Synergistic modification method of soybean protein-casein compound and product

Abstract

The invention discloses a synergistic modification method of a soybean protein-casein compound and a product, and belongs to the technical field of food protein processing and modification.The method comprises the steps that SPI and CS are mixed according to the mass ratio selected from (9: 1) to (1: 9) and dissolved in water, dispersion liquid with the concentration being 4%-8% is prepared, the pH is adjusted to 10-12.0 after hydration, and stirring is conducted; carrying out ultrasonic treatment on the protein liquid under the condition of keeping the alkaline pH unchanged; finally, the pH is adjusted back to be neutral, centrifugation is conducted, and supernate is taken to obtain the modified compound. According to the method, an ultrasonic cavitation effect acts on protein molecules which are expanded in an alkaline manner to generate a synergistic effect, and protein conformation is induced to generate flexible rearrangement, so that the solubility, emulsification, foaming and other interface functions of the compound are synergistically and remarkably improved. The invention provides a green and efficient way for constructing a high-performance plant-animal double-protein ingredient.

Description

Technical Field

[0001] This invention belongs to the field of food protein processing and modification technology, specifically relating to a method and product for the synergistic modification of soybean protein-casein complex. Background Technology

[0002] Soy protein isolate (SPI) and casein (CS) are representative plant-based and animal-based proteins, respectively. SPI has a high protein content and good nutritional value, but its low solubility and tendency to aggregate under neutral conditions result in poor interfacial functions such as emulsification and foaming, limiting its applications. CS, on the other hand, has a flexible structure and good solubility and surface activity. Theoretically, combining SPI and CS can create a system with complementary nutrition and function.

[0003] However, studies have shown that due to significant differences in molecular conformation and surface properties, simple mixing of SPI and CS under normal conditions often results in weak interactions, making them prone to phase separation or competitive aggregation, and difficult to form stable, homogeneous complexes. Furthermore, their functions cannot achieve a synergistic effect of "1+1>2". Therefore, it is necessary to regulate the intermolecular interactions and conformation of these two molecules through physical or chemical means.

[0004] pH shifting is a method that induces reversible denaturation and refolding of proteins under extreme pH conditions, effectively improving protein solubility and function. Ultrasonic treatment utilizes the mechanical force generated by cavitation to disrupt protein aggregates and alter their structure. While there are studies on using pH shifting or ultrasound alone to modify SPI or other plant proteins, and reports of combining the two to treat single proteins, a method for synergistically applying pH shifting and ultrasound under specific alkaline conditions to the SPI / CS complex system to directionally regulate its conformation and synergistically enhance its overall interfacial properties has not yet been publicly disclosed.

[0005] Therefore, there is an urgent need in this field to develop a simple and effective method and product for the synergistic modification of soybean protein-casein complexes. Summary of the Invention

[0006] The purpose of this invention is to address the technical problem of difficulty in synergistically improving the interface function of existing SPI / CS composites by providing a simple and effective synergistic modification method and the resulting high-performance composite.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for the synergistic modification of soybean protein-casein complexes, the key of which lies in ultrasonic treatment of the protein dispersion under alkaline pH conditions. The method includes the following steps: S1. Compounding and pretreatment: Soy protein isolate (SPI) and casein (CS) are mixed in a mass ratio selected from (9:1) to (1:9), dissolved in water, and a protein dispersion with a concentration of 4%-8% (w / v) is prepared. The mixture is stirred and hydrated at low temperature. S2. Alkali treatment: Adjust the pH of the protein dispersion obtained in step S1 to an alkaline range of 8.0-12.0, and continue stirring under this condition; S3. Ultrasonic treatment: Under the condition of maintaining a pH value of 10.0-12.0, the protein solution after step S2 is subjected to ultrasonic treatment; S4. pH Adjustment and Post-processing: Adjust the pH of the protein solution after step S3 to neutral, centrifuge, and collect the supernatant to obtain the modified SPI-CS complex.

[0008] Preferably, in step S1, the mass ratio of SPI to CS is 1:1.

[0009] Preferably, in step S1, the concentration of the protein dispersion is 5% (w / v).

[0010] Preferably, in step S2, the pH value is adjusted to 11.0.

[0011] Preferably, in step S2, the stirring time under alkaline conditions is 1-2 hours.

[0012] Preferably, in step S3, the power of the ultrasonic treatment is 200-600W, and the treatment time is 5-20 minutes.

[0013] Preferably, the ultrasonic treatment has a power of 400W and a treatment time of 10 minutes.

[0014] Preferably, in step S3, the ultrasonic treatment is performed under ice-water bath conditions.

[0015] Preferably, in step S4, the centrifugation conditions are: centrifugation at 4°C and a speed of 7000-12000 r / min for 5-15 minutes.

[0016] Secondly, the present invention provides a soy protein isolate and casein complex prepared by the method of the first aspect. This complex exhibits significantly improved solubility and excellent interfacial functional properties.

[0017] Compared with the prior art, the present invention has the following outstanding advantages: 1. Significant synergistic effect: This invention innovatively performs ultrasonic treatment in an alkaline environment. This condition allows the protein molecular chains to fully extend and enhances charge repulsion. At this time, the cavitation effect of ultrasound can act more efficiently on the extended flexible molecules, destroying the non-covalent interactions within and between molecules, and inhibiting ineffective re-aggregation during subsequent pH adjustment, thereby achieving a synergistic modification effect of "1+1>2".

[0018] 2. Clear Enhancement of Structure and Function: After treatment with the method of this invention, the secondary structure of the SPI-CS complex undergoes a directional transformation, with a decrease in α-helix content and an increase in random coil content, indicating increased protein flexibility and easier exposure of hydrophobic groups. This directly leads to a significant increase in its solubility (up to 92.95%), a significant decrease in equilibrium interfacial tension (up to 40.94 mN / m), and a synergistic enhancement of both emulsifying activity and foaming ability.

[0019] 3. Green and efficient process: The entire method involves only pH adjustment and physical ultrasound, without the need for chemical reagents. The conditions are mild and controllable, which is in line with the trend of clean labeling and is easy to scale up industrially.

[0020] Meanwhile, the soy protein isolate and casein complex prepared by the method of the present invention has the following significant advantages: 1. Functional Synergy: This complex achieves a synergistic effect of solubility and interfacial functions (such as emulsification and foaming) that cannot be achieved by a single protein or simple mixture. Its comprehensive performance indicators (such as solubility >92% and interfacial tension <41 mN / m) significantly exceed the linear sum of the performance of each individual component, demonstrating a true "1+1>2" effect.

[0021] 2. Structural stability: The complex has a smaller hydrodynamic particle size and a higher absolute value of Zeta potential in solution, indicating that it forms a stable and uniform dispersion system that is not prone to aggregation or phase separation.

[0022] 3. Unique conformation: Its specific secondary structure composition (low α-helix, high random coil) is the intrinsic structural basis for its excellent function. This structural state is induced and fixed by the specific process of "ultrasound under alkaline conditions". Attached Figure Description

[0023] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a comparison chart of the solubility of samples from different treatment groups in the embodiments of the present invention; Figure 2 This is a particle size distribution diagram of SPI under different processing methods in the embodiments of the present invention; Figure 3 Zeta potential diagrams of samples from different treatment groups in this embodiment of the invention; Figure 4 This is a graph showing the changes in surface hydrophobicity of samples from different treatment groups in this embodiment of the invention. Figure 5 These are circular dichroism (CD) chromatograms under different processing methods in embodiments of the present invention; Figure 6 This is a graph showing the secondary structure content analysis of samples from different treatment groups in this embodiment of the invention; Figure 7 This is a comparison diagram of the molecular flexibility of samples from different treatment groups in the embodiments of the present invention; Figure 8 This is a comparison chart of the emulsification characteristics of samples from different treatment groups in the embodiments of the present invention; Figure 9 This is a comparison diagram of the foaming characteristics of samples from different treatment groups in the embodiments of the present invention; Figure 10 This is a graph showing the change of interfacial tension over time under different processing methods in embodiments of the present invention. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0025] Example 1: Preparation of synergistic modification of soy protein isolate-casein complex (SPI-CS) This embodiment details the specific operation steps of the synergistic modification method of the present invention, and simultaneously prepares control group and single-treatment group samples for performance comparison.

[0026] 1.1 Raw Materials and Reagents Soy protein isolate (SPI), purchased from Zhengzhou Yukong Biotechnology Co., Ltd.

[0027] Casein (CS) was purchased from Wankang Biotechnology Co., Ltd.

[0028] Sodium hydroxide and hydrochloric acid were both analytical grade.

[0029] The water used in the experiment was deionized water.

[0030] 1.2 Main Instruments and Equipment Magnetic stirrer Precision pH meter Ultrasonic cell disruptor (probe type) high-speed centrifuge 4℃ refrigerator 1.3 Sample preparation steps ① Mixing and Hydration: Accurately weigh 2.5 g each of SPI and CS, place them in a 250 mL beaker, add 100 mL of deionized water to prepare a composite dispersion with a total protein concentration of 5% (w / v) and a SPI:CS mass ratio of 1:1. Place the beaker on a magnetic stirrer and stir continuously at 800 rpm for 2 hours at room temperature (approximately 25°C) to fully wet and disperse the protein powder. Then, seal the beaker and transfer it to a 4°C refrigerator to stand overnight (12 hours) to allow the protein to fully hydrate and obtain a homogeneous initial dispersion.

[0031] ② Synergistic Modification Treatment (PU Group): Take the initial dispersion described above and precisely adjust its pH to 11.0 with 1 mol / L NaOH solution while stirring, and continue stirring for 1 hour under these conditions. Subsequently, while maintaining the pH at 11.0, place the sample beaker in an ice-water bath and process it using an ultrasonic cell disruptor. Set the ultrasonic power to 400 W, use the pulse mode (5 seconds on, 5 seconds off), and the total processing time is 10 minutes. After processing, adjust the pH of the system back to 7.0 with 1 mol / L HCl solution.

[0032] ③ Post-processing: Centrifuge the modified dispersion at 4℃ and 8000 r / min for 10 minutes, collect the supernatant to obtain the synergistically modified SPI-CS complex of the present invention, which can be directly used for subsequent performance characterization (see Example 2), or freeze-dried to obtain a solid powder product for later use.

[0033] ④ Preparation of control samples: Prepare in parallel following the same steps. Control group (C): Initial dispersion, without any modification treatment.

[0034] Sonication group (U): The initial dispersion (pH≈7) was subjected to the same sonication treatment as the PU group (400W, 10min, ice bath).

[0035] Individual pH shift group (P): The initial dispersion was subjected to the same pH shift treatment (pH 11, 1 h, back to 7), but without sonication.

[0036] ⑤ Preparation of control samples for single protein dispersions: Prepare in parallel following the same steps. SPI and CS single protein dispersions with a protein concentration of 5% (w / v) were prepared using the same preparation steps. The SPI and CS single protein dispersions were then treated in parallel according to the same treatment grouping method (C, U, P, PU) for subsequent comparison.

[0037] 1.4. Explanation of Process Parameter Ranges This invention is not limited to the specific parameters mentioned above. Its key process steps can be implemented within the following ranges, all of which can achieve the goal of improving the interfacial function of the SPI-CS complex: Protein system: Single SPI and CS protein dispersions or SPI / CS dual protein dispersions can be prepared. The mass ratio of SPI to CS in the dual protein can be between 9:1 and 1:9, preferably 1:1; the total protein concentration can be between 4% and 8% (w / v), preferably 5%. Suitable protein concentration and ratio are the basis for functional improvement. A protein concentration in the range of 4% to 8% (w / v) can ensure that the protein system has good fluidity, avoid local aggregation due to excessive concentration, and at the same time meet the effective protein content required for subsequent modification treatment.

[0038] Pretreatment: During dispersion by stirring, the speed can be between 500 and 1000 rpm, and the time can be between 1 and 3 hours to ensure sufficient wetting. The hydration process is carried out at 4°C for 12 to 24 hours. Sufficient stirring and hydration ensure uniform molecular dispersion. Controlling the stirring speed and time within the above range is beneficial for breaking down protein powder agglomerates and promoting the entry of water molecules into the protein's internal structure. Low-temperature static hydration can reduce non-specific aggregation between protein molecules and improve the uniformity of the dispersion.

[0039] Ultrasonic treatment: The ultrasonic power can be between 200 and 600 W, and the treatment time can be between 5 and 20 minutes. Continuous or pulsed modes can be used. Treatment should preferably be carried out in an ice-water bath to control the temperature rise. The ultrasonic cavitation effect can effectively disrupt the weak interactions between protein molecules within the above power and time range, causing partial unfolding of the protein structure, thereby improving solubility and reactivity; at the same time, using an ice-water bath can prevent irreversible protein denaturation caused by the local high temperature generated by ultrasound.

[0040] pH shift treatment: The concentrations of the alkali (e.g., NaOH) and acid (e.g., HCl) solutions used to adjust the pH can be 1-3 mol / L. The target pH value can be in the alkaline range of 10.0 to 12.0, preferably 11.0. The continuous stirring time at this pH can be 1 to 2 hours. Under alkaline conditions, the protein molecule charge increases, and electrostatic repulsion is enhanced, which can promote the unfolding of the protein structure and expose the internal hydrophobic groups; subsequently, when the pH is adjusted back to neutral, the protein forms a new molecular conformation during the refolding process, thereby improving its functional properties.

[0041] Synergistic treatment: Maintaining the alkaline pH (10.0-12.0) is crucial for generating the synergistic effect during sonication. After sonication, the system pH is adjusted back to the neutral range (e.g., 6.8-7.2). Sonication at pH (10.0-12.0) further amplifies the unfolding of the protein molecule structure, making it easier for the ultrasonic cavitation effect to act on the internal structure of the protein, thereby enhancing the modification effect. This synergistic effect is conducive to the formation of a more stable protein molecule conformation, improving its solubility and functional properties. After adjusting back to the neutral pH (6.8-7.2), a new stable structure is formed.

[0042] Post-processing: When collecting soluble components by centrifugation, the rotation speed can be 7000 to 12000 r / min, the time can be 5 to 15 minutes, and the temperature is preferably 4℃. Centrifugation can effectively remove undissolved large particle aggregates, while avoiding protein re-aggregation due to excessive centrifugal force.

[0043] Example 2: Performance Characterization of the Modified Composite 2.1 Physical and chemical properties 2.1.1 Solubility The protein concentration of the supernatant was determined using the BCA protein concentration assay kit (Solarbio Technology Co., Ltd., Beijing, China). The sample solubility was calculated using the following formula.

[0044] (1) 2.1.2 Particle size and Zeta potential The sample was diluted to 3 mg / mL, and the particle size distribution and potential changes of the sample were determined using a nanoparticle size and Zeta potential analyzer (ZS90, Malvern Pana Technology Co., Ltd., USA).

[0045] 2.1.3 Surface hydrophobicity Dilute the sample to 1 mg / mL and prepare a 1 mg / mL bromophenol blue reagent. Mix 1 mL of protein solution with 200 μL of bromophenol blue solution and centrifuge at 10000 r / min for 10 min. Using bromophenol blue reagent as a blank control, measure the absorbance at 595 nm and calculate the surface hydrophobicity of the sample according to the following formula.

[0046] Surface hydrophobicity (μg) = 200*(A0-A) / A0(2) 2.2 Structural Characteristics 2.2.1 CD Spectrum The sample was diluted to 0.1 mg / mL with deionized water, using deionized water as a blank control. The CD spectrum was measured using a circular dichroism spectrometer (Chirascan, Applied Photonics Ltd, London, United Kingdom). The spectral scan range was 190–260 nm, the bandwidth was 1 nm, and the sampling time per point was 0.6 s. The secondary structure of the protein was calculated using CDNN software.

[0047] 2.3 Functional Characteristics 2.3.1 Molecular flexibility Dilute the sample to 5 mg / mL for later use. Prepare a 1 mg / mL trypsin solution using 0.05 mol / L Tris-HCl buffer. Mix 50 μL of the trypsin solution with 800 μL of the diluted sample solution and incubate at 37 °C for 15 min. Then, add 800 μL of trichloroacetic acid to terminate the reaction. After centrifugation, measure the absorbance of the supernatant at 280 nm.

[0048] 2.3.2 Emulsifying properties Dilute the protein solution to 10 mg / mL. Mix 15 mL of the protein solution with 5 mL of soybean oil and homogenize using a high-speed homogenizer at 12000 rpm for 2 min. Mix 50 μL of the emulsion with 5 mL of SDS solution (0.1%) and measure its absorbance at 500 nm (A1). After standing for 10 min, measure its absorbance again (A2). The emulsifying activity index (EAI) and emulsifying stability index (ESI) are calculated using the following formulas.

[0049] (3) (4) In the formula, N is the dilution factor and c is the protein concentration (g / mL). The volume fraction of the oil phase (v / v) (25%) is used as the blank control, which is a blank SDS solution.

[0050] 2.3.3 Foaming properties Take 20 mL of sample and record the initial volume V0 in a 100 mL plastic graduated cylinder. Use a high-speed homogenizer to shear at 12000 rpm for 1 min, and record the total volume V1 after shearing. After standing for 30 min, read the total volume V2. Calculate the foaming capacity (FC) and foam stability (FS) of the sample according to the following formula.

[0051] (5) (6) 2.3.4 Interface Features The surface tension of the sample at the air-water interface was determined using an optical contact angle (OCA-25, German Data Physics Instruments Company). The sample was diluted to 2 mg / mL, and then a 5 μL droplet was added using a microsyringe. The change in surface tension was monitored over 2000 s, and the surface tension was calculated using the Yang-Laplace equation.

[0052] 2.4 Statistical Analysis All experiments were independently repeated three times. Data were statistically analyzed using SPSS, and significant differences between groups were assessed using one-way ANOVA. Statistical charts were generated using Origin (2022). A p-value < 0.05 was considered statistically significant.

[0053] The key results of systematic testing of the samples obtained in Example 1 are summarized below: 2.5Physical and chemical properties 2.5.1 Solubility like Figure 1 As shown, the solubility of SPI, CS, and SPI-CS under different treatment conditions exhibited significant differences (P < 0.05). The solubility of untreated SPI was only 36.19%, while the initial solubility of SPI-CS significantly increased to 55.30% after being combined with highly soluble CS (P < 0.05). For the SPI and SPI-CS systems, ultrasound, pH shift, and combined treatment all led to a significant increase in solubility (P < 0.05), with the combined treatment showing the most significant solubilizing effect, achieving a solubility of SPI-CS of 92.95%, significantly higher than the other groups. The synergistic effect of ultrasound and pH shift resulted in a more pronounced enhancement of protein solubility.

[0054] 2.5.2 Particle size and Zeta potential like Figure 2 The effects of different treatment conditions on the particle size of SPI, CS, and SPI-CS are shown. The particle sizes of untreated SPI and CS were 595.87 nm and 546.31 nm, respectively, while the particle size of the SPI-CS mixed protein increased significantly (P<0.05) to 639.63 nm, exhibiting a distinct bimodal distribution. Compared with the control group, the particle sizes of all proteins treated with ultrasound and pH shift were significantly reduced (P<0.05), and the combined treatment led to a further reduction in particle size. After the combined treatment, the original bimodal distribution of the SPI-CS complex became blurred, and the curve shifted significantly towards smaller particle sizes (down to 333.46 nm). The synergistic effect of the combined treatment allowed the protein structure to fully unfold, resulting in smaller particle sizes and a more uniform particle distribution.

[0055] like Figure 3 As shown, all samples exhibited negative Zeta potentials. The untreated SPI had the lowest absolute Zeta potential of -32.48 mV, while the composite system with added CS showed a higher absolute potential of -36.29 mV in the initial state. Compared with the untreated group, the absolute Zeta potentials of the protein samples after combined treatment were significantly increased (P<0.05), with the SPI-CS system showing the highest absolute potential of -39.26 mV. This indicates that the treatment significantly enhanced the stability of the protein system. This may be because pH shifting treatment causes protein structure unfolding, exposing more charged groups inside the protein, enhancing the electrostatic repulsion between protein particles, and inhibiting aggregate formation. Sonication treatment can disrupt the protein structure and alter the surface charge distribution of molecules.

[0056] 2.5.3 Surface hydrophobicity like Figure 4 As shown, the surface hydrophobicity changes of SPI, CS, and the SPI-CS complex under different treatment conditions are illustrated. Under untreated conditions, SPI exhibits the highest surface hydrophobicity, while the SPI-CS complex falls between the two individual components. Compared to the untreated group, the surface hydrophobicity of all samples significantly increased after ultrasonic and pH shift treatments. Among these, the combined treatment further increased the surface hydrophobicity of SPI and SPI-CS proteins (P < 0.05), showing the most significant increase.

[0057] 2.6 Structural Analysis 2.6.1 CD Spectrum like Figure 5 As shown, the CD spectra of SPI, CS, and SPI-CS composite systems under different treatments all exhibited typical protein secondary structure characteristics in the 190–260 nm wavelength range, but their peak intensities differed significantly. Compared with the untreated group, the negative peak intensities of each sample changed after different treatments, indicating that the protein secondary structure composition was rearranged.

[0058] like Figure 6As shown, further evidence indicates that the modified samples exhibited decreased α-helix and β-turn content, while increased β-sheet and random coil content. The combined treatment showed the most significant effect. In SPI-CS, the α-helix content decreased from 9.35% in the control group to 7.35%, and the β-turn content decreased from 19.38% to 18.45%; simultaneously, the β-sheet content increased from approximately 39.56% to 41.29%, and the random coil content increased from approximately 31.71% to 32.91%. Similar trends were observed in both the SPI and CS systems. This suggests that the original ordered structure was disrupted, promoting the formation of β-sheets and random coils, leading to a more disordered conformational shift in the protein.

[0059] 2.7 Functional Characteristics 2.7.1 Molecular flexibility like Figure 7 As shown, there were significant differences in molecular flexibility among different protein systems (P < 0.05). Under control conditions, the molecular flexibility of SPI and CS were 0.82 and 0.78, respectively, while that of SPI-CS was 0.80. Compared with the control group, the molecular flexibility of each protein system was significantly improved after different treatments (P < 0.05), with the combined treatment showing the most significant improvement. The flexibility of SPI-CS increased to 0.87 after combined treatment. This may be because sonication may disrupt non-covalent interactions such as hydrogen bonds and van der Waals forces, thereby breaking down the rigid structure of proteins and increasing molecular flexibility. pH shift treatment causes changes in the secondary structure of proteins, including a decrease in α-helices and an increase in the number of random coils, making the amide bonds in the molecule more easily recognized by trypsin, enhancing the protein-trypsin interaction, and thus improving flexibility.

[0060] 2.7.2 Emulsifying properties like Figure 8 As shown, under untreated conditions, the EAI of SPI was 48.43 m² / g, significantly higher than that of CS (36.26 m² / g), while the EAI of the SPI-CS complex was 47.13 m² / g. After different treatments, the EAI of each protein system significantly increased (P < 0.05). Specifically, the EAI of SPI-CS increased to 54.44 m² / g after co-treatment. This may be because under alkaline conditions, proteins may carry more negative charges, which helps proteins to spread and diffuse more uniformly and effectively at the oil-water interface. Ultrasonic treatment can improve protein solubility and reduce particle size, promote interfacial rearrangement and interactions, and improve the emulsifying properties of proteins. Regarding emulsifying stability, the ESI of SPI-CS after co-treatment was 82.69%, significantly higher than that of the untreated group (49.96%), indicating that the modification treatment significantly enhanced the stability of the interfacial adsorption layer.

[0061] 2.7.3 Foaming Characteristics like Figure 9 As shown, under untreated conditions, the FC and FS of SPI were 71.43% and 53.33%, respectively, significantly higher than those of CS (53.33% and 36.96%), while the FC and FS of SPI-CS were 52.94% and 42.31%, respectively. After modification, the FC and FS of all systems were significantly improved. Among them, the FC and FS of the combined treatment group SPI-CS reached 93.75% and 46.77%, respectively, which were 77.09% and 10.54% higher than those of the untreated group, indicating that the combined treatment significantly enhanced the stability of the foam structure. The significant improvement in foaming performance may be closely related to the protein structural rearrangement induced by the modification treatment.

[0062] 2.7.4 Interfacial Tension like Figure 10 As shown, the interfacial tension of all protein systems decreased rapidly within the initial 0–100 s, then gradually leveled off, reaching equilibrium at approximately 1500–2000 s. After equilibrium was reached, the interfacial tension of the SPI control group was 51.31 mN / m, higher than that of the CS group (46.00 mN / m). Modification treatment further reduced the equilibrium interfacial tension. Specifically, the interfacial tension of SPI-CS after combined treatment decreased to 40.94 mN / m, a 9.38% decrease compared to the control group. The cavitation effect of ultrasonic treatment weakened protein aggregation behavior and promoted its diffusion and adsorption at the interface. pH shift, by altering the charge state and spatial conformation of the protein, exposed more hydrophobic regions. Furthermore, the increased molecular flexibility of the modified protein also enhanced its adsorption force at the interface. The combined treatment showed a significant synergistic effect: pH shift modulated the charged properties of the protein, while ultrasonic cavitation further disrupted its aggregation structure, jointly promoting the reduction of interfacial tension and achieving the lowest equilibrium tension among all samples.

[0063] The results of Examples 1 and 2 together demonstrate that the specific synergistic process of "ultrasonic treatment under alkaline pH conditions" described in this invention is a sufficient and necessary condition for the formation of the unique structure (see 2.6 Structural Analysis) and the achievement of the superior functions (see 2.7 Functional Properties) of the SPI-CS complex. The experimental results of the comparative groups (Group U and Group P) prove that without any of the conditions in the synergistic step (alkaline environment or ultrasound), it is impossible to simultaneously obtain the final product with comprehensive and synergistic improvements in solubility, interfacial tension, emulsification, and foaming properties. Therefore, there is a direct and necessary causal relationship between the synergistic modification method defined in claims 1-9 and the complex with the aforementioned comprehensive superior properties claimed in claim 10.

[0064] The above results indicate that the addition of CS initially improved the dispersibility of SPI through steric hindrance, significantly enhancing the solubility of the composite system. Furthermore, pH shifting combined with ultrasonic treatment synergistically increased the solubility of SPI-CS and induced a transformation from an ordered structure to a flexible disordered conformation, thereby significantly enhancing the spatial flexibility and surface hydrophobicity of the molecules. Force analysis demonstrated that hydrophobic interactions are the main driving force maintaining the dual-protein complex, while the combined treatment further strengthened the system's stability by optimizing charge distribution. Structural rearrangement and interaction regulation led to changes in functional properties, resulting in the modified SPI-CS composite system exhibiting significantly better interfacial functional characteristics in terms of interfacial tension, emulsification stability, and foaming performance compared to the initial composite state and the single component. The ultrasonic and pH shifting treatment provided by this invention is an efficient approach for constructing high-performance plant-animal complementary protein systems, providing a theoretical basis and technical support for developing foods with excellent interfacial functional properties. This synergistic modification method can effectively and synergistically improve the structure of the SPI-CS complex and comprehensively enhance its interfacial functional properties.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synergistic modification of soybean protein-casein complex, characterized in that, Includes the following steps: S1. Compounding and pretreatment: Soy protein isolate (SPI) and casein (CS) are mixed in a mass ratio selected from (9:1) to (1:9), dissolved in water, and a protein dispersion with a concentration of 4%-8% (w / v) is prepared. The mixture is stirred and hydrated at low temperature. S2. Alkali treatment: Adjust the pH of the protein dispersion obtained in step S1 to an alkaline range of 10.0-12.0, and continue stirring under this condition; S3. Ultrasonic treatment: Under the condition of maintaining a pH value of 10.0-12.0, the protein solution after step S2 is subjected to ultrasonic treatment; S4. pH Adjustment and Post-processing: Adjust the pH of the protein solution after step S3 to neutral, centrifuge, and collect the supernatant to obtain the modified SPI-CS complex.

2. The synergistic modification method according to claim 1, characterized in that, In step S1, the mass ratio of SPI to CS is 1:

1.

3. The method according to claim 1 or 2, characterized in that, In step S1, the concentration of the protein dispersion is 5% (w / v).

4. The method according to claim 1, characterized in that, In step S2, the pH value is adjusted to 11.

0.

5. The method according to claim 1, characterized in that, In step S2, the stirring time under alkaline conditions is 1-2 hours.

6. The method according to claim 1, characterized in that, In step S3, the power of the ultrasonic treatment is 200-600W, and the treatment time is 5-20 minutes.

7. The method according to claim 6, characterized in that, The ultrasonic treatment has a power of 400W and a treatment time of 10 minutes.

8. The method according to claim 1, characterized in that, In step S3, the ultrasonic treatment is performed under ice-water bath conditions.

9. The method according to claim 1, characterized in that, In step S4, the centrifugation conditions are: centrifugation at 4°C and a speed of 7000-12000 r / min for 5-15 minutes.

10. A soy protein isolate and casein complex, characterized in that, Prepared by the method according to any one of claims 1 to 9.

Images