A method for preparing a high emulsibility soybean lipophilic protein

Abstract

The application belongs to the technical field of soybean protein deep processing, and particularly relates to a preparation method of high-emulsifying soybean lipophilic protein. The application takes soybean lipophilic protein (SLP) as an object and physically modifies the SLP through alkaline pH deviation combined with cavitation jet processing. The method comprises the following steps: (1) preparing soybean lipophilic protein (2) treating the protein solution through alkaline pH deviation (3) treating through cavitation jet. The application has the advantages that through pH-induced reversible unfolding of the molecular structure, combined with the high-speed shearing, shock wave and turbulent effect generated by the cavitation jet, the original dense spherical structure of the SLP can be effectively broken, and the molecular flexibility and interface adsorption rate are increased. The emulsifying activity and emulsifying stability of the SLP prepared through the method are significantly enhanced, the problem of poor emulsifying performance of natural soybean protein caused by the dense molecular structure is successfully overcome, the functional properties of the SLP are improved, and the application range of the SLP in the food industry is expanded.

Description

Technical Field

[0001] This invention relates to the field of soybean deep processing technology, and mainly to a method for preparing highly emulsifiable soybean lipophilic protein. Background Technology

[0002] In food production and processing, emulsion stability is an important functional property. Through emulsification, a uniformly dispersed multiphase system is formed, effectively improving the smoothness of the texture, the gloss of the appearance, and the overall structural stability of food. This system is often used as a functional carrier for fat-soluble flavor substances, nutrients, pigments, and bioactive components, and is widely applied in beverages, sauces, desserts, baked goods, and protein substitutes, having a decisive impact on the sensory quality and shelf life of the final product.

[0003] Soybean lipophilic protein is an amphiphilic protein isolated from soybean protein isolate. This protein is a complex composed of various proteins, mainly including small amounts of denatured 7S and 11S globulins, and oil body proteins with molecular weights of approximately 16, 18, and 24 kDa. Emulsifying property refers to the ability of a protein to form a stable emulsion between two immiscible phases. In a mixed system, soybean protein spontaneously migrates to the oil-water interface, where its hydrophilic and hydrophobic regions directionally bind to the water and oil phases, respectively, effectively reducing interfacial tension. Simultaneously, protein molecules rearrange and cross-link at the interface, forming a stable adsorption film that prevents the re-aggregation of emulsified droplets, ensuring the homogeneity and stability of the emulsion. However, due to its large molecular weight, dense spatial structure, and compact globular conformation, the migration rate of soybean protein to the oil-water interface during emulsification is significantly limited. This structural characteristic prevents it from rapidly adsorbing and spreading at the interface, thus hindering the rapid formation of a stable interfacial film, and its emulsifying performance needs further improvement. Therefore, it is necessary to further investigate methods for modification and improvement.

[0004] Currently, protein modification methods are mainly categorized into chemical modification, physical modification, enzymatic modification, and genetic engineering modification. Among these, pH shifting is a simple and efficient protein modification strategy. When a protein is in an acidic or alkaline environment, the protonation of amino groups and the deprotonation of carboxyl groups cause the molecular side chains to carry a large number of positive / negative charges, disrupting the covalent bonds and non-covalent interactions that maintain the protein's spatial conformation. This leads to varying degrees of protein alteration, resulting in unfolding and the formation of a more flexible and resilient structure. Furthermore, when the protein's environment shifts from acidic or alkaline to neutral, the imbalance of internal forces triggers structural folding and remodeling, forming a "molten ball" state. This structural transformation is accompanied by changes in functional properties. Among numerous modification methods, cavitation jet treatment has emerged as a physical modification method. This technology can effectively remodel the molecular conformation and folding state of proteins, significantly improving their solubility, regulating aggregation behavior, and enhancing surface activity. Notably, many studies emphasize the synergistic application of multiple modification methods, as they often have a more significant effect on regulating protein structure and functional properties.

[0005] In summary, to improve the surface activity and strong affinity for lipids of soybean lipophilic proteins and optimize their application in stable emulsion systems, this invention aims to overcome the shortcomings of existing technologies by employing cavitation jet and alkaline pH shift treatment as modification methods to achieve targeted regulation of the structure and function of soybean lipophilic proteins, promote the in-depth development of the protein food field, and realize higher value transformation. Summary of the Invention

[0006] To address the aforementioned issues, this invention modifies the structure and function of soybean lipoprotein through cavitation jet combined with alkaline pH shift treatment. The modified soybean lipoprotein exhibits good surface activity and strong affinity for lipids, effectively improving the oxidative stability of emulsions.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0008] A method for preparing highly emulsifiable soybean lipophilic protein, characterized in that the method includes the following steps:

[0009] (1) First, defatted soybean flour was dispersed in deionized water at a ratio of 1:8 (w / v), and the pH was adjusted to 8.0 using 1.0 mol / L NaOH. The mixture was stirred at 25°C for 1 h and then centrifuged for 10 min (4°C, 3000 g). Subsequently, the supernatant was collected and 10 mmol / L Na2SO3 solution was added. The pH was then adjusted to 5.8 using 1 mol / L H2SO4, and the precipitate was collected after centrifugation at 8000 g for 5 min. The remaining supernatant was further adjusted to pH 5.0, heated in a 55°C water bath for 15 min, and 50 mmol / L NaCl solution was added. The pH was adjusted to 5.5 again, and the precipitate was obtained after centrifugation at 3000 g for 10 min. After freeze-drying, SLP was obtained.

[0010] (2) Weigh an appropriate amount of the SLP powder prepared above and add it to deionized water at a mass fraction of 0.5-5% (w / v) to prepare a protein solution with a concentration of 5-500 mg / mL. Stir overnight to allow it to fully hydrate. Then, perform pH adjustment on the obtained solution. Adjust the pH of the SLP solution to 2.0-12.0 with 1 mol / L HCl or 1 mol / L NaOH respectively, and stir magnetically for 30-90 min. (3) Use a cavitation jet machine to treat the above protein solution at pressures of 40, 80 and 120 MPa for 1-10 min respectively. Finally, adjust the pH back to 7 and stir for 1 h.

[0011] According to claim 1, the method for preparing highly emulsifiable soybean lipophilic protein is characterized in that: the preferred addition amount of SPI powder is 1%, and the protein solution concentration is 10 mg / mL.

[0012] According to claim 1, the method for preparing highly emulsifiable soybean lipophilic protein is characterized in that: the preferred pH condition is 12.0, and the preferred stirring time is 60 min.

[0013] According to claim 1, the method for preparing highly emulsifiable soybean lipophilic protein is characterized in that the preferred cavitation jet treatment pressure is 80 MPa and the preferred treatment time is 3 min. Attached Figure Description

[0014] Figure 1 The process for preparing modified soybean lipophilic protein with high emulsification properties involves using low-temperature defatted soybean flour as raw material. Soybean lipophilic protein is obtained by separation and purification through steps such as pH adjustment, stirring, and centrifugation. The protein is then reconstituted, pH adjusted to 12.0, stirred, and subjected to cavitation jet treatment. Finally, the pH is adjusted back to 7.0 and stirred to obtain highly emulsifiable soybean lipophilic protein.

[0015] Figure 2The effects of different pressures (40, 80, and 120 MPa) cavitation jet-assisted pH shifting on the average particle size (A) and zeta potential (B) of SLP were analyzed. Here, SLP represents untreated soybean lipoprotein; pH 12 represents soybean lipoprotein treated with pH shifting; Cj1 represents soybean lipoprotein treated with a 40 MPa cavitation jet; pH 12-Cj1 represents soybean lipoprotein treated with a 40 MPa cavitation jet-assisted pH shifting; Cj2 represents soybean lipoprotein treated with an 80 MPa cavitation jet; pH 12-Cj1 represents soybean lipoprotein treated with an 80 MPa cavitation jet-assisted pH shifting; Cj3 represents soybean lipoprotein treated with a 120 MPa cavitation jet; and pH 12-Cj3 represents soybean lipoprotein treated with a 120 MPa cavitation jet-assisted pH shifting.

[0016] Figure 3 The effect of cavitation jet-assisted pH shifting at different pressures (40, 80, and 120 MPa) on the free thiol content of SLP was analyzed. Here, SLP represents untreated soybean lipoprotein; pH 12 represents soybean lipoprotein after pH shifting treatment; Cj1 represents soybean lipoprotein after cavitation jet treatment at 40 MPa; pH 12-Cj1 represents soybean lipoprotein after pH shifting treatment with cavitation jet at 40 MPa; Cj2 represents soybean lipoprotein after cavitation jet treatment at 80 MPa; pH 12-Cj1 represents soybean lipoprotein after pH shifting treatment with cavitation jet at 80 MPa; Cj3 represents soybean lipoprotein after pH shifting treatment with cavitation jet at 120 MPa; and pH 12-Cj3 represents soybean lipoprotein after pH shifting treatment with cavitation jet at 120 MPa.

[0017] Figure 4 To analyze the effect of pH shift assisted by cavitation jets at different pressures (40, 80, and 120 MPa) on the foam microstructure of SLP, different letters (af) represent significant differences. p <0.05): Wherein, SLP represents untreated soybean lipoprotein; pH 12 represents soybean lipoprotein after pH shift treatment; Cj1 represents soybean lipoprotein after treatment with 40 MPa cavitation jet; pH 12-Cj1 represents soybean lipoprotein after pH shift treatment assisted by 40 MPa cavitation jet; Cj2 represents soybean lipoprotein after treatment with 80 MPa cavitation jet; pH 12-Cj1 represents soybean lipoprotein after pH shift treatment assisted by 80 MPa cavitation jet; Cj3 represents soybean lipoprotein after treatment with 120 MPa cavitation jet; pH12-Cj3 represents soybean lipoprotein after pH shift treatment assisted by 120 MPa cavitation jet. Detailed Implementation

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0019] Example 1:

[0020] (1) Low-temperature defatted soybean flour was dispersed in deionized water at a feed-to-liquid ratio of 1:8 (w / v), and the pH was adjusted to 8.0 using 1.0 mol / L NaOH. The mixture was stirred at 25 °C for 1 h and then centrifuged for 10 min (4 °C, 3000 g). Subsequently, the supernatant was collected and 10 mmol / L Na2SO3 solution was added. The pH was then adjusted to 5.8 using 1 mol / L H2SO4, and the precipitate was collected after centrifugation at 8000 g for 5 min. The remaining supernatant was further adjusted to pH 5.0, heated in a 55 °C water bath for 15 min, 50 mmol / L NaCl solution was added, and the pH was adjusted to 5.5 again. Finally, the precipitate was obtained by centrifugation at 3000 g for 10 min, and then freeze-dried to obtain SLP.

[0021] Example 2:

[0022] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0023] (2) pH shift treatment: Weigh an appropriate amount of SLP powder, add deionized water to prepare a solution with a concentration of 10 mg / mL, and stir overnight to allow it to fully hydrate. Adjust the pH of the SLP solution to 12.0 with 1 mol / L HCl or 1 mol / L NaOH respectively. After magnetic stirring for 1 h, adjust the protein solution to pH 7.0 and stir for another 1 h. The sample is labeled as pH 12.

[0024] Example 3:

[0025] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0026] (2) Cavitation jet treatment: The protein solution was treated with a cavitation jet machine at a pressure of 40 MPa for 3 min, and the sample was named Cj1.

[0027] Example 4:

[0028] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0029] (2) Cavitation jet treatment: The pH of the protein solution was adjusted to 12.0 and maintained for 1 h, and cavitation jet treatment was carried out for 3 min at a pressure of 40 MPa.

[0030] (3) pH shift treatment: the pH was adjusted back to 7.0, stirred and equilibrated for 1 h, and the samples were recorded as pH 12-Cj1.

[0031] Example 5:

[0032] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0033] (2) Cavitation jet treatment: The protein solution was treated with a cavitation jet machine at a pressure of 80 MPa for 3 min, and the sample was named Cj2.

[0034] Example 6:

[0035] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0036] (2) Cavitation jet treatment: The pH of the protein solution was adjusted to 12.0 and maintained for 1 h, and cavitation jet treatment was carried out for 3 min at 80 MPa pressure.

[0037] (3) pH shift treatment: The pH was adjusted back to 7.0, and the mixture was stirred and equilibrated for 1 h. The samples were recorded as pH 12-Cj2.

[0038] Example 7:

[0039] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0040] (2) Cavitation jet treatment: The protein solution was treated with a cavitation jet machine at a pressure of 120 MPa for 3 min, and the sample was named Cj3.

[0041] Example 8:

[0042] (1) Extract soybean lipophilic protein, using the same method as in Example 1.

[0043] (2) Cavitation jet treatment: The pH of the protein solution was adjusted to 12.0 and maintained for 1 h, and cavitation jet treatment was carried out for 3 min at a pressure of 120 MPa.

[0044] (3) pH shift treatment: The pH was adjusted back to 7.0, and the mixture was stirred and equilibrated for 1 h. The samples were recorded as pH 12-Cj3.

[0045] The experimental results were further verified through experiments, and some indicators were measured as follows:

[0046] Test 1: Average particle size and Zeta potential.

[0047] The sample was diluted to 0.1 mg / mL, and the average particle size and zeta potential were determined using a particle size and potential analyzer at 25 °C. During the measurement, the refractive index of the continuous phase was set to 1.46, and the refractive index of the dispersed phase was set to 1.33. The measurements were repeated three times.

[0048] Test 2: Surface hydrophobicity (H0).

[0049] Different sample solutions were diluted to concentrations of 0.08, 0.16, 0.24, 0.32, and 0.40 mg / mL, respectively. Then, 20 μL of 8.0 mmol / L ANS was added to 4 mL of the diluted sample, and the solution was incubated at 25 °C for 15 min. Fluorescence intensity was measured using a fluorescence spectrophotometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. A linear fit was performed with protein concentration as the x-axis and fluorescence intensity as the y-axis, and H0 was calculated based on the slope of the fitted line.

[0050] Test 3: Determination of emulsifying activity (EAI) and emulsifying stability (ESI).

[0051] Take 9 mL of a 10 mg / mL sample and mix it with 1 mL of soybean oil. Homogenize the mixture using a high-speed homogenizer at 10000 r / min for 2 min. Take 40 μL of the resulting emulsion and dilute it 200 times with 0.1% SDS (this solution serves as a blank control). Use a UV spectrophotometer at 500 nm to measure the absorbance of the sample at 0 min and 30 min, respectively. Calculate EAI and ESI using the following formulas: Among them, A0 and A 30 The absorbance values ​​are 0 min and 30 min, respectively; C is the protein concentration (g / mL) in the solution before emulsion formation; 0.1 is the volume fraction of the oil phase in the emulsion; and D is the dilution factor of the sample.

[0052] The results of some of the indicators are as follows:

[0053] Test Result 1: The effects of different treatments on the average particle size and zeta potential of SLP were evaluated using a particle size and potential analyzer. Figure 2Particle size analysis showed that pH shift treatment significantly reduced SLP particle size through electrostatic repulsion (P<0.05). The cavitation jet treatment effect was pressure-dependent: 80 MPa treatment was optimal, as its shear force and high temperature / pressure efficiently dissociated the aggregates; 40 MPa treatment was insufficient, resulting in incomplete dissociation; while 120 MPa treatment, due to overtreatment, caused excessive protein collisions, inducing secondary aggregation and significantly increasing particle size. Zeta potential directly reflects the surface charge distribution of proteins, and its changes effectively characterize the structural changes of SLP. The absolute value of the SLP zeta potential showed an inverse trend with particle size. This is attributed to the unfolding and dissociation of the protein structure under modification conditions, exposing charged groups originally buried within the molecule to the surface, thus significantly increasing the SLP zeta potential (P<0.05).

[0054] Test Result 2: The effect of different treatments on the hydrophobicity (H0) of SLP surface was analyzed using a fluorescence spectrophotometer. Figure 3 Surface hydrophobicity reflects the degree of exposure of hydrophobic groups in protein molecules and indicates structural changes in proteins. SLP had the lowest H0 (832.47 ± 24.16). After cavitation and pH shift treatments, the H0 of the samples significantly increased to varying degrees (P < 0.05). This finding indicates that cavitation and alkaline pH conditions disrupt the intramolecular and intermolecular hydrophobic interactions of SLP molecules. As a result, hydrophobic groups originally buried inside the molecule migrated to the protein surface. Compared with single treatments, the combined treatment further increased the H0 level, indicating that the protein structure was more fully unfolded, and more hydrophobic sites were exposed.

[0055] Test Result 3: The effects of different treatments on the emulsifying activity index (EAI) and emulsifying stability index (ESI) of SLP were determined using a UV spectrophotometer. Figure 4 SLP's EAI (104.39 ± 3.75 m) 2 ·g -1 The lowest values ​​were observed for both the surface hydrophobicity (SLP) and the ESI (72.37 ± 7.8 min). This is likely due to the limited interface of the SLP, restricting its adsorption at the oil-water interface. Increased hydrophobicity contributes to the interfacial activity of the protein. With increased surface hydrophobicity, each treated sample rapidly adsorbed to the oil-water interface. Furthermore, protein emulsification was negatively correlated with rigid structure. The modification treatment resulted in a decrease in protein particle size, a reduction in α-helix content, and an increase in random coil content, leading to a looser and more flexible SLP structure. This facilitated the unfolding of the SLP at the interface and the formation of a stable adsorption layer, ultimately improving its emulsification performance. The pH 12-Cj2 value reached a maximum of 196.11 ± 14.45 min. 2 ·g -1ESI reflects the ability of a protein to form a stable emulsion within a certain time. Compared with SLP, the ESI of the modified proteins was significantly increased, reaching a maximum at pH 12-Cj2 (151.13 ± 8.53 min). This may be because the structural modification enabled SLP to form a more viscoelastic and mechanically strong interfacial film at the oil-water interface, thereby effectively inhibiting droplet coalescence and improving the overall stability of the emulsion.

[0056] The above embodiments are merely preferred embodiments of the present invention, intended to illustrate the technical solution of the present invention in detail, but are not intended to limit the scope of protection of the present invention in any way. Any equivalent substitutions, simple modifications, or improvements made by those skilled in the art based on the present invention without departing from the core idea of ​​the technical solution of the present invention should be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing highly emulsifiable soybean lipophilic protein, characterized in that, The method includes the following steps: First, defatted soybean flour was dispersed in deionized water at a ratio of 1:8 (w / v). The pH was adjusted to 8.0 using 1.0 mol / L NaOH. The mixture was stirred at 25°C for 1 hour and then centrifuged for 10 minutes (4°C, 3000g). The supernatant was collected and 10 mmol / L Na2SO3 solution was added. The pH was then adjusted to 5.8 using 1 mol / L H2SO4. The precipitate was collected after centrifugation at 8000g for 5 minutes. The remaining supernatant was further adjusted to pH 5.0 and heated in a 55°C water bath for 15 minutes. 50 mmol / L NaCl solution was added, and the pH was adjusted to 5.5 again. Finally, the precipitate was obtained by centrifugation at 3000g for 10 minutes and then freeze-dried to obtain SLP. Weigh an appropriate amount of the SLP powder prepared above and add it to deionized water at a mass fraction of 0.5-5% (w / v) to prepare a protein solution with a concentration of 5-500 mg / mL. Stir overnight to allow it to fully hydrate. Then, perform pH shift treatment on the resulting solution. Adjust the pH of the SLP solution to 2.0-12.0 with 1 mol / L HCl or 1 mol / L NaOH respectively, and stir magnetically for 30-90 min. The protein solution was treated with a cavitation jet machine at pressures of 40, 80 and 120 MPa for 1 to 10 minutes, and then the pH was adjusted back to 7 and stirred for 1 hour.

2. The method for preparing highly emulsifiable soybean lipophilic protein according to claim 1, characterized in that: The preferred addition amount of SPI powder is 1%, and the protein solution concentration is 10 mg / mL.

3. The method for preparing highly emulsifiable soybean lipophilic protein according to claim 1, characterized in that: The preferred pH value is 12.0, and the preferred stirring time is 60 min.

4. The method for preparing highly emulsifiable soybean lipophilic protein according to claim 1, characterized in that: The preferred pressure for cavitation jet treatment is 80 MPa, and the preferred treatment time is 3 min.

Images