulin, and became more complete with increasing reaction time. Specifically, the fluorescence intensities of whey protein isolate and the conjugate after wet heating (from 1 h to 5 h) were 3206, 2779, 2449, 2103, 2027, and 1793, respectively. The fluorescence intensity of the ultrasonically treated conjugate was 2306, closest to the fluorescence intensity of samples reacted with wet heating for 2 h or 3 h. The maximum fluorescence emission wavelength of WPI was 343 nm. The fluorescence intensity peaks of the covalent complexes treated with wet heating and ultrasound red-shifted to 344.2, 344.8, 347.8, 347, 348, and 345.4 nm, respectively, indicating that the spatial structure of the protein became more relaxed.

[0076] DSC: DSC is an effective method for analyzing the thermal denaturation temperature and enthalpy change of proteins. Generally, the higher the thermal denaturation temperature of a protein, the better its thermal stability. Figure 3 (D) DSC curves of WPI and its conjugates under different treatment conditions. The peak denaturation temperature of the WPI-inulin conjugate (105.3–116.7 °C) is higher than that of the WPI conjugate (101.2 °C), indicating that the thermal stability of whey protein isolate is improved after covalent binding with inulin. This result may be due to the steric hindrance effect of the sugar chain binding to the protein peptide chain during the Maillard reaction, thereby inhibiting the stretching of the protein peptide chain. In addition, the denaturation temperature of the WPI-inulin conjugate increases significantly with the extension of reaction time. As shown in Table 2, the enthalpy changes of the covalent bonding of WPI and inulin (wet heat treatment for 1–5 h and ultrasonic treatment) are 117.9, 123.8, 124.8, 126.4, 136.8, and 126.2 J / g, respectively. Compared with WPI, the enthalpy change of the WPI-inulin conjugate is significantly increased, indicating that the conjugate requires more energy to unfold the spatial structure of the protein.

[0077]

[0078]

[0079] Emulsifying properties: The emulsifying characteristics of proteins are typically evaluated using EAI and ES. From Figure 4(A) It can be seen that the EAI and ES values ​​of the WPI-inulin conjugate are significantly higher than those of whey protein isolate. This is because the protein structure becomes stretched and loosened after wet heating, which is beneficial for adsorption and unfolding at the oil-water interface. With prolonged heating time, the EAI value of the conjugate gradually increases, but decreases when the heating time exceeds 4 hours. This is mainly because prolonged heating damages the protein structure, causing denaturation and leading to a decrease in the EAI value. When the reaction time increases from 1 hour to 4 hours, the ES values ​​of the WPI-inulin conjugate increase by 11.98%, 17.05%, 20.30%, and 27.23% compared to WPI, respectively, while the conjugate heated for 5 hours shows almost no increase. Compared to WPI, the EAI and ES values ​​of the sample after ultrasonic treatment increase by 44.73% and 34.63%, respectively. In summary, compared to WPI, the WPI-inulin conjugate exhibits improved emulsifying activity and emulsifying stability, which is related to the increased water solubility due to the addition of inulin. It is worth mentioning that ultrasonic treatment has an advantage over wet heating treatment in obtaining excellent emulsification properties, which is related to the highest GD of conjugate c.

[0080] Antioxidant activity: The antioxidant capacity was evaluated by measuring the ability of whey protein isolate, inulin, and their conjugates to scavenge ABTS and DPPH free radicals. Figure 4 As shown in (B), the conjugates exhibited significantly stronger ABTS radical scavenging activity compared to whey protein isolate or inulin alone, indicating that the conjugates can enhance antioxidant activity through the Maillard reaction. Furthermore, the ABTS radical scavenging activity increased in a dose-dependent manner within the concentration range of 1 mg / mL to 5 mg / mL. Among these conjugates, the conjugate subjected to wet heating for 5 h showed the highest ABTS radical scavenging activity compared to other samples, particularly at a concentration of 5 mg / mL, where its scavenging activity reached 45.94%. The ABTS radical scavenging rates of the conjugates at 1–5 h were 26.86%, 36.79%, 37.37%, 41.61%, and 45.94%, respectively, while the scavenging rate of the conjugate subjected to sonication at a concentration of 5 mg / mL was 36.47%. Figure 4(C) shows the DPPH radical scavenging ability of whey protein isolate and the whey protein isolate-inulin conjugate. Clearly, this conjugate exhibits a higher DPPH radical scavenging ability compared to whey protein isolate or inulin alone, consistent with the ABTS results. This is mainly due to the ability of the Amadori rearrangement products (ARPs) and melanoidins generated by the Maillard reaction to scavenge oxygen radicals. Unfortunately, the DPPH radical scavenging ability of the sample was significantly lower than that of the ABTS conjugate; the antioxidant capacity of the conjugate after 5 hours of wet heating reached only 16.72%. At a concentration of 5 mg / mL, the DPPH radical scavenging rates of the conjugate (1–5 h) were 2.3%, 6.04%, 7.12%, 7.41%, and 8.13% higher than WPI, and 2.63%, 6.37%, 7.45%, 7.71%, and 8.46% higher than inulin, respectively.

[0081] Conclusion: In this invention, WPI-inulin conjugates were obtained through wet heating at 80℃ for 1–5 h followed by ultrasonic treatment. The ultrasonic treatment conditions were optimized using response surface methodology to maximize the grafting degree. Furthermore, the formation of WPI-inulin conjugates was confirmed by grafting degree, browning degree, FT-IR, SDS-PAGE, CD, and fluorescence spectroscopy. The emulsifying activity, emulsifying stability, thermal stability, and antioxidant activity of the WPI-inulin conjugates that underwent the Maillard reaction were all improved. Notably, compared to conjugates prepared by wet heating, the ultrasonically treated conjugates exhibited better structural and functional properties. Therefore, using ultrasound-assisted Maillard reactions to prepare protein-polysaccharide covalent complexes may be a promising method to expand the application of proteins in food.

Claims

1. A method for preparing whey protein isolate-inulin conjugates by ultrasound-assisted hydrothermal method, characterized in that: Includes the following steps: Step 1: Prepare whey protein isolate solution and inulin solution, ensuring that the concentrations of the whey protein isolate solution and inulin solution are equal. Then, mix equal volumes of the whey protein isolate solution and inulin solution to obtain the first mixture for later use. Step 2: Adjust the pH of the first mixture from Step 1 to 7.0 using an acidic or alkaline solution to obtain the second mixture; the specific operation of Step 2 is as follows: Adjust the pH of the first mixture to 7.0 using a 0.1 M HCl solution and a 0.1 M NaOH solution. Step 3: Under the conditions of ultrasonic frequency of 20-25KHz and ultrasonic power of 150-450W, the second mixture in step 2 is ultrasonically treated for 30-150 minutes to obtain the third mixture. Step 4: After hydrating the third mixture from Step 3 overnight at 4°C, heat it at 80°C for 1-5 hours to form conjugates with different grafting degrees. Then, immediately place it in an ice bath to stop the reaction and perform dialysis to separate unreacted inulin, obtaining whey protein isolate-inulin conjugates. Freeze-dry the whey protein isolate-inulin conjugates and store them at -18°C. In Step 4, the conjugates are dialyzed for 48 hours using a dialysis bag with a molecular weight of 12000. Step 5: The whey protein isolate-inulin conjugate obtained in Step 4 was subjected to structural and functional characterization tests.

2. The process for the production of whey protein isolate-inulin conjugate by ultrasound assisted wet-heating process as claimed in claim 1 wherein: The specific operation of step 1 is as follows: prepare whey protein isolate solution and inulin solution with milliq water, stir at room temperature for 2 hours to completely dissolve them, and obtain whey protein isolate solution with a concentration of 100 mg / mL and inulin solution with a concentration of 100 mg / mL.

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