Method for rapidly preparing high-emulsibility soybean protein nanofiber by utilizing pulsed electric field in cooperation with water bath

Abstract

The invention discloses a method for rapidly preparing high-emulsibility soybean protein nanofibers by utilizing a pulsed electric field in cooperation with a water bath, and belongs to the technical field of protein modification. The method comprises the following steps: preparing an aqueous solution from soybean protein isolate, adjusting the pH value to 1.5-2.5 to obtain a soybean protein isolate solution with the concentration of 20-30 mg / mL, stirring, and hydrating; the hydrated soybean protein isolate solution is subjected to circulating pretreatment through a pulsed electric field, the electric field intensity of the pulsed electric field is 13-21 kV / cm, and the pulse frequency is 50-125 Hz; stirring the soybean protein isolate solution pretreated by the pulsed electric field in a water bath; after the water bath is finished, cooling the solution to room temperature to obtain a soybean protein nanofiber solution, and freeze-drying to obtain soybean protein isolate nanofiber powder; the soybean protein nanofiber obtained by the method disclosed by the invention is small in diameter (the minimum diameter is 72.2 nm), and the emulsifying property is obviously improved by more than 40%.

Description

Technical Field

[0001] The present invention relates to a method for preparing protein nanofibers, and in particular to a method for rapidly preparing highly emulsifiable soybean protein nanofibers by utilizing a pulsed electric field in conjunction with a water bath, belonging to the technical field of protein fiberization modification. Background Art

[0002] Fibrillation modification is an effective means to improve the functional properties of proteins. Protein nanofibers have been increasingly used as functional materials or components in various fields due to their excellent functional properties, extremely high aspect ratio, a large number of functional groups on the fiber surface, and high stiffness. In addition, fibrillation can improve their physical properties by increasing the interaction between proteins, such as enhancing the stability and thickening of emulsions, which is particularly important in food. This modification can not only enhance the sensory properties of food, but also meet consumers' growing demand for plant-based foods, thereby promoting sustainable food production. In this way, fibrillated soy protein shows great potential in a variety of food applications and has become an important direction for future food science research.

[0003] Currently, the most common protein fibrillation method is to heat for 12 hours or even longer at a high temperature (usually 85°C) in an acidic environment (pH = 2). This method takes a long time, consumes a lot of energy, and has a low fiber conversion efficiency. Chinese invention patent application CN116656767A discloses a method for preparing highly emulsifiable β-lactoglobulin and its fiber-like substance, which uses β-lactoglobulin as a raw material and is obtained by reacting for 16-18 hours at a pH of 1.8-2.2 and a reaction temperature of 80-90°C. Although the β-lactoglobulin fibers obtained by this invention method are suitable for neutral and acidic environments, and have improved emulsification properties, this method essentially only uses high temperature as the driving force for protein fibrillation. Under these conditions, the hydrolysis changes in the protein structure, the generation of aggregated monomers, and their polymerization are still slow and inefficient, and the key problem of long protein fibrillation time and low conversion efficiency has not been solved.

[0004] Chinese utility model patent CN219907679U discloses a device for rapidly fibrillating whey protein by combining enzymatic pretreatment with radiofrequency heating, which improves the preparation efficiency of existing whey protein nanofibers and shortens the preparation time. However, this technology has the following problems: 1. The enzymatic hydrolysis process is difficult to control, which can easily lead to uneven fiber structure. The efficiency of the enzymatic reaction is affected by parameters such as pH, temperature, and enzyme concentration. If the device does not achieve precise dynamic regulation, it may lead to inconsistent protein hydrolysis and too wide a molecular weight distribution of the fibrillated product; 2. The problem of uniformity of radiofrequency heating. Radiofrequency heating is prone to "hot spots" or local overheating, resulting in protein denaturation and aggregation or a decrease in the browning inhibition effect; 3. Insufficient control of the intermolecular forces of proteins. The fibrillation process relies on β-folding stacking, but the rapid temperature rise of radiofrequency heating may destroy the balance of hydrogen bonds or hydrophobic interactions, resulting in short or branched fibers. Summary of the Invention

[0005] To address the shortcomings of traditional soy protein isolate fiberization methods, which are time-consuming, energy-intensive, inefficient, and poorly emulsifying, this invention provides a method for rapidly preparing highly emulsifying soy protein nanofibers using a pulsed electric field in conjunction with a water bath. During the process, the sample is in solution, which is then freeze-dried to a solid powder.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for rapidly preparing highly emulsifiable soybean protein nanofibers using a pulsed electric field in conjunction with a water bath comprises the following steps:

[0008] S1: preparing soy protein isolate into an aqueous solution and adjusting the pH to 1.5-2.5 to obtain a soy protein isolate solution with a concentration of 20-30 mg / mL, stirring, and hydrating;

[0009] S2: cyclically pretreating the hydrated soy protein isolate solution using a pulsed electric field, wherein the pulsed electric field has an electric field strength of 13-21 kV / cm, a pulse frequency of 50-125 Hz, a pulse width of 8-10 μs, a treatment time of 30-90 min, and a cycle of 20-40 s;

[0010] S3: Place the soy protein isolate solution pretreated by the pulsed electric field in a water bath at 80-90° C. while stirring at a speed of 100-300 rpm for 4-10 hours;

[0011] S4: After the water bath is completed, the solution is cooled to room temperature to obtain a soy protein nanofiber solution, which is freeze-dried to obtain soy protein isolate nanofiber powder.

[0012] To further achieve the purpose of the present invention, preferably, in step S1, adjusting the pH to 1.5-2.5 is performed by adding hydrochloric acid.

[0013] Preferably, the concentration of the hydrochloric acid is 1.0-1.5 mol / L.

[0014] Preferably, in step S2, the electric field strength of the pulsed electric field is 19-21 kV / cm.

[0015] Preferably, in step S2, the pulse frequency of the pulse electric field is 75-115 Hz.

[0016] Preferably, in step S2, the pulse electric field treatment time is 45-75 minutes.

[0017] Preferably, in step S3, the water bath heating time is 4-8 hours.

[0018] Preferably, in step S3, the stirring speed is 150-250 rpm.

[0019] Preferably, in step S1, the stirring speed is 500-1000 rpm, and the stirring time is 2-3 hours.

[0020] Preferably, in step S1, the hydration temperature is 2-4°C, and the hydration time is 8-12 hours.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] (1) The pulsed electric field combined with the water bath significantly shortens the fiberization time to 1 / 3-1 / 2 of the traditional method, greatly improving the fiber conversion efficiency by more than 80%;

[0023] (2) The pulsed electric field parameters are easy to control and can be combined with a water bath to avoid low protein fibrillation or excessive protein aggregation. The parameter conditions of the pulsed electric field are closely related to the microscopic aggregation behavior of proteins. Precise control of the pulse parameters can achieve the highest fiber conversion rate in a short time and with low energy consumption.

[0024] (3) Compared with fibers obtained from natural soy protein isolate and other methods, the fibers obtained by the present invention have unique structural and functional properties. Specifically, the fiber diameter is between 70-80 nm, and its emulsification performance is significantly improved by more than 40%. This makes its application scenarios in food processing more diverse and has broad prospects.

[0025] (4) The energy consumption of protein fiberization is reduced by more than 30%. For example, to process 500 mL of soy protein isolate solution (2% g / ml), the energy consumption of heating in an 85°C water bath for 8 hours is about 0.8 kWh, while using the method of the present invention to achieve the same fiber conversion rate only requires about 0.42 kWh. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The fluorescence intensity graphs of Thioflavin of Examples 2, 4, 5 and Comparative Examples 1, 4, 5 are shown.

[0027] Figure 2 The endogenous fluorescence spectra of Examples 1 and 3, Comparative Examples 2 and 3, and natural SPI are shown.

[0028] Figure 3 are laser confocal images, ABCDEF are Example 2, Comparative Example 1, Example 1, Comparative Example 2, Example 5 and Comparative Example 5, respectively. DETAILED DESCRIPTION

[0029] For a better understanding of the present invention, the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments of the present invention are not limited thereto. The embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Soy protein isolate is an important plant-derived protein. It is inexpensive, readily available, and nutritious, making it a crucial component in the food processing industry. However, its large molecular weight, low surface hydrophobicity, and dense, compact conformation of the natural globular protein significantly limit its functional properties, such as emulsification, gelation, and foaming. Fiberization of soy protein isolate significantly overcomes these functional limitations. The successful formation of soy protein isolate fibers is often accompanied by the following key structural and property changes: a significant increase in the β-sheet fraction, a significant increase in Th T fluorescence intensity, and the observation of a fibrous structure by TEM.

[0031] Pulsed electric fields, as a novel non-thermal food processing technology, have attracted widespread attention in recent decades. As a novel non-thermal physical processing method, it is widely used in food sterilization, extraction of natural active ingredients, modification and modification of biomacromolecules, and intensification of chemical reactions.

[0032] Currently, methods using microwaves, radiofrequency heating, ultrasound, and low-frequency magnetic fields to accelerate the fibrillation of soy protein isolate have been used. Testing has found that these methods can slightly improve fiber conversion efficiency, but the effect is not significant. Other studies have used enzymatic hydrolysis to treat protein, but the extent and uniformity of enzymatic hydrolysis cannot be guaranteed, and stability is poor. This may be because, while these strengthening methods, such as microwaves, radiofrequency heating, ultrasound, and low-frequency magnetic fields, can influence protein structure to a certain extent, shifting it in a direction that favors fiber formation, they may not be well-suited to soy protein isolate fibrillation and lack the stable, sustained driving force to sustain fiber formation and elongation.

[0033] The present invention has found that the use of a pulsed electric field in conjunction with a water bath can effectively overcome the problems existing in the prior art. To this end, the present invention adopts a method for rapidly preparing highly emulsifiable soybean protein nanofibers using a pulsed electric field in conjunction with a water bath, comprising the following steps:

[0034] S1: preparing soy protein isolate into an aqueous solution and adjusting the pH to 1.5-2.5 to obtain a soy protein isolate solution with a concentration of 20-30 mg / mL, stirring, and hydrating;

[0035] S2: cyclically pretreating the hydrated soy protein isolate solution using a pulsed electric field, wherein the pulsed electric field has an electric field strength of 13-21 kV / cm, a pulse frequency of 50-125 Hz, a pulse width of 8-10 μs, a treatment time of 30-90 min, and a cycle of 20-40 s;

[0036] S3: Place the soy protein isolate solution pretreated by the pulsed electric field in a water bath at 80-90° C. while stirring at a speed of 100-300 rpm for 4-10 hours;

[0037] S4: After the water bath is completed, the solution is cooled to room temperature to obtain a soy protein nanofiber solution, which is freeze-dried to obtain soy protein isolate nanofiber powder.

[0038] As a preferred technical measure, in step S1, adjusting the pH to 1.5-2.5 can be done by adding hydrochloric acid. The concentration of hydrochloric acid is preferably 1.0-1.5 mol / L. In step S1, the stirring speed is preferably 500-1000 rpm, and the stirring time is preferably 2-3 hours. In step S1, the hydration temperature is preferably 2-4°C, and the hydration time is preferably 8-12 hours.

[0039] In step S2, the electric field intensity of the pulsed electric field is preferably 19-21 kV / cm, the pulse frequency is preferably 75-115 Hz, and the treatment time is preferably 45-75 min.

[0040] In step S3, the water bath heating time is preferably 4-8 hours, and the stirring speed is preferably 150-250 rpm.

[0041] The present invention uses a pulsed electric field in conjunction with a water bath to effectively avoid or improve the above technical problems. First, the fiberization of the pulsed electric field pretreatment does not require an enzymatic hydrolysis step, and the destruction of the peptide bond structure is accelerated by the pulse action. Second, the pulsed electric field has good uniformity. It forms a uniform electric field under the same treatment environment, and all materials in the treatment chamber are treated with the same effect. Third, because the pulsed electric field treatment parameters are easy to adjust accurately, they can be combined with a water bath to achieve effective perturbation of the protein molecular structure. After the pulsed electric field treatment, the water bath is added to the fiberization reaction to further denature and hydrolyze the protein, which has undergone significant structural changes at high temperature, in the shortest possible time. The water bath provides a stable and effective driving force for the subsequent fiber formation. The pulsed electric field provides the maximum effective perturbation of the protein molecules, and the water bath provides the indispensable and effective driving force for fiber formation. Both are indispensable, which is the fundamental reason why the present invention is significantly superior to other methods. At the same time, the fiber obtained by this method has excellent emulsification properties. The three main purposes of improving fiberization efficiency, reducing reaction energy consumption, and improving protein emulsification properties are achieved, which is also the fundamental problem to be solved by the present invention. Tests have shown that the pulsed electric field combined with a water bath can reduce the energy consumption of protein fiberization by more than 30%. For example, to process 500 mL of soy protein isolate solution (2% g / ml), heating in an 85°C water bath for 8 hours consumes approximately 0.8 kWh of energy, while using the method of the present invention to achieve the same fiber conversion rate only requires approximately 0.42 kWh.

[0042] Example 1:

[0043] S1: Prepare soy protein isolate into an aqueous solution and adjust the pH to 1.5 with 1 M hydrochloric acid to obtain a soy protein isolate solution with a concentration of 20 mg / mL. Stir at 700 rpm for 2 h and hydrate at 4°C overnight to fully dissolve the protein.

[0044] S2: The soy protein isolate solution was pretreated using a pulsed electric field. Each 30 s was a treatment cycle. The electric field strength of the pulsed electric field was 19 kV / cm, the pulse frequency was 115 Hz, the treatment time was 75 min, and the pulse width was 10 us.

[0045] S3: The soy protein isolate solution after the pulse electric field treatment was heated in a water bath at 85° C. for 4 h while being stirred at a speed of 200 rpm.

[0046] S4: After the water bath is completed, the solution is cooled to room temperature to obtain the soybean protein nanofiber solution, which is freeze-dried into a solid powder for storage or use.

[0047] Comparative Example 1

[0048] The difference from Example 1 is that the prepared soy protein solution is not treated with a pulsed electric field, but is directly heated in a water bath at 85° C. for 8 h while being stirred at a speed of 200 rpm.

[0049] Example 2

[0050] S1: Prepare soy protein isolate into an aqueous solution and adjust the pH to 2 with 1 M hydrochloric acid to obtain a soy protein isolate solution with a concentration of 25 mg / mL. Stir at 500 rpm for 3 h and hydrate at 2°C for 8 h to fully dissolve the protein.

[0051] S2: The soy protein isolate solution was pretreated using a pulsed electric field, with each treatment cycle lasting 20 seconds. The electric field strength of the pulsed electric field was 13 kV / cm, the treatment time was 30 minutes, the pulse frequency was 75 Hz, and the pulse width was 8 μs.

[0052] S3: The SPI solution after the pulse electric field treatment was heated in a water bath at 80° C. for 8 h while being stirred at 250 rpm.

[0053] S4: After the water bath is completed, the solution is cooled to room temperature to obtain the soybean protein nanofiber solution, which is freeze-dried into a solid powder for storage or use.

[0054] Example 3

[0055] S1: Prepare soy protein isolate into an aqueous solution and adjust the pH to 2.5 with 1 M hydrochloric acid to obtain a soy protein isolate solution with a concentration of 30 mg / mL. Stir at 100 rpm for 3 h and hydrate at 3 ° C for 10 h to fully dissolve the protein.

[0056] S2: The soy protein isolate solution was pretreated using a pulsed electric field, with each 40 s being a treatment cycle, the electric field strength of the pulsed electric field being 21 kV / cm, the treatment time being 45 min, the pulse frequency being 125 Hz, and the pulse width being 10 μs.

[0057] S3: The SPI solution after the pulse electric field treatment was heated in a water bath at 90° C. for 4 h while being stirred at 300 rpm.

[0058] S4: After the water bath is completed, the solution is cooled to room temperature to obtain the soybean protein nanofiber solution, which is freeze-dried into a solid powder for storage or use.

[0059] Comparative Example 2

[0060] The difference from Example 1 is that the prepared soy protein solution is not treated with a pulsed electric field, but is directly heated in a water bath at 85° C. for 4 h while being stirred at a speed of 200 rpm.

[0061] Example 4

[0062] S1: Prepare soy protein isolate into an aqueous solution and adjust the pH to 2.0 with 1 M hydrochloric acid to obtain a soy protein isolate solution with a concentration of 20 mg / mL. Stir at 700 rpm for 3 h and hydrate at 4 ° C for 10 h to fully dissolve the protein.

[0063] S2: The soy protein isolate solution was pretreated using a pulsed electric field, with each 40 s being a treatment cycle, the electric field strength of the pulsed electric field being 21 kV / cm, the treatment time being 90 min, the pulse frequency being 50 Hz, and the pulse width being 10 μs.

[0064] S3: The SPI solution after the pulse electric field treatment was heated in a water bath at 85° C. for 8 h while being stirred at 300 rpm.

[0065] S4: After the water bath is completed, the solution is cooled to room temperature to obtain the soybean protein nanofiber solution, which is freeze-dried into a solid powder for storage or use.

[0066] Example 5

[0067] S1: Prepare soy protein isolate into an aqueous solution and adjust the pH to 2.5 with 1 M hydrochloric acid to obtain a soy protein isolate solution with a concentration of 25 mg / mL. Stir at 500 rpm for 2 h and hydrate at 3 ° C for 12 h to fully dissolve the protein.

[0068] S2: The soy protein isolate solution was pretreated using a pulsed electric field, with each treatment cycle lasting 30 seconds. The electric field strength of the pulsed electric field was 19 kV / cm, the treatment time was 75 minutes, the pulse frequency was 115 Hz, and the pulse width was 8 μs.

[0069] S3: The SPI solution after the pulse electric field treatment was heated in a water bath at 85° C. for 8 h while being stirred at a speed of 100 rpm.

[0070] S4: After the water bath is completed, the solution is cooled to room temperature to obtain the soybean protein nanofiber solution, which is freeze-dried into a solid powder for storage or use.

[0071] Comparative Example 3

[0072] The difference from Example 5 is the pulse electric field treatment parameters: field intensity of 27 kV / cm, frequency of 150 Hz, treatment time of 10 min, and 85° C. water bath time of 8 h.

[0073] Comparative Example 4

[0074] The difference from Example 5 is the pulse electric field treatment parameters: field intensity is 6 kV / cm, frequency is 150 Hz, and treatment time is 10 min.

[0075] Comparative Example 5

[0076] Use 2.4kW, 27.12MHz radio frequency heating for 7 hours.

[0077] The relevant detection methods are described as follows:

[0078] ①Fiber conversion rate determination

[0079] The fiber conversion rate was determined using a 100 kDa ultrafiltration centrifuge tube and the BCA assay. 160 μL of the completed fibrosis reaction sample was diluted 50-fold and brought to a constant volume of 8 mL. The protein concentration of the diluted solution was accurately determined using the BCA assay. 4 mL of the diluted solution was placed in a 4 mL 100 kDa ultrafiltration centrifuge tube and centrifuged at 3000 x g for 30 min at 4°C. After each centrifugation, the filter residue was washed twice with 4 mL of pH 2 hydrochloric acid. The filtrate was collected and combined, and the volume was recorded as V1 (mL). The protein concentration of the filtrate was accurately determined using the BCA assay. The results are shown in Table 1.

[0080]

[0081] C0-protein concentration in the diluted solution (mg / mL); V1-volume of the combined filtrate (mL);

[0082] C1-Protein concentration in filtrate (mg / mL)

[0083] ②Thioflavin (Th T) fluorescence determination

[0084] Prepare a 0.5 mg / mL thioflavin stock solution with phosphate buffer solution at pH 7, filter through a 0.22 μm membrane to remove any undissolved thioflavin, and store at 4°C in the dark. On the day of testing, dilute the stock solution 50-fold with phosphate buffer solution to obtain a working solution. Mix 25 μL of sample with 5 mL of working solution, let stand for 2 minutes, and measure the fluorescence intensity at 440 nm excitation and 482 nm emission. Figure 1 .

[0085] ③ Protein intrinsic fluorescence assay

[0086] After the fibrosis reaction is completed, the sample is diluted 20 times with pH = 2 hydrochloric acid to about 1 mg / mL. Then 100uL of the diluted solution is diluted 30 times with pH = 2 hydrochloric acid. The fluorescence spectrum of the second diluted solution is measured using a fluorescence spectrophotometer at 280nm excitation and 300-450nm emission. The results are shown in Figure 2 .

[0087] ④Circular dichroism spectroscopy

[0088] After the fibrillation reaction, the sample was diluted 100-fold with pH 2 hydrochloric acid to approximately 0.2 mg / mL. 300 mL of the diluted sample was taken and 3 mL of distilled water was added. The protein secondary structure was measured by circular dichroism spectrometry in the wavelength range of 190 nm-260 nm. The results are shown in Table 2.

[0089] ⑤ Particle size determination

[0090] After the fibrillation reaction, the sample was diluted 20 times with pH 2 hydrochloric acid to about 1 mg / mL. The particle size was measured using a Malvern nanoparticle size analyzer at an equilibrium time of 120 s and 25°C. The results were measured three times in parallel. The results are shown in Table 3.

[0091] ⑥ Emulsification index determination

[0092] The emulsifying activity index (EAI) was determined by mixing 2 mL of corn oil with 2 mL of sample and homogenizing at 10,000 rpm for 2 minutes using an IKA high-speed disperser. At 0 minutes after homogenization, 20 μL of sample was aspirated from the bottom of the test tube and diluted 200-fold with a 1 mg / mL sodium dodecyl sulfate (SDS) solution. The absorbance at 500 nm was measured using a UV spectrophotometer, using the SDS solution as a reference. The EAI value was calculated using the following formula (see Table 3).

[0093]

[0094] ⑦ Laser confocal imaging

[0095] 1 ml of emulsion (corn oil volume fraction of 10%) was stained with 40 μL of a mixed fluorescent dye consisting of 0.1 mg / mL Nile red and 0.1 mg / mL Nile blue isopropanol solution. 15 μL of the stained solution was placed on a glass slide and fixed with a coverslip. Fluorescence observation of Nile red and Nile blue was performed using excitation wavelengths of 488 nm and 633 nm, respectively. The results are shown in Figure 2. Figure 3 .

[0096] Effects of pulsed electric field combined with water bath on fiber conversion efficiency

[0097] The fluorescence intensity of thioflavin was determined by conversion rate (Table 1 and Figure 1) confirmed that the pulsed electric field combined with water bath treatment can greatly improve the fiber conversion efficiency by more than 80%, and up to 102%. In terms of improving the fiber rate, compared with radio frequency heating (Comparative Example 5), the present invention still shows obvious advantages, especially the present invention not only achieves a higher fiber conversion rate, but also has a significant advantage in improving the efficiency of the water bath method at the same time, reaching more than 83%, while Comparative Example 5 is only 20%. Through Examples 1, 2, 3, 4, 5 and Comparative Examples 3 and 4, it can be found that the effective treatment parameter range of the pulsed electric field is an electric field intensity of 13-21kV / cm, a pulse frequency of 50-125Hz, and a treatment time of 30-90min; the more optimal parameters are 19-21kV / cm, 75-115Hz, and 45-75min. Based on previous theoretical foundations, the present invention discovered that moderate pulsed electric field intensities, particularly 13–21 kV / cm, can significantly promote protein polarization, disrupt hydrogen bonds and electrostatic interactions, and lead to partial unwinding of secondary structures (α-helices and β-sheets). This alters the dipole orientation and electrostatic shielding effects of protein molecules, causing dipole moment rearrangement under the action of the electric field and disrupting the charge distribution on the protein surface, significantly affecting its stability. Furthermore, moderate field intensities do not accumulate severe thermal effects that could cause random protein aggregation, thereby negating the unique advantage of pulsed electric fields as non-thermal processing. Low- to medium-frequency pulse treatments of 50–125 Hz achieve conformational adjustments and molecular skeleton perturbations through dielectric relaxation and resonance effects. When the treatment time is 30–90 minutes, the effective treatment time of the pulsed electric field is approximately 170 μs. This moderate treatment time balances the treatment effect of the pulsed electric field with the accumulation of thermal effects, achieving excellent results with minimal energy consumption. This achieves a comprehensive balance between pulsed electric field treatment effects, thermal effect control, and energy consumption reduction.

[0098] Table 1 Conversion rate results

[0099]

[0100] Effects of pulsed electric field combined with water bath on protein structure

[0101] As shown in Table 2, after protein fibrillation, its β-sheet content increases, α-helix and random coil decrease, and the structure tends to be ordered. Since β-sheet is the characteristic structure of protein fiber, the amount of β-sheet content in protein can reflect the degree of protein fibrillation. The results show that the samples treated with pulsed electric field in combination with water bath (Example 4 and Example 5) have a higher β-sheet content than Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, reaching 42.4% and 43.3%, respectively, and their structure is more ordered, proving that pulsed electric field in combination with water bath treatment accelerates the fibrillation process and faster protein fiber formation. Figure 2The intrinsic fluorescence shown can indicate changes in the protein's tertiary structure and hydrophobic microenvironment. Compared to Comparative Examples 2 and 3, the pulsed electric field-assisted waterbath treatment (Examples 1 and 3) exacerbated the exposure of hydrophobic groups such as tryptophan and tyrosine, which were originally located within the protein, resulting in a significant decrease in fluorescence intensity. The exposure of hydrophobic amino acids in proteins facilitates hydrophobic polymerization during fibrosis, providing a driving force for the fibrosis process and accelerating its rate.

[0102] Table 2 Secondary structure content

[0103]

[0104] Emulsification properties of soy protein isolate nanofibers prepared by pulsed electric field combined with water bath

[0105] From Table 3, it can be seen that after the pulse electric field combined with water bath treatment (Example 1, Example 2, Example 5), the fiber diameter obtained was significantly reduced, with the lowest being 72.2nm, which was significantly lower than that of Comparative Example 1, Comparative Example 2 and Comparative Example 5. At the same time, it can also be found from Table 3 that the emulsification index of the embodiment (8.6-9.2nm) 2 / g) is significantly greater than that of the comparative example (<5.8m 2 / g). This demonstrates the excellent effectiveness of the present method in improving emulsification. Notably, the emulsification index is negatively correlated with the average fiber particle size. This is because smaller fiber diameters indicate greater flexibility and interfacial coverage, allowing them to better wrap and adhere to the oil droplet interface in the emulsion, thereby providing enhanced emulsion stability and anti-agglomeration capabilities.

[0106] Table 3 Particle size and emulsification index measurement results

[0107]

[0108] Figure 3The laser confocal images are shown in Figures ABCDEF, Example 2, Comparative Example 1, Example 1, Comparative Example 2, Example 5, and Comparative Example 5, respectively. It can be clearly seen from the figure that after the water-oil two-phase solution is homogenized, the diameter of the oil droplets in each embodiment is about 20 μm and the oil droplets are in a single spherical shape, without aggregation. In contrast, in the comparative example, not only is the particle size of the oil droplets significantly larger than that of the embodiment (more than 50 μm), but the aggregation phenomenon between the oil droplets is also obvious, which is in sharp contrast to the embodiment. It should be noted that there are certain differences in the particle size of the oil droplets in Examples 1, 2, and 5, which may be caused by the heterogeneity of the homogenization process, which is unavoidable during the homogenization process. However, this result better proves that the soy protein isolate nanofibers prepared by the method of the present invention have an effective water-oil interface adhesion effect, resist the aggregation of oil droplets, and have a significant emulsification effect. The laser confocal imaging results further confirm the particle size and EAI measurement results, confirming that the nanofibers prepared by the pulsed electric field in a water bath have excellent emulsification properties. Enhanced emulsification properties delay oil-water separation, extending the shelf life of emulsions. They are also more tolerant to changes in pH, temperature, and ionic strength, making them suitable for complex environments. As a natural protein derivative, they can replace artificial emulsifiers and meet consumer demand for healthy, clean-label products. Compared to traditional emulsifiers, soy protein nanofibers are less likely to cause allergies, providing new insights into the development of high-performance, sustainable emulsion systems, with significant potential in health products and green technology.

[0109] In summary, the present invention utilizes a pulsed electric field in conjunction with a water bath to address the challenges of slow soy protein isolate fibrillation kinetics and low fibrillation efficiency. Furthermore, the resulting fibers possess excellent emulsifying properties, making them suitable for use in more complex emulsion systems. Compared to other methods, the present invention offers unique advantages with low energy consumption and significant results.

[0110] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention is described in detail in each embodiment, ordinary technicians in this field should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapidly preparing highly emulsifiable soybean protein nanofibers using a pulsed electric field in conjunction with a water bath, characterized in that The following steps are involved: S1: preparing soy protein isolate into an aqueous solution and adjusting the pH to 1.5-2.5 to obtain a soy protein isolate solution with a concentration of 20-30 mg / mL, stirring, and hydrating; S2: cyclically pretreating the hydrated soy protein isolate solution using a pulsed electric field, wherein the pulsed electric field has an electric field strength of 13-21 kV / cm, a pulse frequency of 50-125 Hz, a pulse width of 8-10 μs, a treatment time of 30-90 min, and a cycle of 20-40 s; S3: Place the soy protein isolate solution pretreated by the pulsed electric field in a water bath at 80-90° C. while stirring at a speed of 100-300 rpm for 4-10 hours; S4: After the water bath is completed, the solution is cooled to room temperature to obtain a soy protein nanofiber solution, which is freeze-dried to obtain soy protein isolate nanofiber powder.

2. The method according to claim 1, characterized in that In step S1, the pH is adjusted to 1.5-2.5 by adding hydrochloric acid.

3. The method according to claim 2, characterized in that The concentration of the hydrochloric acid is 1.0-1.5 mol / L.

4. The method according to claim 1, wherein In step S2, the electric field strength of the pulsed electric field is 19-21 kV / cm.

5. The method according to claim 1, wherein In step S2, the pulse frequency of the pulse electric field is 75-115 Hz.

6. The method according to claim 1, characterized in that In step S2, the pulse electric field treatment time is 45-75 minutes.

7. The method according to claim 1, characterized in that In step S3, the water bath heating time is 4-8 hours.

8. The method according to claim 1, characterized in that In step S3, the stirring speed is 150-250 rpm.

9. The method according to claim 1, characterized in that In step S1, the stirring speed is 500-1000 rpm, and the stirring time is 2-3 hours.

10. The method according to any one of claims 1 to 9, characterized in that In step S1, the hydration temperature is 2-4° C., and the hydration time is 8-12 h.

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