Zein-spi complex protein particles, and preparation method and application thereof

Abstract

The application belongs to the technical field of food processing, and particularly relates to a Zein-SPIs composite protein particle and a preparation method and application thereof. The preparation method of the composite protein particle is as follows: corn alcohol-soluble protein and soybean protein isolate are added into an ethanol aqueous solution for dispersion, then lye is added to adjust the pH of the mixed solution to 9-12, the protein structure is fully unfolded and dissolved through stirring reaction, insoluble substances are removed through centrifugation, an acid solution is added to adjust the pH to neutral, dialysis is performed after ethanol is removed through rotary evaporation, freeze-drying is performed, grinding and sieving are performed, and a Zein-SPIs composite protein particle powder is obtained. The Zein and SPIs are modified through non-covalent interaction, the modification target can be achieved without destroying the protein structure and nutritional ingredients, and the solubility of the composite protein particle and the stability of the Pickering emulsion are significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a Zein-SPIs composite protein particle, its preparation method, and its application. Background Technology

[0002] Pickering emulsions are systems that use solid particles instead of traditional surfactants to stabilize the aqueous and oil phases of an emulsion. Compared with emulsions using traditional surfactants, picking emulsions have the following advantages: 1) good biocompatibility, low toxicity, and minimal harm to humans; 2) high stability, thick interfacial layer, and minimal impact from the external environment; 3) small dosage and low cost; 4) various functional materials can be derived from this emulsion, enriching its application environment and scope. Currently, the solid particles used to stabilize picking emulsions are mainly inorganic particles and chemically synthesized particles. Although these solid particles can stabilize picking emulsions well, their biocompatibility is still relatively poor, limiting their application in food. Protein particles are one of the most commonly used solid particles and are currently a research hotspot in the preparation of Pickering solid particles widely used in food. Due to differences in their sources and extraction and separation methods, biomolecules have significant differences in molecular weight, particle size, and functional properties. How to obtain the desired solid particles through the simplest preparation and modification methods is a pressing problem to be solved in the food industry.

[0003] In recent years, the commercialization of plant proteins has become a hot topic in both scientific research and industrial applications. Among various plant proteins, zein has the outstanding advantage of being widely available, and also possesses characteristics such as reusability, biodegradability, and good biocompatibility. Soy protein isolates (SPIs), with their high yield, are widely used to replace animal proteins such as milk protein and egg white, exhibiting excellent emulsifying, gelling, film-forming, and foaming properties in food processing. However, zein can only dissolve in a specific ratio of ethanol-water solution, exhibiting strong hydrophobicity and poor water solubility; soy protein isolates also cannot be completely dissolved in water. This characteristic greatly limits their application in many areas of the food industry. To overcome this predicament, improve the solubility of zein, and broaden its application range, scholars at home and abroad have conducted extensive research on the modification of zein using chemical, enzymatic, and physical methods. However, the methods currently used all have certain limitations and cannot achieve the ideal balance of both. Summary of the Invention

[0004] In view of the shortcomings and deficiencies of the existing technology, the primary objective of this invention is to provide a method for preparing Zein-SPIs complex protein particles.

[0005] Another object of the present invention is to provide a Zein-SPIs composite protein particle prepared by the above method.

[0006] Another object of the present invention is to provide the application of the above-mentioned Zein-SPIs complex protein particles in Pickering emulsion.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for preparing Zein-SPIs complex protein particles includes the following preparation steps:

[0009] Zein and soy protein isolate (SPIs) were dispersed in an ethanol-water solution. Then, an alkaline solution was added to adjust the pH of the mixed solution to 9-12. The mixture was stirred to fully expand and dissolve the protein structure. The insoluble matter was removed by centrifugation. An acid solution was added to adjust the pH to neutral. The ethanol was removed by rotary evaporation, followed by dialysis to remove salts. The mixture was then freeze-dried, ground, and sieved to obtain Zein-SPIs composite protein particle powder.

[0010] Further, the mass ratio of zein to soy protein isolate is 1:0.1-1.2. More preferably, the mass ratio of zein to soy protein isolate is 1:0.1-0.2. Most preferably, the mass ratio of zein to soy protein isolate is 1:0.2.

[0011] Furthermore, the mass-to-volume ratio (w / v, g / ml) of the zein to the aqueous ethanol solution is 0.5-2%.

[0012] Further, the ethanol-water solution is an ethanol-water solution with an ethanol volume fraction of 40-60%. More preferably, it is an ethanol-water solution with an ethanol volume fraction of 40-50%. Most preferably, it is an ethanol-water solution with an ethanol volume fraction of 50%.

[0013] Furthermore, the alkaline solution is a 1-5 mol / L NaOH solution.

[0014] Furthermore, the stirring reaction time is 0.5-3 hours.

[0015] Furthermore, the centrifugation refers to centrifugation at 5000-10000×g for 10-30 minutes.

[0016] Furthermore, the acid solution is a 1-5 mol / L hydrochloric acid solution.

[0017] Furthermore, the dialysis desalination refers to the removal of salts by dialysis using a 3500 kDa dialysis bag.

[0018] Furthermore, the grinding and sieving refers to passing the material through a 100-mesh sieve after grinding.

[0019] A Zein-SPIs complex protein particle was prepared by the above method.

[0020] Application of the aforementioned Zein-SPIs complex protein particles in Pickering emulsion.

[0021] Furthermore, the method of application is as follows:

[0022] Zein-SPIs complex protein particles were added to deionized water and mixed and dispersed, then added to the oil phase and homogenized to obtain Pickering emulsion.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] (1) This invention uses non-covalent interactions to co-modify zein and soy protein isolates (SPIs). This method can achieve the modification goal without destroying the protein structure and nutritional components. This significantly improves the solubility of the composite protein particles and the stability of Pickering emulsions.

[0025] (2) This invention further establishes the solvent conditions and the mass ratio of Zein to SPIs for the preparation of Zein-SPIs composite protein particles. The emulsion stabilized by the composite protein particles prepared using a 50% ethanol aqueous solution exhibits uniform droplets and high stability. The droplet size of the emulsion at different Zein:SPIs ratios first decreases and then increases in the range of 1:0.1 to 1:1.2, reaching its minimum at a ratio of 1:0.2. At this ratio, the synergistic effect of the two proteins significantly improves the emulsification performance. Attached Figure Description

[0026] Figure 1 and Figure 2 The images show the microscopic and external views of the Pickering emulsions that stabilize the Zein-SPIs complex protein particles obtained under different ethanol concentration solvent conditions in Example 1.

[0027] Figure 3 and Figure 4 The images show the appearance and particle size distribution of the Zein-SPIs composite protein solution after pH adjustment to 12 (A1), neutralization of the mixture solution to neutral (pH=7) (A2), and rotary evaporation to remove ethanol (A3) during step (1) of Example 1.

[0028] Figure 5The images show the appearance of the Zein-SPIs composite protein particles obtained under different Zein and SPIs mass ratios in Example 2, as well as the appearance of single Zein (1:0) and SPIs (0:1) dissolved in pure water.

[0029] Figure 6 The images show gel electrophoresis diagrams of Zein-SPIs composite protein particles and individual Zein and SPIs obtained under different Zein and SPIs mass ratios in Example 2.

[0030] Figure 7 The images show SEM images of Zein-SPIs composite protein particles and single Zein self-assembled particles (after being cycled at the same pH value) obtained under different Zein and SPIs mass ratios in Example 2.

[0031] Figure 8 The figure shows the water contact angle test results of Zein-SPIs composite protein particles and single Zein particles (untreated) obtained under different Zein and SPIs mass ratios in Example 2.

[0032] Figure 9 Microscopic images of stable Pickering emulsions of Zein-SPIs composite protein particles and single Zein particles (untreated) obtained under different Zein and SPIs mass ratios in Example 2. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0034] In the following examples, the solubility of the protein / complex protein particles was determined by gravimetric analysis:

[0035] After dissolving the protein / complex protein particles in water / ethanol, centrifuge (8000×g, 24℃, 10 min) to separate the precipitate. Dry the precipitate (40℃, 24 h). By weighing the precipitate before and after drying and determining the mass of soluble substances, the solubility of the protein / complex protein particles in water / ethanol can be calculated.

[0036] The calculation formula is as follows:

[0037] ×100,

[0038] Where: M1—total protein mass (g);

[0039] M2 — Protein mass (g) in the precipitate after centrifugation.

[0040] Example 1

[0041] (1) Preparation of Zein-SPIs complex protein particles:

[0042] Zein and SPIs were dissolved in an ethanol-water solution at a mass ratio of 1:1, maintaining the concentration of both Zein and SPIs at 1% (w / v). The pH of the mixture was adjusted to 12 using a 5 mol / L NaOH solution, followed by stirring for 1 h to allow the protein structure to fully develop. The protein mixture was then centrifuged at 8000×g and 24℃ for 20 min to remove insoluble components. The mixture was then neutralized to neutral (pH=7) with a 1 mol / L HCl buffer solution and stirred for 1 h. The supernatant was then rotary evaporated at 50℃ to remove ethanol, followed by freeze-drying to obtain a protein complex powder. This powder was then ground and passed through a 100-mesh sieve to obtain Zein-SPIs composite protein particle powder, which was stored in a desiccator for later use.

[0043] The solubility of Zein and SPIs in aqueous ethanol solutions of different concentrations used in this embodiment is shown in Table 1 below.

[0044] Table 1. Solubility of Zein and SPs in ethanol at different concentrations

[0045]

[0046] As shown in Table 1, the solubility of Zein increases with increasing ethanol concentration. In lower concentrations of ethanol-water solutions, the water content is higher, resulting in stronger polarity. The interaction between the hydrophobic groups of Zein and water molecules is weaker, thus hindering its dissolution. As the ethanol concentration increases, the polarity of the solution gradually decreases, making the solution closer to the solubility state of zein. This enhances the interaction between zein and the solvent, leading to increased solubility. Soy protein isolates (SPIs) are hydrophilic biomolecules and are more readily soluble in highly polar water. As the ethanol concentration increases, the polarity of the solution gradually decreases, increasing the polarity difference between the SPIs and the solution. Furthermore, according to the principle of "like dissolves like," the greater the polarity difference, the lower the solubility of the protein in the solution. Therefore, the solubility of SPIs in ethanol concentrations shows a trend of first increasing and then decreasing.

[0047] (2) Preparation of Pickering emulsions with stable Zein-SPIs complex protein particles obtained under different ethanol concentration solvent conditions:

[0048] 0.1 g of the composite protein particles (prepared using 40%, 50%, and 60% ethanol as solvents) were added to 20 mL of deionized water to form composite solutions. 20 mL of the composite solution was mixed with 1 mL of soybean oil, and an O / W Pickering emulsion was prepared using a homogenizer with the following parameters: homogenization speed of 10,000 rpm and homogenization time in an ice-water bath for 1 min. All emulsions were stored at 25°C for subsequent analysis.

[0049] The microscopic and morphological images of the stable Pickering emulsions of Zein-SPIs complex protein particles obtained under different ethanol concentration solvent conditions in this embodiment are shown below. Figure 1 and Figure 2 As shown in the figure (A: 40% ethanol; B: 50% ethanol; C: 60% ethanol). According to... Figure 1 The droplet size analysis reveals that the emulsion stabilized by composite protein particles prepared with 50% ethanol produces more uniform droplets with smaller particle sizes, while the emulsion stabilized by composite protein particles prepared with 60% ethanol produces larger droplets with lower density and poorer stability. This is further supported by... Figure 2 The emulsion appearance diagram shows that the emulsion with the composite protein particles prepared using 50% ethanol is the whitest, indicating that the Zein-SPIs complex forms a stable interfacial film at the oil-water interface, effectively encapsulating and dispersing oil droplets and preventing separation of the oil and water phases, resulting in higher emulsion stability. The second most stable emulsion is prepared with 40% ethanol, followed by 60% ethanol. Therefore, 50% ethanol was used as the solvent to prepare the composite protein particles in subsequent experiments.

[0050] In step (1) of this embodiment, the pH value was adjusted to 12 (A1), the mixture solution was neutralized to neutral (pH=7) (A2), and the appearance and particle size distribution of the composite protein solution after rotary evaporation to remove ethanol (A3) during the preparation of composite protein particles using 50% ethanol as a solvent are shown in the following figures. Figure 3 and Figure 4 As shown.

[0051] according to Figure 3 , Figure 4 As can be observed from the solution and line graph, the average particle size gradually increases with each experimental step, and the amount of precipitate also increases. When the solution pH is 12, the average particle size is about 400 nm. This is because, in an alkaline environment, acidic groups such as carboxyl groups in protein molecules dissociate in large numbers, acquiring more negative charges. The electrostatic repulsion between protein molecules is enhanced, causing them to be fully stretched and forming a relatively loose state, at which point the protein molecules are relatively small. In addition, a strongly alkaline environment also disrupts some non-covalent bonds between protein molecules, such as hydrogen bonds and hydrophobic interactions, thereby causing protein molecules to separate from each other and promoting dissolution, thus resulting in smaller particle sizes.

[0052] As the pH is adjusted from 12 to 7, the average particle size is around 800 nm. The number of charges on the protein molecules decreases, the electrostatic repulsion decreases, and the molecular conformation will shrink and curl to a certain extent, exposing more hydrophobic regions. This makes it easier for protein molecules to approach and aggregate, resulting in an increase in particle size and the appearance of a small amount of precipitation in the solution. At the same time, disulfide bonds may break and recombine during the pH change. The newly formed disulfide bonds may cross-link the protein molecules, further promoting the aggregation of protein molecules and thus increasing the particle size.

[0053] After rotary evaporation, the average particle size increased again, and a large amount of precipitate was produced. This is because the rotary evaporation process, at 50°C, promoted the volatilization of ethanol. However, the volatilization of ethanol led to a gradual increase in the concentration of the protein solution, and the zein, which was originally soluble in ethanol, also re-aggregated due to decreased solubility. This increased the probability of collisions between protein molecules, accelerating protein aggregation and further increasing the particle size. In addition, two peaks appeared in the particle size distribution after rotary evaporation. Studies have shown that SPIs have two peaks in their particle size distribution, namely 7S globulin and 11S globulin, while Zein's particle size distribution is mostly single-peaked. This indicates that the two proteins bind to each other during pH cycling. The change from a single peak to a double peak in Zein is not due to Zein self-assembly.

[0054] Example 2

[0055] Zein and SPIs were dispersed in a 50% ethanol aqueous solution at specified mass ratios (1:0.1, 1:0.2, 1:0.5, 1:1, 1:1.2), while maintaining a constant Zein concentration of 1% (w / v). The pH of the mixture was adjusted to 12 using a 5 mol / L NaOH solution, followed by stirring for 1 h to allow the protein structure to fully develop. The protein mixture was then centrifuged at 8000 × g and 24 °C for 20 min to remove insoluble components. The mixture was then neutralized with a 1 mol / L HCl buffer solution and stirred for 1 h. The supernatant was then rotary evaporated at 50 °C to remove ethanol, followed by freeze-drying to obtain a protein complex powder. This powder was then ground and passed through a 100-mesh sieve to obtain Zein-SPIs composite protein particle powder, which was stored in a desiccator for later use.

[0056] The solubility results of the composite protein particles and single Zein (1:0) and SPIs (0:1) in pure water obtained under different Zein and SPIs mass ratios in this embodiment are shown in Table 2 below.

[0057] Table 2. Solubility of composite protein particles at different Zein / SPIs ratios

[0058]

[0059] Table 2 shows that Zein has a solubility of only 4.5% in pure water, while SPIs have a solubility of 28.4%. This indicates that SPIs are more dispersed in solution than Zein from the outset. As the amount of SPIs in the system increases, the solubility of the complex protein particles also increases, from 27.7% to 34.8%, both higher than that of Zein. This is because Zein is rich in hydrophobic amino acids and has poor solubility; it mainly relies on pH cycling to change the charge state and structure to improve solubility. SPIs, on the other hand, contain more hydrophilic amino acids and have a certain degree of water solubility. During pH cycling, changes in intramolecular and intermolecular electrostatic interactions and hydrogen bonds further enhance the solubility of the complex protein particles, with a more pronounced effect than Zein.

[0060] The composite protein particles achieved a maximum solubility of 34.8%, higher than that of individual Zein and SPIs. This is because when Zein and SPIs form composite particles, intermolecular interactions occur, altering the original intermolecular forces. The hydrophilic groups of SPIs may interact with the hydrophobic groups of Zein, changing the environment around the Zein molecules. Some hydrophobic regions are shielded, reducing hydrophobic aggregation between Zein molecules and thus increasing the solubility of the composite protein particles. Furthermore, the pH cycle has a more complex effect on the structure and charge state of the composite protein particles during the complexation process. This may result in a looser overall structure of the composite protein particles, exposing more hydrophilic groups, and a more uniform charge distribution, thereby enhancing the interaction with water molecules and increasing the solubility of the composite protein particles in water, surpassing that of individual proteins.

[0061] The appearance of the composite protein particles obtained under different Zein and SPIs mass ratios, as well as the solubility and appearance of single Zein (1:0) and SPIs (0:1) in pure water in this embodiment are as follows: Figure 5 As shown. From Figure 5As can be seen, the Zein solution contains a significant amount of precipitate, with some particles suspended in the water, further indicating the poor solubility of Zein in water. The SPIs solution, on the other hand, is relatively clear, indicating better dispersibility, but also contains considerable precipitate, confirming its solubility is consistent with the data in Table 2. Furthermore, the figure clearly shows the solubility of different ratios of Zein-SPIs composite protein particles in water. The clearest solution is achieved with a ratio of 1:0.2. This is primarily because at this ratio, the composite particles have the smallest particle size and the lowest solids content. With smaller particle size and lower solids content, light is not significantly blocked or scattered, allowing it to penetrate the solution smoothly (good light transmittance), resulting in the highest solution clarity. Solutions with other ratios were more turbid and contained a small amount of sediment. This is because when the SPI ratio was below 1:0.2, the solubility was low, and Zein's strong hydrophobic aggregation tendency led to an increase in the particle size of the composite particles. While the solubility increased with the amount of SPI added when the SPI ratio was above 1:0.2, the solid content also increased. Furthermore, excessive SPIs might entangle themselves or excessively bind with Zein, forming larger aggregates and further increasing the particle size. These factors resulted in lower clarity in solutions with other ratios compared to the 1:0.2 ratio.

[0062] The gel electrophoresis images of the composite protein particles and individual Zein and SPIs obtained under different Zein and SPIs mass ratios in this embodiment are shown below. Figure 6 As shown (M: Maker; L1: Zein; L2: SPIs; L3: 1:0.1; L4: 1:0.2; L5: 1:0.5; L6: 1:1; L7: 1:1.2).

[0063] like Figure 6 As shown, Zein and SPIs exhibit different banding patterns, but both have characteristic bands in the 35-40 kDa range. Zein is biased towards 40 kDa, while SPIs are biased towards 35 kDa. Lanes 4-8 show that the two proteins combine to form complex protein particles through pH cycling. A new band appears between the two proteins' specific bands (35-40 kDa), and its position differs from the individual SPIs (L1) and Zein (L2) bands. This suggests that the two proteins most likely combined to form new Zein-SPIs complex protein particles in different proportions. As the amount of SPIs added increases, the color of the electrophoretic bands gradually deepens.

[0064] The formation of these composite particles is due to the unique interactions between the two protein subunits, involving bonding mechanisms such as hydrogen bonds, hydrophobic interactions, and ionic bonds in their chemical structure. The newly formed composite particles exhibit different migration rates in electrophoresis compared to the individual Zein or SPI subunit bands, thus appearing as new bands. This reflects the dynamic changes in the protein chemical structure and subunit composition within the mixed system.

[0065] When Zein is mixed with Spirulina Spectroradiata (SPIs), the environment surrounding Zein changes due to the hydrophilicity of SPIs and the polar groups they contain. At a higher SPI ratio (Zein:SPIs = 1:1.2), some hydrophobic regions of Zein may be surrounded or protected by the hydrophilic regions of SPIs, leading to changes in intermolecular interactions. The subunits may undergo some degree of depolymerization or rearrangement, manifesting as the appearance of new bands in electrophoresis with a certain degree of intensity reduction.

[0066] The polar groups (such as carboxyl and amino groups) in SPIs interact with the hydrophobic regions of Zein through hydrogen bonds and ionic bonds. The degree and manner of these interactions change with the mixing ratio. At a Zein:SPIs ratio of 1:0.1, the proportion of Zein is relatively large, and the interactions are likely primarily intermolecular, with relatively weaker interactions with SPIs. However, when the ratio becomes 1:1.2, SPIs are abundant and can interact fully with Zein, inducing the unfolding of Zein subunit structures and exposing more active sites. Simultaneously, the subunits of SPIs also undergo conformational changes due to this interaction.

[0067] SEM images of the composite protein particles and single Zein self-assembled particles (after being cycled at the same pH value) obtained under different Zein and SPIS mass ratios in this embodiment are shown below. Figure 7 As shown (A1: Zein self-assembly, A2-A6 are Zein:SPIs=1:0.1, 1:0.2, 1:0.5, 1:1, 1:1.2 respectively).

[0068] according to Figure 7A1 shows that the structure of Zein after self-assembly is relatively regular and uniform, mostly exhibiting angular particles with an orderly and somewhat regular arrangement. The surface of the particles is uniformly distributed with smaller protrusions, exhibiting a certain degree of roughness. Furthermore, the boundaries of the self-assembled Zein particles are clear, distinguishing them from their surroundings. Compared to A1, the particle morphology of A2 (1:0.1) begins to change, with some relatively smaller particles attaching to the surface of larger particles. This may be due to the addition of SPIs, which begin to interact with Zein, altering the surface properties and aggregation state of the particles. The morphology of A3 (1:0.2) particles changes further, with a rougher surface and an irregular distribution, lacking the orderly arrangement of Zein during self-assembly. The boundaries between particles become blurred, showing a certain degree of aggregation. This indicates that with the increase of the SPI ratio, SPIs have a greater impact on the complex protein particles, binding more to Zein and affecting Zein aggregation. The particles of A4 (1:0.5) and A5 (1:1) exhibited a more pronounced spherical structure with some wrinkles and depressions on the surface, indicating that the interaction between the two proteins was further strengthened by the increase of SPIs, leading to significant changes in the particle surface structure and aggregation state. The particles of A6 (1:1.2) became more irregular in morphology, with increased surface wrinkles and depressions, and the fusion of particles was more prominent, forming larger aggregates, indicating a more complex interaction between the two proteins. This is consistent with the results of electrophoresis showing the formation and ratio of complex protein particles.

[0069] SEM images revealed that the composite protein particles with a Zein:SPIs ratio of 1:0.2 were the smallest, suggesting a possible equilibrium between the two proteins. SPIs carry a negative charge, unlike the surface charge of Zein. Appropriate amounts of SPIs interact with Zein via electrostatic and moderate hydrophobic interactions. These interactions promote a tighter, more ordered structure between protein molecules, preventing excessive aggregation and resulting in smaller particle sizes. When the SPIs ratio is too low (1:0.1), Zein's strong hydrophobic aggregation tendency may lead to larger aggregates; conversely, when the SPIs ratio is too high (1:1.2), SPIs may become entangled or excessively bind to Zein, resulting in even larger aggregates. Therefore, at a ratio of 1:0.2, the amount of SPIs effectively prevents excessive self-aggregation of Zein without triggering excessive binding and aggregation, maintaining a relatively small particle size.

[0070] The water contact angle test results of the composite protein particles and single Zein particles (untreated) obtained under different Zein and SPIS mass ratios in this embodiment are as follows: Figure 8 As shown (A1: unprocessed Zein, A2-A6 are Zein:SPIs=1:0.1, 1:0.2, 1:0.5, 1:1, 1:1.2 respectively).

[0071] like Figure 8 As shown, Zein exhibits strong hydrophobic properties, with a three-phase contact angle reaching 124°, which is the direct reason why Zein cannot effectively stabilize Pickering emulsions. The surface chemical composition and microstructure of the composite protein particles combined with different proportions of Spines (SPIs) change, resulting in a decrease in contact angles. The overall trend of the contact angle is first decreasing, then increasing, and then decreasing again. The lower angles of A2 (1:0.1) and A3 (1:0.2) may be due to the introduction of a small amount of soy protein isolate, whose surface active groups interact with Zein, altering the surface polarity and hydrophilicity of the composite, making the liquid easier to spread and reducing the contact angle. Combined with the SEM results above, the microstructures of composite protein particles with different proportions differ. A2 and A3 particles are relatively small, and their structural characteristics facilitate liquid penetration and spreading. Their surfaces have more micropores or rough structures, increasing the contact area between the liquid and solid, generating capillary action, and reducing the contact angle. Meanwhile, because the two proteins possess different types and quantities of hydrophilic groups, their exposure and distribution differ at different ratios. In A6 (1:1.2), the proportion of SPIs is higher, resulting in a large amount of hydrophilic groups exposed on the surface of the composite protein particles. This leads to a strong affinity for water, causing the liquid to spread on the surface and reducing the contact angle. Furthermore, the salt ions added during pH cycling may also cause a decrease in the contact angle. Metal ions in the salt adsorb onto the surface of the composite protein particles, thereby altering the surface chemical composition and charge distribution. If ion adsorption increases the number of hydrophilic groups on the surface, the hydrophilicity of the particles increases, and the contact angle decreases.

[0072] Microscopic images of stable Pickering emulsions of Zein-SPIs composite protein particles and single Zein particles (untreated) obtained under different Zein and SPIs mass ratios in this embodiment are shown below. Figure 9 As shown.

[0073] according to Figure 9Microscopic images of the emulsion show that as the ratio of Zein to Spiral Integrities (SPIs) changes from 1:0.1 to 1:1.2, the overall trend of emulsion particle size is first decreasing and then increasing, with the smallest droplet size at 1:0.2. When the ratio changes from 1:0.1 to 1:0.2, the relative content of SPIs increases, resulting in more uniform and compact adsorption of the composite protein particles on the droplet surface, enhanced interfacial film stability, and thus better prevention of droplet aggregation, resulting in the smallest droplet size. Increasing the SPI ratio from 1:0.5 onwards, the droplet size gradually increases. This may be because as the SPI ratio further increases, the interaction between the two proteins becomes more complex, potentially leading to competitive adsorption, protein aggregation, or changes in interfacial film properties, resulting in decreased interfacial film stability and increased droplet aggregation, thus gradually increasing the droplet size.

[0074] Furthermore, there is a certain interaction between Zein and SPIs. When the ratio is appropriate, a synergistic effect may occur, making their adsorption on the emulsion surface more orderly and compact, further reducing surface tension. Conversely, when the ratio is inappropriate, the interaction between the proteins may be interfered with, affecting their adsorption and arrangement on the surface, resulting in a less significant decrease in surface tension or fluctuations. In this system, a Zein:SPIs ratio of 1:0.2 may be the optimal synergistic point. At this ratio, the two proteins cooperate optimally, allowing the resulting composite protein particles to exert the best effect in stabilizing emulsion droplets and reducing particle size. Deviating from this ratio is not conducive to the formation of stable small-diameter droplets.

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing Zein-SPIs complex protein particles, characterized in that: The preparation steps include the following: Zein-SPIs complex protein particles were dispersed in an ethanol-water solution, and then an alkaline solution was added to adjust the pH of the mixed solution to 9-12. The reaction was stirred to allow the protein structure to fully expand and dissolve. The insoluble matter was removed by centrifugation, and the pH was adjusted to neutral by adding an acid solution. After removing the ethanol by rotary evaporation, the particles were dialyzed to remove salt, freeze-dried, ground, and sieved to obtain Zein-SPIs complex protein particle powder.

2. The method for preparing Zein-SPIs composite protein particles according to claim 1, characterized in that: The mass ratio of zein to soy protein isolate is 1:0.1-1.

2.

3. The method for preparing Zein-SPIs composite protein particles according to claim 2, characterized in that: The mass-to-volume ratio of the zein to the aqueous ethanol solution is 0.5-2%.

4. The method for preparing Zein-SPIs composite protein particles according to claim 1, characterized in that: The ethanol-water solution used is an ethanol-water solution with an ethanol volume fraction of 40-60%.

5. The method for preparing Zein-SPIs composite protein particles according to claim 1, characterized in that: The alkaline solution is a 1-5 mol / L NaOH solution, the stirring reaction time is 0.5-3 h, the centrifugation refers to centrifugation at 5000-10000×g for 10-30 min, and the acid solution is a 1-5 mol / L hydrochloric acid solution.

6. The method for preparing Zein-SPIs composite protein particles according to claim 1, characterized in that: The aforementioned dialysis desalination refers to the removal of salts by dialysis using a 3500 kDa dialysis bag.

7. The method for preparing Zein-SPIs composite protein particles according to claim 1, characterized in that: The grinding and sieving process refers to passing the material through a 100-mesh sieve after grinding.

8. A Zein-SPIs complex protein particle, characterized in that: It is prepared by the method described in any one of claims 1-7.

9. The application of the Zein-SPIs complex protein particles as described in claim 8 in Pickering emulsion.

10. The application of a Zein-SPIs complex protein particle according to claim 9 in Pickering emulsion, characterized in that: The application method is as follows: Zein-SPIs complex protein particles were added to deionized water and mixed and dispersed, then added to the oil phase and homogenized to obtain Pickering emulsion.

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