Ultrasonic phosphoric acid acetylated mung bean protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food processing, and particularly relates to ultrasonic phosphoric acid acetylated mung bean protein as well as a preparation method and application thereof. According to the ultrasonic phosphoric acid acetylated mung bean protein preparation method provided by the invention, the mung bean protein is prepared by utilizing the combined action of three methods of ultrasonic assistance, phosphorylation modification and acetylation modification; the emulsifying activity of the prepared ultrasonic phosphoric acid acetylated mung bean protein reaches 149 mL / g and the emulsifying stability reaches 75%; the double emulsion system prepared on the basis of the ultrasonic phosphate acetylated protein shows the following outstanding properties: the average particle size of liquid drops is as low as 0.6 mu m, the entrapment efficiency is as high as 71%, the liquid drop distribution uniformity is remarkably improved, and meanwhile, the physical stability of the emulsion is effectively enhanced due to the moderate increase of the viscosity of the system.

Description

Technical Field

[0001] This invention belongs to the technical field of food processing, and specifically relates to an ultrasonically phosphorylated mung bean protein, its preparation method and application. Background Technology

[0002] As a high-quality plant-based protein resource, mung bean protein has shown broad application prospects in the fields of food, health products, and biomaterials due to its rich amino acid content, low allergenicity, and potential functional properties.

[0003] However, existing processes for preparing mung bean protein still face technical bottlenecks. Traditional methods often employ acid-base treatment combined with centrifugation, but mung bean protein has a wide isoelectric point distribution (pH 4.5-5.2). Single pH control is insufficient to simultaneously precipitate different protein components, leading to solubility mutations in some proteins under acidic or alkaline conditions, resulting in incomplete precipitation or over-dissolution, thus reducing the extraction rate of mung bean protein. Furthermore, drastic pH changes during acid-base treatment can easily damage the tertiary structure of proteins, causing irreversible aggregation of exposed hydrophobic groups without effective protection, resulting in weak stability and low emulsification properties of the obtained mung bean protein. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a mung bean protein, its preparation method, and its applications. This invention employs safe and effective methods such as ultrasound, acetylation, and phosphorylation to regulate the functional properties of mung bean protein, enhancing its solubility and stability; it also improves the extraction rate of mung bean protein. The combined effect of ultrasound-assisted modification, phosphorylation modification, and acetylation modification enhances the stability and emulsifying properties of mung bean protein.

[0005] This invention provides a highly emulsifiable and stable ultrasonically phosphorylated mung bean protein, which is ultrasonically phosphorylated mung bean protein UPA-MBPI. It is obtained by using crude mung bean protein as raw material, which is modified by pH shift, phosphorylated with sodium tripolyphosphate, acetylated with acetic anhydride, and then ultrasonically treated.

[0006] This invention also discloses a method for preparing the above-mentioned ultrasonically phosphorylated mung bean protein, comprising the following steps: (1) The crude mung bean protein was subjected to pH shift modification treatment to obtain pH shift modified mung bean protein MBPI. The pH shift modification operation is as follows: first, the pH value of the crude mung bean protein solution is adjusted to 8.0-9.5, the supernatant is collected after centrifugation, the pH of the supernatant is adjusted to 4.0-5.0, the precipitate is collected, the precipitate is washed and centrifuged, then reconstituted, the pH is adjusted to 7.0, dialyzed, and dried to obtain mung bean protein MBPI; (2) Phosphorylation modification of mung bean protein MBPI: The mung bean protein MBPI obtained in (1) was dissolved in water, the pH was adjusted to 8.0-9.5, sodium tripolyphosphate was added, the pH was adjusted to 4.0-5.0, the precipitate was collected and dried to obtain phosphorylated mung bean protein P-MBPI; (3) Ultrasonic acetylation modification of phosphorylated mung bean protein P-MBPI: dissolve the phosphorylated mung bean protein P-MBPI in (2) in water, adjust the pH to 8.0-9.5, add acetic anhydride, sonicate, adjust the pH to 4.0-5.0, collect the precipitate, dry it, and obtain ultrasonic phosphorylated mung bean protein UPA-MBPI.

[0007] Preferably, in (1), the concentration of the mung bean crude protein solution is 0.1-0.3 g / mL.

[0008] As a preferred embodiment, in (2), after adjusting the pH to 8.0-9.5 with sodium hydroxide solution, 0.5-1.5g of sodium tripolyphosphate is added, and then the pH is adjusted to 4.0-5.0 with hydrochloric acid solution.

[0009] As a preferred embodiment, in (3), the pH is adjusted to 8.0-9.5 with sodium hydroxide solution, 0.5-1.5g of acetic anhydride is added, ultrasonic treatment is performed for 20-30min, and the pH is adjusted to 4.0-5.0 with hydrochloric acid solution.

[0010] The present invention also provides a dual emulsion containing the above-mentioned ultrasonically phosphated mung bean protein UPA-MBPI; specifically comprising an inner aqueous phase W1, an oil phase O, and an outer aqueous phase W2; wherein the inner aqueous phase W1 and the oil phase O are prepared at a mass ratio of 1:(2-8) to obtain a primary emulsion W1 / O, and the mass ratio of the primary emulsion W1 / O to the outer aqueous phase W2 is 2:3; in the inner aqueous phase W1, glycerol accounts for 1%-4%, trehalose accounts for 5%-15%, and ultrasonically phosphated mung bean protein UPA-MBPI accounts for 10%-20%; in the oil phase O, the mass-volume ratio of emulsifier to oil is 3 g:(45-55) mL; in the outer aqueous phase W2, the mass-volume ratio of whey protein isolate: compounding materials:distilled water is (2.5-8.0) g:(0.15-0.5) g:100 mL.

[0011] The above-mentioned double emulsion is prepared by the following steps: S1 Preparation of internal aqueous phase W1: Weigh glycerol, trehalose, and ultrasonically phosphorylated mung bean protein UPA-MBPI, dissolve them in distilled water, stir and mix well to obtain internal aqueous phase W1; S2 Preparation of oil phase O: Add emulsifier to oil, wherein the mass-volume ratio of emulsifier to oil is 3g:(45-55)mL, stir and mix, cool to room temperature, and obtain oil phase O; S3 Preparation of external aqueous phase W2: Mix whey protein isolate and compound material to prepare a solution, wherein the mass-volume ratio of whey protein isolate: compound material: distilled water is (2.5-8.0) g: (0.15-0.5) g: 100 mL, stir and refrigerate overnight to obtain external aqueous phase W2; S4 Preparation of primary emulsion W1 / O: At 35-60℃, the inner aqueous phase W1 is added dropwise to the oil phase O while stirring, wherein the mass ratio of the inner aqueous phase W1 to the oil phase O is 1:(2-8). Under ice bath conditions, the emulsion is sheared at a speed of 10000-16000 r / min for 5-15 min to obtain primary emulsion W1 / O. S5 Preparation of dual emulsion: The primary emulsion W1 / O and the external aqueous phase W2 are mixed at a mass ratio of 2:(1.5-5), and then sheared at a speed of 6000-10000 r / min for 2-8 min using a high-speed shearing machine to obtain the dual emulsion W1 / O / W2.

[0012] Preferably, in (2), the emulsifier is selected from any one of polyglycerol ricinoleate, lecithin, sucrose fatty acid ester, Span 80, and mono- and diglyceride fatty acid esters; The oil is selected from any one of sunflower seed oil, soybean oil, or medium-chain triglycerides.

[0013] Preferably, in (3), the compound material is selected from any one of sodium caseinate SC, sodium alginate-calcium carbonate, and β-cyclodextrin β-CD.

[0014] The present invention has the following advantages and effects compared with the prior art: (1) The present invention uses safe and effective methods such as ultrasound, acetylation and phosphorylation to regulate the functional properties of mung bean protein, thereby enhancing the solubility of mung bean protein and improving its stability; (2) To improve the extraction rate of mung bean protein, the stability and emulsifying properties of mung bean protein were improved by using a combination of three methods: ultrasound-assisted extraction, phosphorylation modification, and acetylation modification. Data from the examples show that the emulsifying property value can reach 149 mL / g and the emulsifying stability value can reach 75%. (3) The double emulsion obtained by the mung bean protein preparation method provided by the present invention has significant advantages: its minimum particle size reaches 0.6 μm and its encapsulation rate is as high as 71%. This characteristic indicates that the emulsion droplet distribution is more uniform, and the increase in viscosity within the system effectively enhances stability. Attached Figure Description

[0015] Figure 1 The image shows a macroscopic view of mung bean protein solutions, from left to right: crude mung bean protein solution, phosphorylated mung bean protein solution, acetylated mung bean protein solution, phosphorylated acetylated mung bean protein solution, and ultrasonically phosphorylated acetylated mung bean protein solution. Figure 2Particle size distribution of mung bean protein under different treatment conditions; Figure 3 The surface hydrophobicity of mung bean protein under different treatment conditions; Figure 4 Determination of the foaming properties of mung bean protein under different treatment conditions; Figure 5 Determination of the emulsifying properties and emulsifying stability of mung bean protein under different treatment conditions; Figure 6 This is a double-sided microparticle size diagram; Figure 7 The image shows macroscopic views of two emulsions, from left to right: macroscopic views of emulsions prepared from mung bean protein, phosphorylated mung bean protein, acetylated mung bean protein, phosphorylated acetylated mung bean protein, and ultrasonically phosphorylated acetylated mung bean protein. Figure 8 A graph showing the stability index of a dual emulsion; Figure 9 This is a diagram showing the encapsulation rate of the double emulsion. Detailed Implementation

[0016] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0017] Example 1 This embodiment provides methods for preparing five proteins: mung bean protein, phosphorylated mung bean protein, acetylated mung bean protein, phosphorylated acetylated mung bean protein, and ultrasonically phosphorylated acetylated mung bean protein.

[0018] The specific steps for processing mung bean protein, phosphorylated mung bean protein, and ultrasonically phosphorylated acetylated mung bean protein are as follows: (1) Prepare a mung bean crude protein solution by taking commercially available mung bean crude protein. The concentration of the mung bean crude protein solution is 0.1 g / mL. Adjust the pH to 9.0 with a 2 mol / L sodium hydroxide solution. Centrifuge at 4℃ and 10000 r / min for 20 min. Collect the supernatant. Adjust the pH of the supernatant to 4.5 with a 2 mol / L hydrochloric acid solution. Let it stand for 30 min. Centrifuge at 8000 r / min for 15 min. Collect the precipitate. Wash the precipitate three times with deionized water. Centrifuge again after washing. Redissolve the precipitate with 3-5 times the amount of deionized water. Adjust the pH to 7.0. Dialyze and freeze dry to obtain mung bean protein MBPI. (2) Dissolve 20g of mung bean protein MBPI in 200mL of deionized water, adjust the pH to 9.0 with 2mol / L sodium hydroxide solution, add 1g of sodium tripolyphosphate (STP), adjust the pH to 4.5 with 2mol / L hydrochloric acid solution, let stand, centrifuge at 8000r / min for 10min, collect the precipitate, dialyze, freeze dry to obtain phosphorylated mung bean protein P-MBPI; (3) Dissolve the phosphorylated mung bean protein P-MBPI in (2) in deionized water, adjust the pH to 9.0 with 2 mol / L sodium hydroxide solution, add 1 g acetic anhydride, sonicate at 25℃ and 300 W for 30 min, adjust the pH to 4.5 with 2 mol / L hydrochloric acid solution, let stand, centrifuge at 8000 r / min for 10 min, take the precipitate, dialyze, freeze dry, and obtain sonicated phosphorylated mung bean protein UPA-MBPI.

[0019] The preparation methods for acetylated mung bean protein and phosphorylated acetylated mung bean protein are as follows: Preparation of acetylated mung bean protein: Mung bean protein MBPI was dissolved in deionized water, the pH was adjusted to 9.0 with 2 mol / L sodium hydroxide solution, 1 g acetic anhydride was added, the pH was adjusted to 4.5 with 2 mol / L hydrochloric acid solution, the mixture was allowed to stand, centrifuged at 8000 r / min for 10 min, the precipitate was collected, dialyzed, and freeze-dried to obtain acetylated mung bean protein A-MBPI; Preparation of phosphorylated acetylated mung bean protein: Phosphorylated mung bean protein P-MBPI was dissolved in deionized water, and the pH was adjusted to 9.0 with 2 mol / L sodium hydroxide solution. 1 g of acetic anhydride was added, and the pH was adjusted to 4.5 with 2 mol / L hydrochloric acid solution. After standing, the mixture was centrifuged at 8000 r / min for 10 min, the precipitate was collected, dialyzed, and freeze-dried to obtain phosphorylated acetylated mung bean protein PA-MBPI.

[0020] Example 2 A method for preparing ultrasonically phosphorylated mung bean protein includes the following steps: (1) Prepare a mung bean crude protein solution by taking commercially available mung bean crude protein. The concentration of the mung bean crude protein solution is 0.2 g / mL. Adjust the pH to 8.0 with a 2 mol / L sodium hydroxide solution. Centrifuge at 4℃ and 8000 r / min for 25 min. Collect the supernatant. Adjust the pH of the supernatant to 4.0 with a 2 mol / L hydrochloric acid solution. Let it stand for 25 min. Centrifuge at 5000 r / min for 20 min. Collect the precipitate. Wash the precipitate three times with deionized water. Centrifuge again after washing. Redissolve the precipitate with 5 times the amount of deionized water. Adjust the pH to 7.0. Dialyze and freeze dry to obtain mung bean protein MBPI. (2) Dissolve 20g of mung bean protein MBPI in 200mL of deionized water, adjust the pH to 8.0 with 2mol / L sodium hydroxide solution, add 0.5g of sodium tripolyphosphate (STP), adjust the pH to 4.0 with 2mol / L hydrochloric acid solution, let stand, centrifuge at 8000r / min for 10min, collect the precipitate, dialyze, freeze dry to obtain phosphorylated mung bean protein P-MBPI; (3) Dissolve the phosphorylated mung bean protein P-MBPI in (2) in deionized water, adjust the pH to 8.0 with 2 mol / L sodium hydroxide solution, add 0.5 g acetic anhydride, sonicate at 25℃ and 300 W for 30 min, adjust the pH to 4.0 with 2 mol / L hydrochloric acid solution, let stand, centrifuge at 8000 r / min for 10 min, take the precipitate, dialyze, freeze dry, and obtain sonicated phosphorylated acetylated mung bean protein UPA-MBPI.

[0021] Example 3 (1) Prepare a mung bean crude protein solution by taking commercially available mung bean crude protein. The concentration of the mung bean crude protein solution is 0.3 g / mL. Adjust the pH to 9.5 with a 2 mol / L sodium hydroxide solution. Centrifuge at 8℃ and 12000 r / min for 15 min. Collect the supernatant. Adjust the pH of the supernatant to 5.0 with a 2 mol / L hydrochloric acid solution. Let stand for 35 min. Centrifuge at 10000 r / min for 10 min. Collect the precipitate. Wash the precipitate 3 times with deionized water. Centrifuge again after washing. Redissolve the precipitate with 4 times the amount of deionized water. Adjust the pH to 7.0. Dialyze. Freeze dry to obtain mung bean protein MBPI. (2) Dissolve 20g of mung bean protein MBPI in 200mL of deionized water, adjust the pH to 9.5 with 2mol / L sodium hydroxide solution, add 1.5g of sodium tripolyphosphate (STP), adjust the pH to 5.0 with 2mol / L hydrochloric acid solution, let stand, centrifuge at 6000r / min for 20min, collect the precipitate, dialyze, freeze dry to obtain phosphorylated mung bean protein P-MBPI; (3) Dissolve the phosphorylated mung bean protein P-MBPI in (2) in deionized water, adjust the pH to 9.0 with 2 mol / L sodium hydroxide solution, add 1 g acetic anhydride, sonicate at 30℃ and 350 W for 25 min, adjust the pH to 4.5 with 2 mol / L hydrochloric acid solution, let stand, centrifuge at 10000 r / min for 5 min, take the precipitate, dialyze, freeze dry, and obtain sonicated phosphorylated mung bean protein UPA-MBPI.

[0022] Example 4 Each lyophilized sample from Example 1 was prepared into a 5 mg / mL protein solution. The prepared protein solutions were dispensed into 20 mL glass bottles and placed at room temperature. Macroscopic images of the protein solutions were then taken and recorded.

[0023] like Figure 1 As shown, compared with mung bean protein prepared by other methods, the protein solution of ultrasonically phosphorylated mung bean protein UPA-MBPI is clearer, indicating that the solution has high solubility, which is also consistent with the subsequent particle size characterization results.

[0024] Example 5 The particle size, surface hydrophobicity, foaming properties, emulsifying properties, and emulsion stability of five proteins obtained—mung bean protein, phosphorylated mung bean protein, acetylated mung bean protein, phosphorylated acetylated mung bean protein, and ultrasonically phosphorylated acetylated mung bean protein—were determined. Unless otherwise specified, the freeze-dried samples in this example refer to the freeze-dried samples prepared in Example 1.

[0025] (1) Particle size test A 5 mg / mL protein solution was prepared from the lyophilized sample, and the particle size distribution of the protein was determined using a nanoparticle size potentiometer. The results are as follows. Figure 2 As shown in Table 1, the refractive index of the protein particles is 1.492, and the measurement temperature is 25℃. The instrument detects the diffusion of Brownian motion particles through dynamic light scattering and uses its built-in software to process and convert it into particle size distribution.

[0026] Table 1. Particle size data for five proteins / mung bean protein Phosphorylated mung bean protein Acetylated mung bean protein Phosphorylated mung bean protein Ultrasonic phosphorylated mung bean protein Particle size (μm) 154 ± 2.1 a ]] 126 ± 3.0 b ]] 130 ± 2.5 b ]] 105 ± 2.0 c ]] 95 ± 2.3 d ]]

[0027] The results showed that the particle size of the above proteins, from largest to smallest, was as follows: mung bean protein > acetylated mung bean protein > phosphorylated mung bean protein > phosphorylated acetylated mung bean protein > sonicated phosphorylated acetylated mung bean protein. Among them, the particle size of mung bean protein was 154 μm, the particle size of acetylated mung bean protein and phosphorylated mung bean protein were similar at 130 μm, the particle size of sonicated phosphorylated acetylated mung bean protein was the smallest at 95 μm, followed by phosphorylated acetylated mung bean protein at 105 μm.

[0028] The aforementioned experimental data demonstrate that ultrasonic-assisted phosphorylation of mung bean protein can disrupt non-covalent bonds such as hydrogen bonds and hydrophobic interactions between protein particles, thereby altering the protein's aggregation state. This study indicates that ultrasonic-assisted phosphorylation can effectively reduce protein particle size. Compared to other proteins, this treatment exhibits better emulsifying properties, primarily due to the smaller protein particle size and more flexible molecular structure.

[0029] (2) Surface hydrophobicity test Prepare protein solutions of 5 mg / mL from lyophilized samples, resulting in concentrations of 0.005%, 0.01%, 0.015%, and 0.02% (m / v). Add 50 μL of ANS fluorescent probe to each 5 mL sample. Measure the exogenous fluorescence intensity at an excitation wavelength of 390 nm and an emission wavelength of 400-650 nm. The surface hydrophobicity of the modified proteins is represented as the initial slope of the graph showing the relationship between maximum fluorescence intensity and protein concentration. The results are shown below. Figure 3 And as shown in Table 2 below.

[0030] Table 2 shows the hydrophobicity data. / mung bean protein Phosphorylated mung bean protein Acetylated mung bean protein Phosphorylated mung bean protein Ultrasonic phosphorylated mung bean protein Surface hydrophobicity 5220 ± 110 a ]] 4950 ± 121 c ]] 4690 ± 112 b ]] 4120 ± 130 a ]] 3590 ± 101 d ]]

[0031] The results showed that among the five proteins, mung bean protein had the lowest surface hydrophobicity at only 5220, while sonicated phosphorylated mung bean protein exhibited the highest surface hydrophobicity at 3590. This indicates that the treatment enhanced the ionic interaction between protein molecules and water molecules, and the carboxyl and amino groups of the protein were ionized, thus making them more prone to exposure to hydrophilic environments. The polarity of the hydrophobic regions of the protein increased, resulting in a decrease in surface hydrophobicity.

[0032] (3) Foaming property test Prepare a 5 mg / mL protein solution. Take 30 mL of the protein solution and treat it under a shear press at 10000 rpm for 2 min. Transfer the foam and solution to a graduated cylinder and measure the volume to determine the foaming property. The results are as follows: Figure 4 And as shown in Table 3 below.

[0033] Table 3 Foaming data / mung bean protein Phosphorylated mung bean protein Acetylated mung bean protein Phosphorylated mung bean protein Ultrasonic phosphorylated mung bean protein Foaming property (%) 37 ± 1.12 c ]] 81 ± 1.51 b ]] 82 ± 1.25 b ]] 89 ± 2.0 a ]] 98 ± 1.52 a ]]

[0034] The foaming properties of mung bean protein were measured. The foaming properties of ultrasonically phosphorylated mung bean protein were relatively poor, while those of ultrasonically phosphorylated mung bean protein were the best, reaching 98%. This indicates that the mung bean protein treated with ultrasonic phosphorylated protein has a more flexible structure and lower protein aggregation, which allows the protein molecules to unfold and increases the interaction between protein molecules.

[0035] (4) Detection of emulsifying properties (EAI) and emulsifying stability (ESI) A certain amount of sample protein was dissolved in PBS solution, and soybean oil was added to the protein solution. The mixture was homogenized at 10000 rpm for 3 min. The protein emulsion was collected from the bottom of the solution and mixed with 0.1% SDS. The absorbance (A0) was measured at 500 nm. After standing for 10 minutes, the absorbance (A10) was measured again. The emulsifying property (EAI) and emulsifying stability (ESI) were calculated, and the results are as follows: Figure 5 And as shown in Table 4 below.

[0036] The calculation formula is as follows:

[0037]

[0038] In the formula: DF = dilution factor; C = sample concentration; φ = 0.25.

[0039] Table 4. Emulsifying Integrity (EAI) and Emulsifying Stability (ESI) Data for Five Proteins / mung bean protein Phosphorylated mung bean protein Acetylated mung bean protein Phosphorylated mung bean protein Ultrasonic phosphorylated mung bean protein EAI (mL / g) 91 ± 2.21 c ]] <![CDATA[112±2.11 b ]]> <![CDATA[108±1.65 b ]]> <![CDATA[137±1.90 a ]]> <![CDATA[149±1.85 a ]]> ESI (%) <![CDATA[21±1.3 c ]]> <![CDATA[40±1.52 b ]]> <![CDATA[42±0.8 b ]]> <![CDATA[66±2.0 a ]]> <![CDATA[75±0.92 a ]]>

[0040] Emulsifying properties and emulsifying stability were determined. Ultrasonic phosphorylated mung bean protein emulsion exhibited higher emulsifying properties and the highest emulsifying stability.

[0041] The basic characterization of five proteins—mung bean protein, phosphorylated mung bean protein, acetylated mung bean protein, phosphorylated acetylated mung bean protein, and sonicated phosphorylated acetylated mung bean protein—was determined.

[0042] The following conclusions were drawn: Mung bean protein exhibited the lowest emulsifying activity and stability, at 91 mL / g and 20%, respectively. Phosphorylated and acetylated mung bean protein showed relatively similar emulsifying activities and stability. Ultrasonic treatment of acetylated phosphate mung bean protein resulted in the highest and most stable emulsifying activity and stability, reaching 149 mL / g and 75%, respectively. Acetylated phosphate mung bean protein followed, with 137 mL / g and 66%, respectively. While the macroscopic images of these proteins showed little difference, the emulsion TSI value of ultrasonically treated acetylated phosphate mung bean protein was the lowest, indicating that this treatment was the most effective in stabilizing the emulsion.

[0043] Example 6 Preparation of a dual emulsion The preparation of a double emulsion based on mung bean protein, which has undergone acetylation, phosphorylation, and ultrasonic treatment, includes the following steps: (1) Preparation of the inner aqueous phase (W1) of the double emulsion: Weigh 2% (w / v) glycerol, 10% (w / v) trehalose and 15% (w / v) different types of mung bean protein prepared in Example 1 and dissolve them in distilled water. Stir on a magnetic stirrer for 30 min to mix them thoroughly and set aside. (2) Preparation of oil phase (O): According to the mass of oil phase, add 6% (w / v) of emulsifier polyglycerol ricinoleate (PGPR) to sunflower seed oil, wherein PGPR: oil = 3:50. Stir at 40°C in a water bath for 30 minutes to mix it thoroughly, cool to room temperature, and obtain oil phase O for later use. (3) Preparation of external aqueous phase (W2): Weigh 5% (w / v) whey protein isolate (WPI) and 0.25% (w / v) SC according to the mass of the aqueous phase, mix them, use distilled water as solvent to prepare the solution, place it on a magnetic stirrer and stir for 2 hours, then put it in a 4°C refrigerator and refrigerate overnight. (4) Preparation of W1 / O primary emulsion: In a 40℃ water bath, the inner aqueous phase is added dropwise to the oil phase while stirring. The mass ratio of the inner aqueous phase (W1) to the oil phase (O) is 1:4. Then, under the condition of 0℃ ice bath, it is sheared at a speed of 13000r / min for 10min to obtain W1 / O primary emulsion. (5) Preparation of W1 / O / W2 double emulsion: The primary W1 / O emulsion and the external aqueous phase are mixed at a mass ratio of 2:3 and then sheared at a speed of 8000 r / min for 5 min using a high-speed shearing machine. The W1 / O / W2 double emulsion is then prepared.

[0044] The particle size and particle size distribution of the W1 / O / W2 dual emulsion were determined using a laser particle size analyzer (Malvern 2000). Monitoring was conducted at 0, 7, and 15 days of sample storage, with the following parameters set: refractive indices of sunflower seed oil and water of 1.475 and 1.333, respectively. The emulsion was added to the dispersion medium using a dropper, ensuring the sample opacity was approximately 5%. The volume-weighted average particle size (D4 / 3) was used to characterize the emulsion particle size. Each sample was measured in triplicate. The particle size results for the dual emulsion are shown below. Figure 6 And as shown in Table 5 below.

[0045] Table 5 Emulsion Particle Size Data lotion mung bean protein Phosphorylated mung bean protein Acetylated mung bean protein Phosphorylated mung bean protein Ultrasonic phosphorylated mung bean protein Particle size (μm) <![CDATA[1.6±0.034 a ]]> <![CDATA[1.12±0.05 b ]]> <![CDATA[1.1±0.045 b ]]> <![CDATA[0.8±0.03 c ]]> <![CDATA[0.6±0.04 c ]]>

[0046] The results showed that the smallest droplet size of the ultrasonically phosphorylated mung bean protein emulsion was 0.6 μm, indicating a smaller droplet size and a more uniform droplet distribution. When other conditions remained constant, emulsions with smaller droplet sizes were generally more stable. This is mainly because smaller emulsion droplets have a larger surface area, allowing for better adsorption at the oil-water interface.

[0047] Example 7 Determination of the stability of dual emulsions The prepared emulsion was dispensed into 20mL glass bottles and placed at room temperature. Macroscopic images of the emulsion were then photographed and recorded, such as... Figure 7 The results show that, macroscopically, the emulsions do not differ significantly.

[0048] The Turbiscan Lab stability analyzer can accurately evaluate and predict changes in the clarification layer, sediment layer, and droplet size of emulsions without diluting the sample. It employs a pulsed near-infrared light source (wavelength 880nm) and a vertical scanning principle. Two synchronous optical detectors detect transmitted and backscattered light passing through the sample, respectively. By measuring the intensity of backscattered and transmitted light, it provides a curve showing the thickness change over time. The specific operation involves adding sample to a designated sample vial of the Turbiscan Lab stability analyzer to the same height, with parameters set to scan every 2 minutes for 3 hours, then scan again for 1 hour after 24 hours, scanning every 2 minutes, at a temperature of 25°C.

[0049] The calculation formula is as follows:

[0050] In the formula: Φ = volume fraction of particles; scan i (h) = Backscattered light intensity at position h of the sample during the i-th scan; scan i-1 (h) = Backscattered light intensity at position h of sample during the (i-1)th scan; H = Overall height of the sample, mm.

[0051] An increase in the TSI value indicates a decrease in the physical stability of the emulsion. Figure 8 Data shows that the emulsion with ultrasonically phosphorylated mung bean protein had the lowest TSI value, indicating that this treatment had the best effect on stabilizing the emulsion with mung bean protein.

[0052] Example 8 Determination of the encapsulation rate of dual emulsions Encapsulation loading rate is the most direct method for evaluating the ability of dual emulsions to encapsulate active substances. The encapsulation loading rate of the dual emulsions prepared in Example 4 was determined using the following method: In the preparation of the double emulsion, a certain mass of marker (e.g., 1% (w / v) NaCl, where 1% is the mass fraction relative to the inner aqueous phase) is added to the inner aqueous phase. After preparation, the conductivity meter is used to measure the encapsulation rate. Each sample is in triplicate, and the average value is taken.

[0053] The calculation formula is as follows:

[0054] Table 6. Encapsulation rate of emulsions lotion mung bean protein Phosphorylated mung bean protein Acetylated mung bean protein Phosphorylated mung bean protein Ultrasonic phosphorylated mung bean protein Load factor (%) <![CDATA[36±0.8 d ]]> <![CDATA[48±1.1 c ]]> <![CDATA[43±1.4 c ]]> <![CDATA[63±0.90 a ]]> <![CDATA[71±1.2 a ]]>

[0055] Table 6 combined Figure 9 The results showed that the encapsulation rate of the ultrasonic phosphorylated mung bean protein emulsion was the best, at 71%, indicating that ultrasonic phosphorylated treatment can effectively improve the encapsulation rate of the emulsion. This is also related to the emulsifier, which increases the viscosity of the system. High viscosity restricts the movement of droplets, thereby maintaining the stability of the emulsion.

[0056] In summary, this invention provides a method for preparing mung bean protein with high emulsifying properties and high stability. Specifically, it refers to the combined use of three methods—ultrasound-assisted synthesis, phosphorylation modification, and acetylation modification—to prepare mung bean protein, resulting in an emulsifying activity of 149 mL / g and an emulsifying stability of 75%.

[0057] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A kind of ultrasonically phosphorylated mung bean protein, characterized in that, The mung bean protein is ultrasonically phosphorylated mung bean protein UPA-MBPI, which is obtained by using crude mung bean protein as raw material, through pH shift modification, sodium tripolyphosphate phosphorylation, acetylation of acetic anhydride, and then ultrasonic treatment.

2. The method for preparing the ultrasonically phosphorylated mung bean protein according to claim 1, characterized in that, The steps include the following: (1) The crude mung bean protein was subjected to pH shift modification treatment to obtain pH shift modified mung bean protein MBPI. The pH shift modification operation is as follows: first, the pH value of the crude mung bean protein solution is adjusted to 8.0-9.5, the supernatant is collected after centrifugation, the pH of the supernatant is adjusted to 4.0-5.0, the precipitate is collected, the precipitate is washed and centrifuged, then reconstituted, the pH is adjusted to 7.0, dialyzed, and dried to obtain mung bean protein MBPI; (2) Phosphorylation modification of mung bean protein MBPI: The mung bean protein MBPI obtained in (1) was dissolved in water, the pH was adjusted to 8.0-9.5, sodium tripolyphosphate was added, the pH was adjusted to 4.0-5.0, the precipitate was collected and dried to obtain phosphorylated mung bean protein P-MBPI; (3) Ultrasonic acetylation modification of phosphorylated mung bean protein P-MBPI: dissolve the phosphorylated mung bean protein P-MBPI in (2) in water, adjust the pH to 8.0-9.5, add acetic anhydride, sonicate, adjust the pH to 4.0-5.0, collect the precipitate, dry it, and obtain ultrasonic phosphorylated mung bean protein UPA-MBPI.

3. The method as described in claim 2, characterized in that, (1) The concentration of the mung bean crude protein solution is 0.1-0.3 g / mL.

4. The method as described in claim 2, characterized in that, In (2), after adjusting the pH to 8.0-9.5 with sodium hydroxide solution, 0.5-1.5g of sodium tripolyphosphate is added, and then the pH is adjusted to 4.0-5.0 with hydrochloric acid solution.

5. The method as described in claim 2, characterized in that, (3) Adjust the pH to 8.0-9.5 with sodium hydroxide solution, add 0.5-1.5g of acetic anhydride, sonicate for 20-30min, and adjust the pH to 4.0-5.0 with hydrochloric acid solution.

6. A dual emulsion, characterized in that, It contains the ultrasonically phosphorylated mung bean protein as described in claim 1.

7. The dual emulsion as described in claim 6, characterized in that, Includes internal aqueous phase W1, oil phase O, and external aqueous phase W2; In this process, the internal aqueous phase W1 and the oil phase O are prepared in a mass ratio of 1:(2-8) to obtain the primary emulsion W1 / O, and the mass ratio of the primary emulsion W1 / O to the external aqueous phase W2 is 2:

3. The internal aqueous phase W1 contains 1%-4% glycerol, 5%-15% trehalose, and 10%-20% ultrasonically phosphorylated mung bean protein UPA-MBPI. In the oil phase O, the mass-to-volume ratio of emulsifier to oil is 3 g: (45-55) mL; In the external aqueous phase W2, the mass-volume ratio of whey protein isolate: compound materials: distilled water is (2.5-8.0) g: (0.15-0.5) g: 100 mL.

8. A method for preparing the dual emulsion according to any one of claims 6-7, characterized in that, The steps include the following: S1 Preparation of internal aqueous phase W1: Weigh glycerol, trehalose, and ultrasonically phosphorylated mung bean protein UPA-MBPI, dissolve them in distilled water, stir and mix well to obtain internal aqueous phase W1; S2 Preparation of oil phase O: Add emulsifier to oil, wherein the mass-volume ratio of emulsifier to oil is 3g:(45-55)mL, stir and mix, cool to room temperature, and obtain oil phase O; S3 Preparation of external aqueous phase W2: Mix whey protein isolate and compound material to prepare a solution, wherein the mass-volume ratio of whey protein isolate: compound material: distilled water is (2.5-8.0) g: (0.15-0.5) g: 100 mL, stir and refrigerate overnight to obtain external aqueous phase W2; S4 Preparation of primary emulsion W1 / O: At 35-60℃, the inner aqueous phase W1 is added dropwise to the oil phase O while stirring, wherein the mass ratio of the inner aqueous phase W1 to the oil phase O is 1:(2-8). Under ice bath conditions, the emulsion is sheared at a speed of 10000-16000 r / min for 5-15 min to obtain primary emulsion W1 / O. S5 Preparation of dual emulsion: The primary emulsion W1 / O and the external aqueous phase W2 are mixed at a mass ratio of 2:(1.5-5), and then sheared at a speed of 6000-10000 r / min for 2-8 min using a high-speed shearing machine to obtain the dual emulsion W1 / O / W2.

9. The preparation method as described in claim 8, characterized in that, In (2), the emulsifier is selected from any one of polyglycerol ricinoleate, lecithin, sucrose fatty acid ester, Span 80, and mono- and diglyceride fatty acid esters; The oil is selected from any one of sunflower seed oil, soybean oil, or medium-chain triglycerides.

10. The preparation method according to claim 8, characterized in that, (3) The compound material is selected from any one of sodium caseinate SC, sodium alginate-calcium carbonate, and β-cyclodextrin β-CD.

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