Lactalbumin gel with shear recovery and pH response functions as well as preparation method and application of whey protein gel

Abstract

The invention belongs to the technical field of whey protein gel, and discloses shear recovery and pH response type whey protein gel, a preparation method and application, and the whey protein gel comprises whey protein isolate and citric acid. The gel does not need an additional gel accelerator, and citric acid is adopted to replace hydrochloric acid, so that toxic substances are prevented from being generated, the product safety is improved, and the production cost is reduced; the prepared gel has a remarkable shear thinning characteristic, and can recover the original gel state after being placed for 24 hours. The gel particles are uniform in particle size (200-300 nm) and high in system stability (the absolute value of Zeta potential is 35-50 mV), have a three-dimensional fibrous network structure and excellent water binding capacity (up to 82.3%) at the same time, can meet various scene requirements of dynamic viscosity adjustment or food system viscosity recovery and the like, and are simple and controllable in process, high in repeatability and convenient for industrial large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of whey protein gel technology, and in particular to a whey protein gel with shear recovery and pH response, its preparation method and application. Background Technology

[0002] Whey protein is a globular protein with wide availability and strong functionality, making it one of the most widely used natural proteins in the food industry, biomimetic materials, and controlled-release systems. Under acidic and hot conditions, whey protein undergoes conformational changes, exposes its hydrophobic surface, aggregates, and forms a network structure, thereby generating a protein gel with certain mechanical strength and rheological properties.

[0003] Currently, the main methods for preparing whey protein gels include the following: (1) Ion-induced gelation method: Calcium ions are often added to promote protein aggregation; (2) Enzymatic cross-linking method: such as treatment with microbial transglutaminase (TGase); (3) Thermally induced gelation method: The spatial structure of proteins is changed by high temperature, so that they form a three-dimensional network; (4) Acid-induced gelation method: utilizing the aggregation of proteins near the equilibrium point under acidic conditions.

[0004] However, these methods generally suffer from drawbacks such as difficulty in control, the need for numerous additives, and unstable gel strength. Especially in heat-induced systems, protein aggregation is sensitive to pH, ionic strength, and the heat treatment curve; even slight deviations can lead to significant differences in gel structure. Furthermore, existing protein gel preparation methods often rely on specific ions, enzymatic cross-linking, or heat treatment, but these generally suffer from complex operations, unstable gel performance, or difficulty in scaling up. Moreover, the aforementioned gels are primarily formed by covalent bonds, lacking shear reversibility or pH responsiveness, thus limiting their application. Therefore, developing a method for preparing whey protein gels that requires no additional gelation promoters, offers controllable conditions, is highly reproducible, has a simple process, and exhibits shear and pH responsiveness is of great significance.

[0005] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a whey protein gel with shear recovery and pH response, its preparation method, and its application.

[0007] The technical solution adopted by this invention to solve its technical problem is: A whey protein gel with shear recovery and pH responsive properties, the whey protein gel comprising whey protein isolate and citric acid; wherein the whey protein isolate has a protein content of 89%, an ash content of 2%, and a moisture content of 4.5%.

[0008] Furthermore, the whey protein isolate is whey protein isolate WPI 9410.

[0009] The method for preparing citric acid-induced whey protein gel as described above includes the following steps: (1) Dissolve whey protein isolate in distilled water to obtain whey protein isolate solution; (2) After adjusting the pH of the whey protein isolate solution prepared in step (1) with citric acid solution, stir magnetically to obtain the solution; (3) Adjust the pH of distilled water with citric acid solution to obtain citric acid-distilled water, and set aside for later use; (4) The solution from step (2) is brought to a final volume using the citric acid-distilled water from step (3), and stored overnight; (5) The solution obtained in step (4) is heated in a water bath and magnetically stirred, and then placed in an ice bath to obtain whey protein gel prepared with citric acid.

[0010] Furthermore, in step (1), the mass concentration of whey protein isolate in the whey protein isolate solution is 2-8%, preferably 4%.

[0011] Further, the citric acid solution in step (2) is a saturated citric acid solution; the pH of the solution in step (1) is adjusted to 1.0-3.5, preferably 2.5, and then magnetically stirred at 300 rpm / min for 2-4 hours.

[0012] Furthermore, in step (3), the pH of the citric acid-distilled water is 2.5.

[0013] Furthermore, in step (4), when adjusting the volume: the solution in step (2) prepared from 20 g of whey protein isolate in each step (1) is adjusted to 500 mL with citric acid-distilled water in step (3) and stored overnight at 4°C for later use.

[0014] Further, in step (5), the water bath heating temperature is 75-95℃, preferably 85℃, and the mixture is continuously magnetically stirred at 300 rpm / min. After heating for 4-24 h (preferably 6 h), the mixture is immediately taken out and placed in an ice bath to terminate the reaction. After cooling to room temperature, a whey protein gel with significant shear thinning properties is obtained.

[0015] Furthermore, the whey protein gel particles are reduced in size to 200-300 nm and are uniformly distributed; the absolute value of the zeta potential is increased to 35-50 mV.

[0016] The application of whey protein gel in dynamic viscosity regulation scenarios, as described above.

[0017] The advantages and positive effects of this invention are as follows: 1. The whey protein gel prepared by this invention has significant shear-thinning properties, which can meet the requirements of dynamic viscosity adjustment scenarios and solve the problem that traditional whey protein isolate gels lack this property.

[0018] 2. The whey protein gel prepared by this invention is a solid gel under pH ≤ 2.5 conditions. When the pH increases, the gel softens and can partially recover when the pH is adjusted back to 2.5, exhibiting pH response characteristics. In the low pH environment of the stomach, it is a soft gel and can be used for intestinal targeted delivery of substances.

[0019] 3. This invention uses citric acid instead of hydrochloric acid, avoiding the risk of producing toxic substances such as chloropropanol by reacting hydrochloric acid with lipids, while also broadening the adaptability of raw materials and reducing production costs.

[0020] 4. The whey protein gel particles prepared by this invention have a smaller particle size of 200-300 nm and a uniform distribution; the absolute value of the zeta potential is increased to 35-50 mV, the electrostatic repulsion is significantly enhanced, and the stability of the system is greatly improved.

[0021] 5. The whey protein gel prepared by this invention has a three-dimensional fibrous network structure with a fiber diameter of 10~50 nm. With the optimization of heating time, the fiber crossover density is increased and the distribution is more uniform, which lays the structural foundation for the excellent performance of the gel.

[0022] 6. The whey protein gel prepared by this invention has a synergistic effect of disulfide bonds, hydrogen bonds and hydrophobic interactions, which increases the water retention capacity of the gel from 5.65% to 82.3%, effectively retaining water and meeting the requirements of different application scenarios for gel moisturizing and texture.

[0023] 7. The whey protein gel prepared by this invention does not require additional gelation promoters. It can be prepared simply by adjusting the pH with citric acid and heating in a water bath. The conditions are controllable and highly reproducible. The required raw materials are readily available and the equipment is conventional, making it convenient for industrial-scale production.

[0024] 8. The purpose of this invention is to solve the problem that whey protein gel cannot respond to shear and pH changes in the prior art, and to meet the requirements of dynamic viscosity adjustment and different pH; to solve the problems of hydrochloric acid reacting with lipids to produce toxic substances such as chloropropanol and poor raw material adaptability in acid-induced technology, thereby reducing production costs and improving product safety.

[0025] 9. The gel of this invention is composed of whey protein isolate (WPI 9410) and citric acid. This invention eliminates the need for additional gelation promoters, using citric acid instead of hydrochloric acid to avoid the generation of toxic substances, improve product safety, and reduce production costs. The prepared gel exhibits significant shear-thinning properties and can recover its original gel state after 24 hours. The gel particles are uniform in size (200~300 nm), exhibit strong system stability (Zeta potential absolute value 35~50 mV), and possess a three-dimensional fibrous network structure and excellent water retention (up to 82.3%). It can meet the needs of various scenarios such as dynamic viscosity adjustment or viscosity recovery in food systems. The process is simple, controllable, and highly repeatable, facilitating large-scale industrial production. Attached Figure Description

[0026] Figure 1 The images shown are of the appearance of the citric acid-induced whey protein gel in this invention. The first column of images, from top to bottom, are the original gel images of the products prepared in Examples 1, 2, 3, 4, and 5 before shearing. The second column of images, from top to bottom, are the gel images of the products prepared in Examples 1, 2, 3, 4, and 5 after shearing. Figure 2 These are images showing the appearance of citric acid-induced whey protein gel before and after shear recovery in this invention. The first column of images, from top to bottom, shows the gel images of the product prepared in Example 1 before shearing, 0 min after shearing, and 24 h after shear recovery. The second column of images, from top to bottom, shows the gel images of the product prepared in Example 2 before shearing, 0 min after shearing, and 24 h after shear recovery. The third column of images, from top to bottom, shows the gel images of the product prepared in Example 3 before shearing, 0 min after shearing, and 24 h after shear recovery. The fourth column of images, from top to bottom, shows the gel images of the product prepared in Example 4 before shearing, 0 min after shearing, and 24 h after shear recovery. The fifth column of images, from top to bottom, shows the gel images of the product prepared in Example 5 before shearing, 0 min after shearing, and 24 h after shear recovery. Figure 3 The images show the appearance of the citric acid-induced whey protein gel pH response in this invention. From left to right, the images are: the initial pH=2.5 gel appearance of the product prepared in Example 5; the gel appearance of the product prepared in Example 5 adjusted to pH=7.0 with 5 M NaOH solution; and the gel appearance of the product prepared in Example 5 adjusted from pH=7.0 back to pH=2.5 with saturated citric acid solution. Figure 4The figures show the particle size-potential diagrams of the citric acid-induced whey protein gels in this invention; the upper figure shows the particle size diagrams of Comparative Examples 1-2 and Examples 1-5, and the lower figure shows the potential diagrams of Comparative Examples 1-2 and Examples 1-5. Figure 5 The images show transmission electron microscopy (TEM) images of citric acid-induced whey protein gels in this invention; from left to right, the images represent the products obtained in Examples 1, 3, and 5. Figure 6 This is a diagram showing the surface hydrophobicity of the citric acid-induced whey protein gel in this invention; Figure 7 This is a Tht plot of the citric acid-induced whey protein gel in this invention; Figure 8 This is a diagram showing the intermolecular interaction forces of the citric acid-induced whey protein gel in this invention. Figure 9 This is a water-holding capacity diagram of the citric acid-induced whey protein gel in this invention; Figure 10 This is a diagram of the free thiol groups in the citric acid-induced whey protein gel of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0028] The various experimental operations involved in the specific embodiments are all conventional techniques in the art. For parts not specifically annotated herein, those skilled in the art can refer to various commonly used reference books, scientific and technological literature, or related instructions and manuals prior to the filing date of this invention for implementation. The following embodiments 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 embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] A citric acid-induced whey protein gel, the gel comprising whey protein isolate (WPI 9410) and citric acid; wherein the whey protein isolate has a protein content of 89%, an ash content of 2%, and a moisture content of 4.5%.

[0030] The method for preparing citric acid-induced whey protein gel as described above includes the following steps: (1) Dissolve whey protein isolate (WPI 9410) in distilled water; (2) After adjusting the pH of the whey protein isolate solution prepared in step (1) with citric acid solution, stir magnetically; (3) Adjust the pH of the distilled water with citric acid solution and set aside; (4) Use the citric acid-distilled water from step (3) to make up the volume of the solution from step (2), and store it overnight; (5) The solution obtained in step (4) is heated in a water bath and magnetically stirred, and then placed in an ice bath to obtain whey protein gel prepared with citric acid.

[0031] Preferably, the mass of whey protein isolate (WPI 9410) in step (1) is 20 g.

[0032] Preferably, the citric acid solution in step (2) is a saturated citric acid solution, the pH of the solution in step (1) is adjusted to 2.5, and then magnetically stirred at 300 rpm / min for 2 hours.

[0033] Preferably, the pH of the citric acid-distilled water in step (3) is 2.5.

[0034] Preferably, the solution prepared in step (4) is diluted to 500 mL with citric acid-distilled water at pH 2.5 from step (3) and stored overnight at 4°C for later use.

[0035] Preferably, in step (5), the water bath heating temperature is 85°C, and the mixture is continuously magnetically stirred at 300 rpm / min. After heating for different times (2 h, 3 h, 4 h, 5 h, 6 h), the mixture is immediately taken out and placed in an ice bath to terminate the reaction. After cooling to room temperature, a whey protein gel with significant shear thinning into a liquid state is obtained.

[0036] The application of citric acid-induced whey protein gels as described above in gelation-related scenarios.

[0037] Specifically, the relevant preparation and testing methods are as follows: 1. Experimental Materials Whey protein isolate WPI 9410 was purchased from Hilmar Ltd.

[0038] Citric acid, purchased from Maclean Biotech Co., Ltd.

[0039] 2. Experimental equipment Magnetic stirrer.

[0040] Example 1 A whey protein gel with shear recovery and pH responsive properties, the gel comprising whey protein isolate (WPI9410) and citric acid; wherein the whey protein isolate has a protein content of 89%, an ash content of 2%, and a moisture content of 4.5%.

[0041] The preparation method of the above whey protein gel includes the following steps: (1) Dissolve 20 g of whey protein isolate (WPI 9410) in an appropriate amount of distilled water to obtain a whey protein isolate solution with a whey protein isolate mass concentration of 4%; (2) Adjust the pH of the whey protein isolate solution prepared in step (1) to 2.5 with saturated citric acid solution, and then stir magnetically for 2 hours at 300 rpm / min; (3) Adjust the pH of distilled water to 2.5 with citric acid solution to obtain citric acid-distilled water, and set aside for later use; (4) Use citric acid-distilled water with pH 2.5 from step (3) to make up the volume of the solution in step (2) to 500 mL and store it overnight at 4°C; (5) The solution obtained in step (4) was heated in a water bath at 85°C and continuously stirred magnetically at 300 rpm / min. After heating for 2 h, it was immediately taken out and placed in an ice bath to terminate the reaction, resulting in a whey protein gel that was significantly shear-thinned into a liquid state.

[0042] Example 2 The preparation steps of Example 2 are the same as those of Example 1, except that the water bath heating time in Example 2 is 3 h.

[0043] Example 3 The preparation steps of Example 3 are the same as those of Example 1, except that the water bath heating time in Example 3 is 4 h.

[0044] Example 4 The preparation steps of Example 4 are the same as those of Example 1, except that the water bath heating time in Example 4 is 5 h.

[0045] Example 5 The preparation steps of Example 5 are the same as those of Example 1, except that the water bath heating time in Example 5 is 6 h.

[0046] Comparative Example 1 The preparation steps for Comparative Example 1 are the same as those for Example 5, except that the pH of the solution in Comparative Example 1 is not adjusted. Specifically, the steps for Comparative Example 1 are as follows: (1) Dissolve 20 g of whey protein isolate (WPI 9410) in an appropriate amount of distilled water to obtain a whey protein isolate solution with a whey protein isolate mass concentration of 4%; (2) The solution obtained in step (1) was magnetically stirred at 300 rpm / min for 2 hours; (3) Dilute the solution from step (2) to 500 mL with distilled water and store it overnight at 4°C; (4) Heat the solution obtained in step (3) in a water bath at 85°C and continuously stir magnetically at 300 rpm / min. After heating for 5 h, take it out and put it in an ice bath to terminate the reaction. Take it out for later use.

[0047] Comparative Example 2 The preparation steps of Comparative Example 2 are the same as those of Example 5, except that step (4) is omitted in the solution preparation process of Comparative Example 2.

[0048] The relevant tests are as follows: 1. The gel of Example 5 has a more robust appearance, while also exhibiting shear-thinning to a liquid state and reverting to a gel state after 24 hours. The results are as follows... Figure 1-3 As shown. The whey protein gel heated for 2 hours exhibited a slightly solid-like state. With prolonged heating, the gel structure changed from being easily collapsed to maintaining a certain shape, and it exhibited shear thinning and thixotropic recovery. The gel morphology of Example 1 was irregular and the texture was soft; the gel morphology of Example 2 was more regular than that of Example 1, but still relatively soft; with prolonged heating, the gel morphology of Example 5 was the most regular, the texture was the hardest, and the color gradually deepened. Prior art (CN120501181A) discloses a transparent milk beverage and its preparation method, and the gel prepared therefrom. Carrageenan, xanthan gum, and water were added to a beaker at 55°C, thoroughly mixed, and then trehalose and potassium citrate were added, mixed again, and then WPI was added and mixed further. The resulting mixture was then subjected to ultra-high temperature sterilization for 5 seconds and filtered through a sieve to obtain a gel with an appearance similar to that of the present invention. However, the present invention only requires whey protein and citric acid, without the need for excess hydrosols and buffer salts, which is significantly superior to the prior art.

[0049] 2. Particle size and zeta potential were measured for Comparative Examples 1-2 and Examples 1-5, and the results are as follows: Figure 4 As shown. The particle size of Comparative Example 1 was 38.19 nm, significantly smaller than that of Examples 1-5 (200~300 nm range), and there was a significant difference between the groups (P<0.05). This is because only thermal denaturation exposes a limited number of hydrophobic groups, forming smaller aggregates. Adjusting the pH to 2.5 weakens electrostatic repulsion, and the hydrophobic effect causes the protein conformation to unfold, forming larger aggregates. The particle size of Comparative Example 2 was 512 nm, significantly larger than that of Examples 1-5 (200~300 nm range), and there was a significant difference between the groups (P<0.05). This indicates that heating at 85°C can fully depolymerize and rearrange whey protein molecules, forming a finer and more uniform particle system. The heating time (2~6 hours) has no significant effect on the final particle size, possibly because the protein molecules always carry a strong positive charge, and the electrostatic repulsion continuously inhibits the fusion between aggregates.

[0050] The isoelectric point of Comparative Example 1 was approximately 4.35, and its Zeta potential was 38.6 mV at pH=2, indicating strong electrostatic repulsion. Comparative Example 2 had a Zeta potential of 27.8 mV at pH=2. Examples 1-5 showed a rightward shift in their isoelectric points, and the absolute value of their Zeta potentials significantly increased to 35-50 mV at pH=2, indicating that heating exposed the charged groups on the protein surface more fully, greatly enhancing electrostatic repulsion and significantly improving system stability. Furthermore, the Zeta potential curves of all examples showed a consistent trend, indicating that heating time (2-6 hours) had no significant effect on the protein's charged properties and pH stability.

[0051] Meanwhile, by comparing Example 5, Comparative Example 1 and Comparative Example 2, it can be seen that step (4) and the two steps of pH 2.5 in the method of the present invention have a synergistic effect and can synergistically improve the relevant properties of the prepared whey protein gel.

[0052] 3. Transmission electron microscopy (TEM) scans were performed on Examples 1, 3, and 5. The results are as follows: Figure 5 As shown, a fibrous network structure can be clearly observed, with fiber diameters ranging from approximately 10 to 50 nm and lengths reaching several micrometers. This fibrous network is a three-dimensional framework formed by the cross-linking of whey protein isolate molecules through non-covalent bonds (hydrogen bonds, hydrophobic interactions, etc.) after thermal denaturation. The left image is a TEM image of Example 1, showing a relatively loose fiber distribution and larger network pores, demonstrating structural heterogeneity. The middle image is a TEM image of Example 3, showing a higher fiber interweaving density and smaller network pores, indicating a more compact gel structure. The right image is a TEM image of Example 5, showing interwoven fibers forming a three-dimensional network with a relatively uniform overall distribution, with only a small amount of fiber aggregation in certain areas.

[0053] 4. The surface hydrophobicity of Comparative Examples 1-2 and Examples 1-5 was measured.

[0054] The testing method was as follows: The surface hydrophobicity (H0) of the examples and comparative examples was determined using an exogenous fluorescent probe method (8-aniline-1-naphthalenesulfonic acid, ANS). Examples 1-5 and Comparative Examples 1-2 were diluted with citric acid-distilled water at pH 2.5 to a protein concentration of 0.05–0.2 mg / mL. 4 mL of the sample was mixed with 80 μL of ANS solution (8-aniline-1-naphthalenesulfonic acid, 8 mM) and reacted in the dark for 2 min. The maximum fluorescence emission intensity of the sample in the range of 400–600 nm was measured using a fluorescence spectrophotometer at an excitation wavelength of 365 nm. The excitation and emission slit widths were both 5 nm, the voltage was 400 V, and the scan rate was 1200 nm / min. Each sample was measured three times. The relative fluorescence intensity at the maximum emission peak was plotted against the protein concentration (mg / mL), and the slope of the straight line was H0.

[0055] The results are as follows Figure 6 As shown, different heating times significantly affected the surface hydrophobicity of whey protein isolate gels. The surface hydrophobicity H0 value of Comparative Example 1 was 978; the surface hydrophobicity H0 value of Comparative Example 2 was 1763; the surface hydrophobicity H0 value of Example 1 increased to 2012; the H0 value of Example 2 further increased to 2800; the H0 value of Example 3 reached a peak of 3074; while the H0 values ​​of Examples 4 and 5 decreased to 1904 and 1864, respectively. This is because under prolonged acidic heating, WPI exhibits a dynamic competition between the exposure of hydrophobic groups and the masking of hydrophobic aggregation. Within 4 hours of heating, thermal denaturation fully exposes the hydrophobic groups of the protein, continuously enhancing surface hydrophobicity; however, after heating for more than 4 hours, excessive aggregation leads to the encapsulation of hydrophobic sites, significantly reducing surface hydrophobicity.

[0056] Meanwhile, by comparing Example 5, Comparative Example 1 and Comparative Example 2, it can be seen that step (4) and the two steps of pH 2.5 in the method of the present invention have a synergistic effect and can synergistically improve the relevant properties of the prepared whey protein gel.

[0057] 5. ThT fluorescence spectra were measured for comparative examples 1-2 and examples 1-5.

[0058] The test method was as follows: 8 mg of thioflavone T (ThT) was dissolved in 10 mL of 10 mM phosphate buffer (pH=7, 0.2 mol / L NaCl) to prepare a 0.8 mg / mL solution. The solution was stirred and filtered through a 0.2 μm filter to remove undissolved ThT. The solution was then diluted 50 times to prepare the working solution, which was stored in the dark and at low temperature. 50 μL of the samples from Examples 1-5 were thoroughly mixed with 5 mL of the ThT working solution. This process was performed under dark conditions, and the solution was detected using a fluorescence spectrophotometer. Detection conditions: excitation wavelength 460 nm, emission wavelength scanning range 490 nm-700 nm, slit width 5 nm, voltage 700 V, and scan speed 1200 nm / min.

[0059] The results are as follows Figure 7As shown, significant differences in fluorescence intensity were observed between Examples 1-5. Comparative Example 1 exhibited the lowest fluorescence intensity peak at only 66.8 au; Comparative Example 2 showed a slightly increased peak fluorescence intensity of 76.8 au; after pH adjustment and heating at 85°C, the fluorescence intensity of Examples 1-5 all increased significantly, with peak values ​​ranging from 150 to 180 au, with Example 5 showing the highest peak at 181.5 au. ThT fluorescence intensity is positively correlated with the degree of fibrous structure formation. Combined with the fibrous network observed under transmission electron microscopy, this indicates that heating caused whey protein to form a large number of ordered fibrous aggregates. Example 5 exhibited the highest fluorescence intensity, corresponding to its dense and uniform fibrous network under transmission electron microscopy, reflecting the highest β-sheet structure among the protein molecules; while Comparative Examples 1 and 2 showed the lowest fluorescence intensity, consistent with their lack of ordered fibrous structure formation.

[0060] Meanwhile, by comparing Example 5, Comparative Example 1 and Comparative Example 2, it can be seen that step (4) and the two steps of pH 2.5 in the method of the present invention have a synergistic effect and can synergistically improve the relevant properties of the prepared whey protein gel.

[0061] 6. The intermolecular interaction forces of Examples 1-5 were measured.

[0062] The test method was as follows: The sample (2 g) from the example was mixed with 18 mL of four different solvents in a 50 mL centrifuge tube. The four solvents were: 0.6 M NaCl (S1), 0.6 M NaCl + 1.5 M urea (S2), 0.6 M NaCl + 8 M urea (S3), and 0.6 M NaCl + 8 M urea + 10 mM DTT (S4). The mixture was allowed to stand at room temperature for 2 hours, and then centrifuged at 10,000 rpm / min for 15 minutes at 4°C. Finally, the protein content in the supernatant was determined using a BCA kit. The contribution of each intermolecular force during gel formation was defined as the difference in protein solubility between adjacent solvents: S1 represents ionic bonds; S2-S1 represents hydrogen bonds; S3-S2 represents hydrophobic interactions; and S4-S3 represents disulfide bonds.

[0063] The results are as follows Figure 8As shown, the proportion of bonding types in whey protein isolate gels under different heating times exhibited a clear pattern. Disulfide bonds had the highest proportion among all groups, reaching 65.939% in Example 3, slightly higher than Example 2 (64.638%), Example 4 (62.658%), and Example 5 (61.815%). The proportion of hydrophobic interactions increased with heating time, reaching 11.733% in Example 5 and 10.144% in Example 4, both significantly higher than Example 1 (5.445%), Example 2 (5.083%), and Example 3 (7.08%). The proportion of hydrogen bonds reached a relatively high level in Example 4 (6.763%) and Example 5 (6.35%). The proportion of ionic bonds gradually decreased, from 30.866% (Example 1) to 20.102% (Example 5).

[0064] Combining transmission electron microscopy and ThT fluorescence analysis, the high hydrogen bonds, hydrophobic interactions, and disulfide bonds in Example 3 synergistically formed a dense and ordered fiber network; while in Example 5, although hydrophobic interactions accounted for the highest proportion, the proportion of ionic bonds decreased, corresponding to its structural characteristics of local fiber aggregation.

[0065] 7. The water-holding capacity of Examples 1-5 was measured.

[0066] The test method is as follows: Take about 5 g of the example sample into a 50 mL centrifuge tube, record the mass of the centrifuge tube as M1, and the total mass of the sample and the centrifuge tube as M2. Centrifuge at 10000 × g for 15 min at 4℃. After discarding the supernatant, record the mass of the precipitate and the centrifuge tube as M3.

[0067]

[0068] The results are as follows Figure 9As shown, different heating times significantly affected the water-holding capacity of whey protein isolate gels. Example 1 had a water-holding capacity of only 5.65%, Example 2 increased to 41.9%, Example 3 further increased to 73.8%, Example 4 reached 79.9%, and Example 5 reached 82.3%. The improvement in water-holding capacity was closely related to the density and bonding of the gel network. The low water-holding capacity of Example 1 was related to its loose fiber network and weak intermolecular interactions (e.g., hydrophobic interactions accounted for only 5.445%). With prolonged heating time, the network synergistically constructed by disulfide bonds and hydrophobic interactions between protein molecules gradually became denser (e.g., in Example 3, disulfide bonds accounted for 65.939% and hydrophobic interactions accounted for 7.08%), and the network pores could effectively trap water, thus increasing the water-holding capacity. The water-holding capacity of Examples 4 and 5 remained at a high level because their fiber networks were continuously optimized, and the intermolecular forces (e.g., in Example 5, hydrophobic interactions accounted for 11.733%) were further enhanced, resulting in stable water trapping capacity. The prior art (CN110338261A) discloses a method for improving the properties of whey protein gel by citric acid crosslinking. In this method, when the protein concentration is 4%, the water retention of the gel is less than 50%. However, the present invention enables the water retention of the gel at a whey protein concentration of 4% to reach more than 80%, which is significantly better than the prior art.

[0069] The existing technology (CN110338261A) uses whey protein at a concentration of 10% (w / w) and citric acid at a final concentration of 0.6% (w / w). After heating at pH 7.0 and 50°C for 6 hours and then freeze-drying, whey protein with excellent gelation properties is obtained. In contrast, the present invention uses whey protein isolate at a concentration of 4% (w / w), adjusts the pH to 2.5 with saturated citric acid solution, and then heats at 85°C for 6 hours to obtain whey protein isolate fibers, which exhibit excellent gelation properties. The whey protein isolate gel of the present invention has significant shear-thinning characteristics and can recover its gel state after 24 hours after shearing. It also possesses a fibrous network structure and excellent water retention, significantly superior to the existing technology.

[0070] 8. Free thiol (SH) groups were tested in Examples 1-5. F Determination of content.

[0071] The test method was as follows: 0.5 mL (protein concentration of 10 mg / mL) of the example sample was mixed with 4.5 mL of 8 M urea buffer (1.04 g Tris, 6.9 g glycine, 0.12 g EDTANa2 and 48 g urea were brought to a final volume of 100 mL of ultrapure water, pH=8.0), and then 50 μL of 4 mg / mL Ellman's reagent (0.2 g of 5,5'-dithiobis-2-nitrobenzoic acid was brought to a final volume of 50 mL with Tris-glycine buffer) was added. After thorough mixing, the mixture was incubated in the dark for 30 min, and the absorbance was measured at a wavelength of 412 nm.

[0072]

[0073] Where A 412 denoted as absorbance, D as sample dilution factor, and C as protein concentration in the sample.

[0074] The results are as follows Figure 10 As shown, different heating times significantly affected the free thiol content of whey protein isolate gel. There was no significant difference in free thiol content in Examples 1-3, approximately 9.57 μmol / g, 9.39 μmol / g, and 9.25 μmol / g, respectively; however, the free thiol content in Examples 4 and 5 decreased significantly, to 8.04 μmol / g and 8.01 μmol / g, respectively. Free thiol groups are key groups involved in disulfide bond formation. The decrease in free thiol content in Examples 4 and 5 corresponds to changes in the proportion of disulfide bonds (e.g., 62.658% in Example 4 and 61.815% in Example 5), indicating that with prolonged heating time (more than 4 hours), more free thiol groups participate in disulfide bond formation, leading to a decrease in free thiol content. In contrast, the high and stable free thiol content in Examples 1-3 reflects a relative balance between the disulfide bond formation rate and the thiol release rate at this stage, which is also closely related to the formed fibrous network structure (e.g., the dense and ordered fibrous network in Example 3).

[0075] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A whey protein gel with shear recovery and pH responsive properties, characterized in that: The whey protein gel comprises whey protein isolate and citric acid; wherein the whey protein isolate has a protein content of 89%, an ash content of 2%, and a moisture content of 4.5%.

2. The whey protein gel according to claim 1, characterized in that: The whey protein isolate is whey protein isolate WPI 9410.

3. The method for preparing citric acid-induced whey protein gel as described in claim 1 or 2, characterized in that: Includes the following steps: (1) Dissolve whey protein isolate in distilled water to obtain whey protein isolate solution; (2) After adjusting the pH of the whey protein isolate solution prepared in step (1) with citric acid solution, stir magnetically to obtain the solution; (3) Adjust the pH of distilled water with citric acid solution to obtain citric acid-distilled water for later use; (4) The solution from step (2) is brought to a final volume using the citric acid-distilled water from step (3), and stored overnight; (5) The solution obtained in step (4) is heated in a water bath and magnetically stirred, and then placed in an ice bath to obtain whey protein gel prepared with citric acid.

4. The preparation method according to claim 3, characterized in that: In step (1), the whey protein isolate solution has a mass concentration of 2-8%.

5. The preparation method according to claim 3, characterized in that: The citric acid solution in step (2) is a saturated citric acid solution; the pH of the solution in step (1) is adjusted to 1.0-3.5, and then magnetically stirred at 300 rpm / min for 2 hours.

6. The preparation method according to claim 3, characterized in that: In step (3), the pH of the citric acid-distilled water is 1.0-3.

5.

7. The preparation method according to claim 3, characterized in that: When adjusting the volume in step (4): the solution in step (2) prepared from 20 g of whey protein isolate in step (1) is adjusted to 500 mL with citric acid-distilled water in step (3) and stored overnight at 4°C for later use.

8. The preparation method according to claim 3, characterized in that: In step (5), the water bath heating temperature is 75-95℃, and the magnetic stirring is continuously performed at 300 rpm / min. After heating for 4-24 h, the mixture is immediately taken out and placed in an ice bath to terminate the reaction. After cooling to room temperature, a whey protein gel with significant shear thinning properties is obtained.

9. The preparation method according to claim 3, characterized in that: The whey protein gel particles are reduced in size to 200-300 nm and are uniformly distributed; the absolute value of the zeta potential is increased to 35-50 mV.

10. The application of the whey protein gel as described in claim 1 in dynamic viscosity adjustment scenarios.

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