Method for modifying legume protein isolate based on metal-phenolic network and application thereof

Abstract

The application takes soybean, pea, mung bean, broad bean and chickpea protein isolate as the object, EGCG and Ca 2+ , Mg 2+ or Fe 3+ are coordinated and assembled into metal-phenolic network (MPNs) with a 2:1 molar ratio, and a complex is prepared by mixing reaction with the protein at pH 9.0. With the increase of metal coordination ability, the structure of MPNs tends to be dense; hydrogen bond, electrostatic, hydrophobic and coordination synergistically regulate the protein conformation. The absolute value of the particle size and the zeta potential of the complex is improved, the antioxidant activity is increased from 40% to 55% to 75% to 95%, the gel strength and emulsion stability are enhanced, and the response of SPI and MPI is the most significant. The method is green and mild, the complex has structural stability, interfacial activity and antioxidant performance, and can be applied to the fields of emulsion, gel food, plant-based substitutes and functional food.

Description

Technical Field

[0001] This invention belongs to the field of protein modification technology, specifically involving the functional modification of plant proteins. Using soy protein isolate, pea protein isolate, mung bean protein isolate, broad bean protein isolate, and chickpea protein isolate as modification targets, epigallocatechin gallate (EGCG) is used to construct a metal-phenolic network with calcium, magnesium, and iron ions. Through hydrogen bonding, electrostatic interactions, hydrophobicity, and coordination synergistic effects, the protein conformation and aggregation behavior are regulated, significantly improving its structural stability, interfacial activity, and antioxidant properties. The modified legume protein isolates can be used as plant-based emulsifiers, gel enhancers, and functional ingredients, and are widely applied in emulsion systems, gel foods, plant-based substitutes, and functional foods. Background Technology

[0002] Legumes are a key protein source for human and animal nutrition, with representative sources including soybeans, peas, broad beans, mung beans, and chickpeas. Legume proteins are rich in protein, have a balanced amino acid profile, and offer advantages such as low cost, high yield, and low environmental impact, holding an important position in the global plant protein market. However, compared to animal proteins, legume proteins generally suffer from functional defects such as low solubility, poor emulsification, and insufficient gel strength, and are more sensitive to pH, ionic strength, and temperature. For example, broad bean protein has a dense molecular structure and a high proportion of hydrophobic amino acids, easily forming large, insoluble particles with weak functions; while pea protein isolate and chickpea protein have high nutritional value, their functional properties still cannot meet the diverse needs of the food industry.

[0003] To improve the functional properties of legume proteins, researchers have explored various modification methods. Physical modification (such as ultrasound, high pressure, and extrusion) offers limited effectiveness, and excessive heat treatment can easily lead to protein denaturation and aggregation. Chemical modification (such as glycosylation and deamidation) is complex and carries certain safety risks. Enzymatic modification is costly and requires stringent reaction conditions. Each of these methods has its limitations, necessitating the development of a novel, green, efficient, and controllable strategy for legume protein modification.

[0004] Metal-Phenolic Networks (MPNs) are supramolecular network structures formed by the self-assembly of polyphenol ligands and metal ions through dynamic coordination bonds. MPNs combine the antioxidant and antibacterial properties of polyphenols with the functional activity of metal ions, and exhibit good biocompatibility. In recent years, MPNs have been preliminarily applied in the field of protein modification: studies have shown that iron ion-EGCG metallophenolic networks can improve the structural and functional properties of soy protein isolate; pH-regulated MPNs can optimize the texture and mechanical properties of soy protein gels; and walnut meal isolate can have its solubility significantly improved by coordinating with metallophenolic networks. However, existing research has mostly focused on single soy protein systems, lacking systematic comparison and in-depth analysis of the structural responses and functional changes of different legume proteins (such as pea, mung bean, broad bean, and chickpea proteins) under the action of MPNs. The differences in modification effects caused by the varying 7S / 11S globulin ratios of different legume proteins remain unclear.

[0005] In view of this, this invention uses soy protein isolate, pea protein isolate, mung bean protein isolate, broad bean protein isolate, and chickpea protein isolate as model proteins, and MPNs constructed by EGCG with calcium, magnesium, and iron ions as a modification system. It systematically studies the regulatory laws of MPNs on the structural and functional properties of different legume proteins, aiming to make up for the lack of horizontal comparison of different legume proteins in the existing technology, and to provide a new technical path for the targeted modification and high-value utilization of legume proteins. Summary of the Invention

[0006] To address the limitations of existing legume protein modification technologies, such as the limited effectiveness of physical modification, safety concerns associated with chemical modification, high costs and stringent reaction conditions for enzymatic modification, and the fact that current MPNs modification research largely focuses on single soybean protein systems and lacks systematic comparison and targeted regulation of different legume proteins, this invention provides a method and application for modifying legume isolated proteins based on metal-phenolic networks.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: (1) Preparation of legume protein isolate: Soybeans, peas, mung beans, broad beans, and chickpeas were crushed and passed through a 60-mesh sieve. The resulting bean flour was mixed with n-hexane at a mass-to-volume ratio of 1:3 for defatting, and the process was repeated three times. The defatted bean flour was dispersed in deionized water at a ratio of 1:10 (w / v), and the pH was adjusted to 8.5 with NaOH. The mixture was stirred and extracted for 2 hours. The extract was centrifuged at 10,000×g for 30 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with 2 mol / L hydrochloric acid, and the mixture was allowed to stand to precipitate the protein. The precipitate was centrifuged at 6,000×g at 4°C for 20 minutes, and the supernatant was discarded. The precipitate was washed three times with deionized water to remove soluble impurities. After washing, the protein precipitate was adjusted to neutral pH with 2 mol / L NaOH and then freeze-dried to obtain soy protein isolate (SPI), pea protein isolate (PPI), mung bean protein isolate (MPI), broad bean protein isolate (FPI), and chickpea protein isolate (CPI). The protein content was determined to be no less than 90% by the Kjeldahl method. (2) Preparation of metal-phenol network (MPNs) solution: EGCG was dissolved in deionized water and fully dissolved under light-protected conditions to prepare a 48 mM EGCG solution; calcium ions, magnesium ions, and iron ions were dissolved in deionized water to prepare a 48 mM metal ion solution. The EGCG solution and the metal ion solution were mixed at a volume ratio of 2:1 and reacted under magnetic stirring at 25°C and 1000 rpm in the dark to fully crosslink EGCG with metal ions, forming calcium ion-EGCG MPNs, magnesium ion-EGCG MPNs, and iron ion-EGCG MPNs solutions, respectively. The pH of the MPNs solution was adjusted to 9.0 with 1 M NaOH solution. (3) Preparation of Legume Protein-MPNs Complex: Disperse the SPI, PPI, MPI, FPI or CPI obtained in step (1) in deionized water to prepare a 1% (w / v) protein solution. After stirring continuously for 2 hours, let it stand overnight at 4°C to ensure that the protein is fully hydrated. Add 100 μL of the MPNs solution obtained in step (2) (the mass ratio of protein to MPNs is 50:1) to 5 mL of protein solution, seal with plastic wrap, and stir for 60 minutes under light-proof conditions. During the reaction, maintain the pH of the system at 9.0 to obtain the Legume Protein-MPNs Complex solution. After freeze-drying, the Legume Protein-MPNs Complex powder is obtained.

[0008] This invention systematically investigated the regulatory effects of metal-phenolic networks (MPNs) on five legume isolates. TEM showed that the microstructure of MPNs gradually became denser with increasing metal coordination ability (calcium ions < magnesium ions < iron ions), and the iron ion-EGCG system formed dense lamellar aggregates. FTIR confirmed that MPNs altered protein conformation through hydrogen bonding, electrostatics, hydrophobicity, and synergistic coordination, with SPI and MPI showing the most significant responses to MPNs. Antioxidant activity showed that MPNs increased the protein free radical scavenging rate from 40%–55% to 75%–95%, with the enhancement effect following the order of calcium ions < magnesium ions < iron ions. This invention significantly improves the structural stability and antioxidant properties of legume proteins through multiple synergistic modifications by MPNs, providing a new strategy for their application in functional foods and plant-based products. Attached Figure Description

[0009] Figure 1 The figure below is a summary of this experiment; Figure 2 This is a TEM image of the metal-polyphenol network from Example 1. Figure 3 The FTIR spectrum of the protein-metal-polyphenol complex in Example 2 is shown below. Figure 4 This is a graph showing the antioxidant properties of the protein-metal-polyphenol complex in Example 3. Detailed Implementation

[0010] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0011] (1) Preparation of legume protein isolate: Soybeans, peas, mung beans, broad beans, and chickpeas were crushed and passed through a 60-mesh sieve. The resulting bean flour was mixed with n-hexane at a mass-to-volume ratio of 1:3 for defatting, and the process was repeated three times. The defatted bean flour was dispersed in deionized water at a ratio of 1:10 (w / v), and the pH was adjusted to 8.5 with NaOH. The mixture was stirred and extracted for 2 hours. The extract was centrifuged at 10,000×g for 30 minutes, and the supernatant was collected. The pH was adjusted to 4.5 with 2 mol / L hydrochloric acid, and the mixture was allowed to stand to precipitate the protein. The precipitate was centrifuged at 6,000×g at 4°C for 20 minutes, and the supernatant was discarded. The precipitate was washed three times with deionized water to remove soluble impurities. After washing, the protein precipitate was adjusted to neutral pH with 2 mol / L NaOH and then freeze-dried to obtain soy protein isolate (SPI), pea protein isolate (PPI), mung bean protein isolate (MPI), broad bean protein isolate (FPI), and chickpea protein isolate (CPI). The protein content was determined to be no less than 90% by the Kjeldahl method. (2) Preparation of metal-phenol network (MPNs) solution: EGCG was dissolved in deionized water and fully dissolved under light-protected conditions to prepare a 48 mM EGCG solution; calcium ions, magnesium ions, and iron ions were dissolved in deionized water to prepare a 48 mM metal ion solution. The EGCG solution and the metal ion solution were mixed at a volume ratio of 2:1 and reacted under magnetic stirring at 25°C and 1000 rpm in the dark to fully crosslink EGCG with metal ions, forming calcium ion-EGCG MPNs, magnesium ion-EGCG MPNs, and iron ion-EGCG MPNs solutions, respectively. The pH of the MPNs solution was adjusted to 9.0 with 1 M NaOH solution. (3) Preparation of Legume Protein-MPNs Complex: Disperse the SPI, PPI, MPI, FPI or CPI obtained in step (1) in deionized water to prepare a 1% (w / v) protein solution. After stirring continuously for 2 hours, let it stand overnight at 4°C to ensure that the protein is fully hydrated. Add 100 μL of the MPNs solution obtained in step (2) (the mass ratio of protein to MPNs is 50:1) to 5 mL of protein solution, seal with plastic wrap, and stir for 60 minutes under light-proof conditions. During the reaction, maintain the pH of the system at 9.0 to obtain the Legume Protein-MPNs Complex solution. After freeze-drying, the Legume Protein-MPNs Complex powder is obtained.

[0012] Example 1: The morphology of MPNs was characterized and analyzed using a JEM-2100 transmission electron microscope (TEM) under the conditions of 100 kV accelerating voltage and 500 nm resolution.

[0013] See results Figure 2EGCG–Ca MPN exhibits a relatively compact and porous network structure, characterized by irregularly distributed aggregates and large pore spaces, indicating relatively weak intermolecular cross-linking. In contrast, EGCG–Mg MPN displays a more compact and uniform morphology, with smaller pore sizes and enhanced connectivity between aggregates. This indicates enhanced intermolecular interactions, which may be attributed to more effective coordination between magnesium ions and the phenolic hydroxyl groups in EGCG, thereby promoting cross-linking and structural stability. Notably, EGCG–Fe MPN exhibits significantly different morphological characteristics, manifesting as larger, denser, and partially fused aggregates with a lamellar structure. The significant increase in aggregate size and structural compactness indicates stronger intermolecular association. This can be attributed to the strong coordination ability between Fe³⁺ and polyphenolic compounds, thereby forming a stable metal-polyphenol network and initiating widespread aggregation. Overall, these results indicate that different metal ions play a key role in regulating the microstructure of EGCG assemblies. With the enhancement of coordination ability (calcium ion < magnesium ion < iron ion), the system structure gradually becomes more compact and exhibits stronger aggregation characteristics.

[0014] Example 2: Structural characterization of protein-metal-polyphenol complexes: Structural changes were analyzed using FTIR.

[0015] FTIR Measurement: Fourier transform infrared spectroscopy (FTIR) was performed using a Nicolet IS50 spectrometer (Waltham, Thermo Scientific, USA). The freeze-dried MPNs powder was thoroughly ground and mixed with potassium bromide (KBr), and then granulated using a granulator. The infrared spectral acquisition range was set to 4000–400 cm⁻¹. −1 The number of scans was 32, and the resolution was controlled at 4 cm. −1 .

[0016] See results Figure 3 Significant intermolecular interactions were observed between MPNs and all five legume proteins, inducing marked changes in protein secondary structure and molecular conformation. Amide A band (3500-3300 cm⁻¹) was observed. −1 The protein exhibits a slight red shift related to NH stretching, with the addition of MPN indicating hydrogen bonding between MPN and the protein, accompanied by a reduction in amino groups. Furthermore, the protein's secondary structure is predominantly characterized by the amide I band (1700-1600 cm⁻¹). −1This is due to the C=O stretching vibration of the protein backbone, combined with CN stretching, CCN deformation, and the bending mode of NH4+ in plants. As shown in the figure, after the five proteins were complexed with the three MPNs, the characteristic wavenumbers of their amide I band shifted to varying degrees, indicating an electrostatic interaction between the proteins and MPNs. The amide I peaks of SPI-MPNs and PPI-MPNs (1700-1600 cm⁻¹) are also shown. -1 The blue shift indicates an increase in C=O bond vibrational frequency, which can be attributed to the coordination interactions between carbonyl groups and MPNs in the protein backbone. Simultaneously, the absorption intensity of the amide I band is enhanced, with MPI-MPNs, FPI-MPNs, and CPI-MPNs showing a significant red shift, indicating protein unfolding and enhanced intramolecular hydrogen bonding within the β-sheet. In the amide II band region (1600–1500 cm⁻¹),... -1 The blue shift observed in SPI-MPNs and FPI-MPNs confirms a direct interaction between MPNs and the amide NH group. PPI-MPNs, MPI-MPNs, and CPI-MPNs exhibit a red shift, which may stem from other intermolecular forces such as electrostatic repulsion and hydrophobic interactions between MPNs and the protein. In summary, MPI and SPI demonstrate strong structural sensitivity and binding ability to MPNs, followed by PPI and CPI, while FPI shows the weakest interaction with MPNs. These results confirm that MPNs significantly alter the secondary structure and conformational characteristics of legume proteins through multiple non-covalent interactions, including metal coordination, electrostatic repulsion, hydrophobic interactions, and hydrogen bonding, thereby multi-regulated protein structure.

[0017] Example 3: Functional properties of protein-metal-polyphenol complex: antioxidant properties.

[0018] Using DPPH and ABTS •+ The antioxidant activity of purified protein and the Proteins-MPNs complex was evaluated using a free radical scavenging method. To determine DPPH, a DPPH working solution (0.2 mM, 95% ethanol) was first prepared. 100 µL of purified protein and Proteins-MPNs dispersions were mixed with an equal volume of DPPH working solution. After reacting in the dark at room temperature for 30 minutes, the absorbance of the mixture was measured at 517 nm. The blank group consisted of a mixture of ethanol and the sample, while the control group consisted of a mixture of ethanol and DPPH. (For ABTS) •+ To prepare ABTS, equal volumes of 7 mmol / L ABTS and 2.45 mmol / L potassium persulfate were mixed and left in the dark for 12–16 h.•+ Dilute ABTS with PBS buffer (0.01 M, pH 7.0). •+ The solution was prepared so that its absorbance at 734 nm reached 0.70 ± 0.02. 20 μL of purified protein and Proteins-MPNs dispersion were then added to 180 μL of diluted ABTS. •+ Mix the solutions and react in the dark for 6 min. Measure the absorbance of the mixture at 734 nm using a microplate reader, with PBS as a blank.

[0019] See results Figure 4 The DPPH free radical scavenging method and ABTS were used. •+ The antioxidant properties of the protein-MPNs complex were evaluated using a free radical scavenging assay. As shown in the figure, the antioxidant activity of the protein-MPNs complex was significantly enhanced after the addition of MPNs compared to the pure protein sample, increasing from 40%-55% to 75%-95%. This indicates that the addition of MPNs significantly enhances the free radical scavenging ability of the protein. The improved antioxidant performance is mainly attributed to the hydroxyl-rich polyphenols and their coordination interactions with metal ions. These interactions synergistically enhance the system's electron supply and hydrogen atom transfer capabilities, thereby achieving efficient free radical scavenging. Combining the results of DPPH and ABTS free radical scavenging experiments, the introduction of MPNs significantly improves the antioxidant activity of five legume proteins, and the improvement effect is closely related to the metal coordination type, exhibiting an EFe0 effect. 3+ ECa 2+ >EMg 2+ Among the results, the SPI and MPI system showed the most significant improvement in antioxidant activity after being compounded with EFe, while the CPI system, although initially exhibiting lower activity, also demonstrated excellent antioxidant performance after compounding. This result, corroborated by particle size, zeta potential, and thermodynamic analysis, further indicates that the stronger interaction between EFe and proteins can significantly improve the antioxidant properties of the system, providing important evidence for its application in active packaging, functional foods, and other fields.

Claims

1. A method for modifying legume isolated proteins based on a metal-phenol network, characterized in that, Includes the following steps: (1) Preparation of legume protein isolate: After crushing and defatting the legume raw materials, the protein was extracted by alkali dissolution and acid precipitation, and then centrifuged, washed, neutralized and freeze-dried to obtain legume protein isolate; (2) Preparation of metal-phenol network (MPNs) solution: Epigallocatechin gallate (EGCG) and metal ion solution were mixed at a molar ratio of 2:1 and stirred under light-protected conditions to fully crosslink EGCG with metal ions to form MPNs solution; (3) Preparation of legume protein isolate-MPNs complex: The legume protein isolate obtained in step (1) was dispersed in deionized water to prepare a 1% (w / v) protein solution. After full hydration, the MPNs solution obtained in step (2) was added, the pH was adjusted to 9.0, and the reaction was stirred in the dark for 60 minutes to obtain the legume protein isolate-MPNs complex.

2. The method according to claim 1, characterized in that, The legume raw material mentioned in step (1) is selected from any one of soybean, pea, mung bean, broad bean or chickpea.

3. The method according to claim 1, characterized in that, The defatting in step (1) involves mixing pulverized and sieved soybean flour with n-hexane at a mass-to-volume ratio of 1:3 and repeating the defatting process three times. The alkaline dissolution and acid precipitation extraction involves dispersing defatted soybean flour in deionized water at a ratio of 1:10 (w / v), adjusting the pH to 8.5, extracting for 2 hours, centrifuging to collect the supernatant, and adjusting the pH to 4.5 with hydrochloric acid to precipitate the protein.

4. The method according to claim 1, characterized in that, The protein content of the legume protein isolate described in step (1) is not less than 90%.

5. The method according to claim 1, characterized in that, The metal ions mentioned in step (2) are selected from any one of Ca²⁺, Mg²⁺ or Fe³⁺, and the concentration of both the EGCG solution and the metal ion solution is 48 mM.

6. The method according to claim 1, characterized in that, The stirring reaction conditions described in step (2) are a temperature of 25°C, a rotation speed of 1000 rpm, and a reaction in the dark.

7. The method according to claim 1, characterized in that, The amount of MPNs solution added in step (3) is 2% (v / v) of the protein solution volume, and the mass ratio of protein to MPNs is 50:1; the hydration conditions are that the protein solution is continuously stirred for 2 hours and then left to stand overnight at 4°C.

8. The legume isolate protein-MPNs complex prepared by the method according to any one of claims 1-7, characterized in that, In the complex, EGCG and metal ions form a metal-phenolic network structure through coordination. This network binds to legume isolate protein through hydrogen bonds, electrostatic interactions, hydrophobic interactions, and coordination synergy.

9. The legume isolate protein-MPNs complex according to claim 8, characterized in that, The microstructure of the complex gradually becomes denser as the coordination ability of metal ions increases; the antioxidant activity of the complex is 40% to 95% higher than that of unmodified legume protein isolate.

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