Preparation method of high-emulsibility sweet potato protein

Abstract

The invention provides a modification method for modifying sweet potato protein through pulsed electric field coupling acetylation. The modification method comprises the following steps: preparing sweet potato protein; performing pulsed electric field pretreatment on the sweet potato protein; preparing a pulsed electric field assisted acetic anhydride modified sweet potato protein; the modified sweet potato protein solution serves as a water phase, soybean oil is added to serve as an oil phase, and the sweet potato protein emulsion is prepared through a high-speed homogenizer. According to the method, firstly, sweet potato protein is pretreated through a pulsed electric field technology, then functional characteristics of the sweet potato protein are improved through acetic anhydride acylation treatment, the solubility, emulsibility and emulsification stability of the sweet potato protein are remarkably improved, and a new method is provided for application of plant protein.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and in particular to a method for modifying sweet potato protein. Background Art

[0002] In recent years, Pickering emulsions have shown wide application potential in many fields such as food and biopharmaceuticals due to their unique stability and functional properties. Emulsions can not only be used as food additives in the processing of products such as ice cream and mayonnaise to improve the texture and taste of the products; they can also be used as oil substitutes to replace animal fats and hydrogenated oils, while maintaining the taste and flavor of food. Improve the nutritional and health value of food. In addition, emulsions can also embed bioactive substances, and by forming a stable Pickering emulsion system, these substances can be transported and protected, thereby improving their bioavailability and stability. Plant protein can be used as an excellent emulsifier due to its advantages such as natural renewability, environmental protection, high safety, and rich in various nutrients. It plays an important role in the preparation and stabilization of emulsions in industries such as food and biopharmaceuticals.

[0003] In China, sweet potatoes are mainly used for starch production, and a large amount of protein-containing wastewater is generated during the production process. Effective use of the protein in these wastewaters can not only avoid resource waste and reduce pollution, but also improve the utilization rate of sweet potato resources. However, during the extraction and processing of proteins, proteins are easily affected by process conditions and undergo adverse changes such as denaturation, which weakens their functional properties. Sweet potato protein (SPP) can be used as an ideal protein source in food processing because of its rich nutrition, balanced amino acid composition, antioxidant activity and medical and health value. However, with the continuous advancement of food technology, the original emulsification properties of sweet potato protein can no longer meet the needs of the modern food industry, and it is urgently needed to be improved through effective modification methods to broaden its application potential in the food industry.

[0004] At present, commonly used protein modification methods include heat treatment, ultrasonic treatment, radiation treatment, enzyme treatment, glycosylation, acylation, esterification and phosphorylation. However, the effects of these single treatment methods often have limitations, which are difficult to fully meet the needs of food processing, resulting in the application of sweet potato protein in the food industry being still limited. In this regard, composite modification technology shows huge application potential, and by significantly improving the functional properties of protein, its application range in the food industry is widened. However, there is no relevant report on the improvement of the emulsifying properties of sweet potato protein by pulse electric field coupling acetylation. Therefore, the present invention adopts pulse electric field coupling acetylation modified sweet potato protein to expand its application range by improving its emulsifying properties, thereby improving the added value of the sweet potato industry. Summary of the invention

[0005] In order to solve the problems of sweet potato protein in practical applications, the present invention combines pulsed electric field pretreatment to assist in acetylation modification of sweet potato protein, thereby enhancing the emulsification of sweet potato protein. The method combines the advantages of pulsed electric field treatment in minimizing nutritional loss, improving food safety and high efficiency and energy saving with the advantages of acetylation treatment in simple operation, low cost and significant effect, thereby significantly improving the modification effect of protein.

[0006] To achieve the above object, the present invention provides a method for preparing highly emulsifiable sweet potato protein, comprising the following steps: (1) Extract sweet potato protein from sweet potato waste liquid.

[0007] (2) The sweet potato protein prepared in step (1) is prepared into a solution, stirred evenly, and subjected to pulse electric field pretreatment to obtain a pulse electric field pretreated sweet potato protein solution.

[0008] (3) subjecting the protein solution in step (2) to an acylation reaction, stirring at a uniform speed and adding acetic anhydride in small amounts and multiple times to obtain pulsed electric field coupled acetylated modified sweet potato protein.

[0009] (4) The modified protein obtained in step (3) is prepared into a solution, with soybean oil as the oil phase and an oil-water ratio of 1:1, and homogenized using a high-speed homogenizer to obtain a pulsed electric field coupled acetylated modified protein emulsion.

[0010] Furthermore, step (1) of extracting sweet potato protein comprises the following steps: the sweet potato waste liquid is placed at 4°C for 12 h. The supernatant is taken and the pH is adjusted to 4.0 with HCl, and centrifuged at 12,000 rpm at 4°C to retain the protein precipitate at the bottom. The precipitation step is repeated twice, and the protein precipitate is dissolved in deionized water and adjusted to neutral by adding NaOH (1 mol / L).

[0011] Furthermore, in the protein purification process in step (1), the protein solution was purified by ultrafiltration using a 10,000 Da membrane and then freeze-dried for 48 h. The sample was ground into powder and passed through a 100-mesh sieve and stored at -20°C for later use.

[0012] Furthermore, in step (2), the sweet potato protein is mixed with deionized water to prepare a 5.0% (w / v) sweet potato protein solution, stirred at room temperature for 2 h, stored in a refrigerator at 4° C. overnight, and the solution is adjusted to pH 8.5 with NaOH.

[0013] Furthermore, in step (2), the sweet potato protein solution is reacted at different electric field strengths of 5 to 15 KV / cm for 3 min, and the overall temperature is controlled below 35°C.

[0014] Furthermore, in step (3), 10% to 30% acetic anhydride is added to the sweet potato protein solution after pulse electric field pretreatment, gradually added in portions, and stirred in a water bath at 30°C for 1 h; during the reaction, the pH of the solution is maintained at 8.0-9.0 by adding 1 mol / L NaOH. After the reaction is completed, the pH is adjusted to 7.0 and placed in an ice water bath to terminate the reaction.

[0015] Furthermore, in step (3), the modified sweet potato protein solution is dialyzed with deionized water at 4°C for 24-48 hours, and the dialyzed molecular weight is 8-14 kDa to remove excess acetic anhydride molecules. After freezing at -20°C for 24-48 hours, it is freeze-dried for 48 hours to obtain pulsed electric field coupled acetylated modified protein. After grinding, it is sieved with 100 mesh, sealed and stored at -20°C; Furthermore, in step (4), the pulse electric field coupled acetylated modified sweet potato protein solution is an aqueous phase with a concentration of 2.0%.

[0016] Furthermore, in step (4), a high-speed homogenizer is used to homogenize at 12,000 rpm for 2 min.

[0017] Beneficial Effects Compared with the prior art, the present invention has the following beneficial effects: first, the present invention uses sweet potato as the main raw material. Sweet potato is a common and widely used resource in the food industry, which has the characteristics of sufficient source and low cost, and is helpful to promote the efficient utilization of agricultural resources. The present invention aims to explore the potential application value of sweet potato protein as an emulsifier and provide theoretical support for the optimization of its functional properties. Through the treatment method of the present invention, the spatial structure of sweet potato protein is changed, thereby improving the functional properties of the protein (such as solubility, emulsification and emulsion stability). Unlike a single modification method, the present invention combines pulsed electric field pretreatment with acetylation treatment to achieve dual modification of sweet potato protein. Appropriate pulsed electric field pretreatment can destroy the non-covalent bonds that maintain the spatial structure of the protein, promote the structural unfolding of the protein and the exposure of the active groups, thereby enhancing the efficiency of the acetylation reaction, and further improving the charge density and electronegativity of the protein surface. These structural changes significantly improve the functional properties of the protein, including solubility, emulsification and emulsion stability. The modified sweet potato protein can be used as a delivery system in food, showing a wide range of application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the effect of pulsed electric field treatment on the average particle size of sweet potato protein in Examples 1-3; Figure 2 is a schematic diagram showing the effect of acetylation treatment on the average particle size of sweet potato protein in Example 4-6; Figure 3Schematic diagram of the effect of pulse electric field coupled acetylation treatment on the average particle size of sweet potato protein in Examples 7-15; Figure 4 is a schematic diagram of the effect of pulse electric field treatment on the potential of sweet potato protein in Example 1-3; Figure 5 is a schematic diagram of the effect of acetylation treatment on the potential of sweet potato protein in Example 4-6; Figure 6 is a schematic diagram of the effect of pulse electric field coupled acetylation treatment on the potential of sweet potato protein in Example 7-15; Figure 7 is a schematic diagram of the effect of pulsed electric field treatment on the solubility of sweet potato protein in Examples 1-3; Figure 8 is a schematic diagram of the effect of acetylation treatment on the solubility of sweet potato protein in Example 4-6; Fig. 9 is a schematic diagram of the effect of pulsed electric field coupled acetylation treatment on the solubility of sweet potato protein in Examples 7-15; Fig.10 These are optical microscope images of the emulsions of Comparative Example 1 and some embodiments. DETAILED DESCRIPTION

[0019] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0020] The sweet potato variety used in the following examples is "Jishu 25".

[0021] Method for extracting sweet potato protein from sweet potato waste liquid: The sweet potato waste liquid was placed at 4°C for 12 h, the supernatant was taken and the pH was adjusted to 4.0 with HCl, and centrifuged at 12000 rpm at 4°C to retain the protein precipitate at the bottom. Repeat the precipitation step twice, dissolve the protein precipitate in deionized water and add NaOH (1 mol / L) to neutralize Comparative Example 1 Sweet potato protein was mixed with deionized water to prepare a 5.0% (w / v) sweet potato protein solution, stirred at a constant speed for 2 h at room temperature, stored in a refrigerator at 4°C overnight, and the solution was adjusted to pH 8.5 with NaOH to obtain unmodified sweet potato protein (represented by Native in the figure).

[0022] Example 1 Mix sweet potato protein with deionized water to prepare a 5.0% (w / v) sweet potato protein solution, stir at room temperature for 2 h, store in a refrigerator at 4°C overnight, and adjust the solution to pH 8.5 with NaOH; react the sweet potato protein solution at an electric field strength of 5 KV / cm for 3 min, and immediately cool it in an ice water bath after the treatment. Freeze at -20°C for 24-48 h and then freeze-dry for 48 h to obtain pulsed electric field modified protein. Grind the protein and pass it through a 100-mesh sieve, seal it and store it at -20°C.

[0023] Example 2 The pulse electric field modified protein was prepared by referring to the method of Example 1, except that the pulse electric field intensity was adjusted to 10 KV / cm, and the other conditions were the same as in Example 1.

[0024] Example 3 The pulsed electric field modified protein was prepared by referring to the method of Example 1, except that the pulsed electric field intensity was adjusted to 15 KV / cm, and the other conditions were the same as in Example 1.

[0025] Example 4 Sweet potato protein was mixed with deionized water to prepare a 5.0% (w / v) sweet potato protein solution, stirred at room temperature for 2 h, stored in a refrigerator at 4°C overnight, and the solution was adjusted to pH 8.5 with NaOH; 10% (g / g Pro) acetic anhydride was added to the sweet potato protein solution, gradually added in batches, and stirred in a water bath at 30°C for 1 h; during the reaction, the solution pH was maintained at 8.0-9.0 by adding 1 mol / L NaOH. After the reaction was completed, the solution pH was adjusted to 7.0 and placed in an ice water bath to terminate the reaction. The protein solution was dialyzed at 4°C for 24-48 h to remove excess acetic anhydride. After freezing at -20°C for 24-48 h, it was freeze-dried for 48 h to obtain acetylated modified protein. The protein was ground and passed through a 100-mesh sieve, sealed and stored at -20°C.

[0026] Example 5 The acetylated modified protein was prepared by referring to the method of Example 4, except that the amount of acetic anhydride added was adjusted to 20% (g / g Pro), and the other conditions were the same as Example 4.

[0027] Example 6 The acetylated modified protein was prepared by referring to the method of Example 4, except that the amount of acetic anhydride added was adjusted to 30% (g / g Pro), and the other conditions were the same as Example 4.

[0028] Example 7 (1) Mix sweet potato protein with deionized water to prepare a 5.0% (w / v) sweet potato protein solution, stir it at room temperature for 2 h, store it in a refrigerator at 4°C overnight, and adjust the solution to pH 8.5 with NaOH; (2) The sweet potato protein solution was reacted at an electric field strength of 5 KV / cm for 3 min and immediately cooled in an ice water bath after the treatment; (3) Add 10% (g / g Pro) acetic anhydride to the sweet potato protein solution after 5 KV / cm pulse electric field pretreatment, gradually add it in portions, and stir in a water bath at 30°C for 1 h; during the reaction, the pH of the solution is maintained at 8.0-9.0 by adding 1 mol / L NaOH. After the reaction is completed, adjust the pH of the solution to 7.0 and place it in an ice water bath to terminate the reaction. The protein solution is dialyzed at 4°C for 24-48 h to remove excess acetic anhydride. Freeze at -20°C for 24-48 h and then freeze-dry for 48 h to obtain pulse electric field coupled acetylated modified protein. Grind the protein and pass it through a 100 mesh sieve, seal it and store it at -20°C.

[0029] Example 8 The pulsed electric field-assisted acylation-modified sweet potato protein was prepared by referring to the method of Example 7, except that the amount of acetic anhydride added was adjusted to 20% (g / g Pro), and the other conditions were the same as Example 7.

[0030] Example 9 The pulsed electric field-assisted acylation-modified sweet potato protein was prepared according to the method of Example 7, except that the amount of acetic anhydride added was adjusted to 30% (g / g Pro), and the other conditions were the same as Example 7.

[0031] Example 10 The pulsed electric field assisted acylation modified sweet potato protein was prepared by referring to the method of Example 7, except that the pulsed electric field treatment intensity was adjusted to 10 KV / cm, and the other conditions were the same as Example 7.

[0032] Embodiment 11 The pulsed electric field assisted acylation modified sweet potato protein was prepared by referring to the method of Example 7, except that the pulsed electric field treatment intensity was adjusted to 10 KV / cm and the amount of acetic anhydride added was 20% (g / g Pro), and the other conditions were the same as in Example 7.

[0033] Example 12 The pulsed electric field assisted acylation modified sweet potato protein was prepared by referring to the method of Example 7, except that the pulsed electric field treatment intensity was adjusted to 10 KV / cm and the amount of acetic anhydride added was 30% (g / g Pro), and the other conditions were the same as in Example 7.

[0034] Example 13 The pulsed electric field assisted acylation modified sweet potato protein was prepared by referring to the method of Example 7, except that the pulsed electric field treatment intensity was adjusted to 15 KV / cm, and the other conditions were the same as Example 7.

[0035] Embodiment 14 The pulsed electric field assisted acylation modified sweet potato protein was prepared by referring to the method of Example 7, except that the pulsed electric field treatment intensity was adjusted to 15 KV / cm and the amount of acetic anhydride added was 20% (g / g Pro), and the other conditions were the same as in Example 7.

[0036] Embodiment 15 The pulsed electric field assisted acylation modified sweet potato protein was prepared by referring to the method of Example 7, except that the pulsed electric field treatment intensity was adjusted to 15 KV / cm and the amount of acetic anhydride added was 30% (g / g Pro), and the other conditions were the same as in Example 7.

[0037] Test methods and results 1.1 Determination of protein particle size and potential Malvern laser particle size analyzer was used to measure the protein particle size and zeta potential of SPP samples.

[0038] 1.2 Determination of protein solubility Prepare a 2.0 mg / mL SPP solution, adjust the pH, and vortex for 10 min at room temperature. Centrifuge the sample at 4000 g for 15 min, and mix 1 mL of the supernatant with 5 mL of Coomassie Brilliant Blue staining solution. Measure the sample solubility using a UV-spectrophotometer at a wavelength of 595 nm. Calculate the solubility based on the BSA standard curve.

[0039] 1.3 Determination of protein emulsification and emulsion stability The protein solution (2%) and soybean oil were mixed in a 1:1 (v / v) ratio and homogenized at 12000 rpm for 2 min to obtain an emulsion. First, 50 μL of the emulsion was drawn from the bottom of the emulsion after standing for 0 min and 10 min, respectively, and added to 5 mL of diluent (0.1 g / 100 mL SDS), and quickly mixed using a vortex stirrer. Then, the absorbance of the diluted sample at 500 nm was measured using a UV-visible spectrophotometer.

[0040] EAI(m 2 / g) = (2.303×2×A 0 ×N) / (c×Ф×10000) ESI (min) = 10×A0 / (A0-A10) Where N is the dilution factor, c is the solution protein concentration (g / mL), and Ф is the oil volume fraction. 0and A 10 are the absorbance values ​​at 0 min and 10 min, respectively.

[0041] 1.4 Observation of protein emulsion microstructure The microstructure of the protein emulsion was observed under an optical microscope at a 40x objective.

[0042] 1.5 Results 1.5.1 Results of determination of protein particle size and potential according to Figure 1-Figure 6 The experimental results in the results show that the pulsed electric field coupled acetylation modification treatment has a significant effect on the particle size and zeta potential of sweet potato protein. After the composite modification treatment, the particle size of sweet potato protein was significantly reduced from 1980.54±35.9 nm to 905.6±18.7 nm, and the absolute value of the zeta potential was greatly increased from -20.12±3.47 mV to -82.28±3.69 mV. This result shows that the composite modification treatment not only effectively reduces the particle size of the protein, but also enhances its surface charge density. Compared with single acetylation modification or pulsed electric field treatment, the effect of pulsed electric field coupled acetylation composite treatment is superior. This synergistic effect may be due to the fact that the pulsed electric field pretreatment partially unfolds the protein structure through the electroporation effect, exposing more reaction sites, thereby enhancing the modification efficiency of the acetylation agent; at the same time, the acetylation modification further optimizes the surface charge distribution of the protein by introducing acetyl groups.

[0043] 1.5.2 Protein solubility determination results according to Figure 7-Figure 9 The experimental results show that after the pulse electric field coupled acetylation treatment modification, the solubility of sweet potato protein is significantly improved from 68.82±3.3% to 87.49±2.77%. Compared with the single acetylation treatment, the pulse electric field pretreatment promotes the disaggregation and redistribution of protein molecules, exposing more reaction sites, thereby significantly improving the modification efficiency of the acetylation reagent, allowing more acetyl groups to react with the protein. This dual modification not only effectively reduces the particle size of the protein, but also increases the surface charge of the protein (such as the increase in the absolute value of the zeta potential), thereby enhancing the interaction between the protein and water molecules and inhibiting the aggregation tendency of the protein. Therefore, the pulse electric field coupled acetylation composite treatment not only optimizes the structural characteristics of the protein and improves its solubility, but also provides an important theoretical basis and technical support for the application of sweet potato protein in the food industry.

[0044] 1.5.3 Results of protein emulsification and stability and emulsion microstructure Table 1 is the effect of pulse electric field coupled acetylation on the emulsification and stability of sweet potato protein in Examples 1-15 According to the results in Table 1, this synergistic effect can significantly improve the modification effect, which is significantly higher than single acetylation or pulse electric field treatment, making the pulse electric field coupled acetylation modification method show superior ability in improving the emulsification performance of protein. The emulsification and emulsion stability increased from 23.24±2.52 m 2 / g and 32.06±4.05 min increased to 67.19±2.81 m 2 / g and 158.09±11.21 min. Under the action of the pulsed electric field, its complex spatial conformation changes, and the hydrophilic residues that were originally hidden in the folding are exposed. These hydrophilic residues increase the interaction between the protein and the water molecules, directly enhancing the emulsification ability of the protein. At the same time, due to this change in the protein structure, the distribution of the protein in the emulsion system is more reasonable, providing the emulsion system with lasting stability. The introduction of acyl groups through acetylation addition reaction has many contributions to the improvement of the emulsification performance of proteins. On the one hand, as an emulsifier, the strength of the interaction between protein molecules and the oil phase and the water phase directly affects the emulsification effect. The introduction of acetyl groups enhances this interaction, allowing protein molecules to play a better role at the oil-water interface. On the other hand, the modification treatment increases the specific surface area and electrostatic repulsion by reducing the protein particle size and increasing the absolute value of the potential, accelerating the adsorption of proteins at the oil-water interface, and promoting the rapid formation of emulsified films, thereby improving the emulsification performance.

[0045] observe Fig.10 From the microstructure of the emulsion, it can be seen that after the pulse electric field coupled acetylation modification treatment, the size of the emulsion droplets is significantly reduced and the distribution is more uniform, showing the advantages of the synergistic effect of the dual effect. The pulse electric field causes the protein molecular chain to unfold and expose more active sites, and the acyl groups introduced by the acetylation reaction further modify the protein structure and properties. Unmodified protein emulsions are prone to aggregation during storage because of the interaction between protein molecules, which leads to aggregation and destroys the stability of the emulsion. The pulse electric field coupled acetylation modification can effectively prevent this situation. On the one hand, the pulse electric field changes the spatial conformation of the protein, making it easier to disperse in the emulsion and reducing aggregation caused by hydrophobic effects; on the other hand, the acyl groups introduced by acetylation change the surface charge distribution of the protein, increase electrostatic repulsion, and hinder molecular aggregation during storage. Pulse electric field pretreatment allows the protein structure to stretch and increase the contact area with the oil droplets; acetylation modification enhances the interaction between the protein and the oil droplets, making them tightly attached. This stable adsorption optimizes the stability of the emulsified system, avoids the aggregation of oil droplets, maintains the uniform dispersion of the emulsion, and improves its functionality in the fields of food and cosmetics.

Claims

1. A method for preparing highly emulsifiable sweet potato protein, characterized in that: The following steps are involved: (1) Preparation of sweet potato protein: extracting sweet potato protein by acid precipitation and alkali dissolution method, and purifying by ultrafiltration; (2) Pulsed electric field pretreatment of sweet potato protein; (3) Acetylation further modified the pulsed electric field pretreated sweet potato protein; (4) The modified sweet potato protein solution is mixed with soybean oil, and the oil-water mixed solution is passed through a high-speed homogenizer to prepare an emulsion.

2. The method for preparing a highly emulsifiable sweet potato protein according to claim 1, characterized in that: The preparation of the sweet potato protein comprises placing the sweet potato waste liquid at 4° C. for 12 h, taking the supernatant and adjusting the pH to pH 4.0 with HCl, centrifuging at 12,000 rpm at 4° C., and retaining the protein precipitate at the bottom; repeating the precipitation step twice, dissolving the protein precipitate in deionized water and adding 1 mol / L NaOH to adjust to neutrality.

3. The method for preparing a highly emulsifiable sweet potato protein according to claim 2, characterized in that: The obtained neutral sweet potato protein solution was purified by ultrafiltration using a 10,000 Da membrane and then freeze-dried for 48 h.

4. The method for preparing a highly emulsifiable sweet potato protein according to claim 1, characterized in that: The pulse electric field pre-treats the sweet potato protein by treating a 5% sweet potato protein solution at different electric field intensities of 5 to 15 KV / cm for 3 min.

5. The method for preparing a highly emulsifiable sweet potato protein according to claim 1, characterized in that: The acetylated sweet potato protein is prepared by gradually adding acetic anhydride in a ratio of 10% to 30% (g / g Pro) to the sweet potato protein solution treated with a pulse electric field, and reacting at 30° C. for 1 h.

6. The method for preparing a highly emulsifiable sweet potato protein according to claim 5, characterized in that: During the acetic anhydride addition reaction, the pH was maintained constant in the range of 8.0-9.0 by adding 1 mol / L NaOH.

7. The method for preparing a highly emulsifiable sweet potato protein according to claim 1, characterized in that: The ratio of oil phase to water phase was 1:1 during emulsion preparation, and homogenization was performed at 12,000 rpm for 2 min.

Images