Rice protein amyloid fibril and preparation method and application thereof

Abstract

This invention provides rice protein amyloid fibers, their preparation method, and applications, relating to the field of protein fiber technology. The preparation method includes reducing a rice protein dispersion with a food-grade reducing agent to obtain a rice protein solution; hydrating the rice protein solution at pH 1.0-3.0 and 0℃-6℃ for 8-16 hours, followed by separation of the hydrated solution; dissolving a composite calcium source in the hydrated solution with stirring; and then heat-treating at 80℃-90℃ to obtain rice protein amyloid fibers. This invention, by breaking the disulfide bonds in rice protein molecules with a reducing agent and hydrating them in an acidic environment, promotes the formation of amyloid fibers under the action of calcium ions, effectively improving the solubility, emulsification, foaming properties, and rheological properties of rice protein. Furthermore, the method is simple, environmentally friendly, and safe, requiring no complex mechanical equipment, thus facilitating industrial production and providing a new approach for the high-value utilization of rice protein.

Description

Technical Field

[0001] This invention relates to the field of protein fiber technology, and in particular to a rice protein starch-like fiber, its preparation method, and its application. Background Technology

[0002] With increasing consumer focus on healthy eating, green foods, and sustainable protein sources, plant proteins have gained widespread attention due to their wide availability, rich nutrition, and environmental friendliness. Rice protein (RP), as an important plant protein, possesses characteristics such as low allergenicity, uniform amino acid composition, and high biocompatibility, showing promising application prospects in functional foods, foods for special medical purposes, and plant-based foods. However, natural rice protein has a relatively dense structure and high degree of aggregation, resulting in relatively poor solubility, emulsification, and foaming properties, which significantly limits its further application in the food industry. Therefore, there is an urgent need to provide a solution to address these issues. Summary of the Invention

[0003] The purpose of this invention is to provide a rice protein amyloid fiber, its preparation method, and its application. By using a reducing agent to break the disulfide bonds in the rice protein molecule and hydrating it in an acidic environment, the formation of rice protein amyloid fibers is promoted under the action of calcium ions. This effectively improves the solubility, emulsification, foaming properties, and rheology of rice protein. Moreover, the preparation method is simple, green, and safe, requiring no complex mechanical equipment, which is conducive to industrial production and provides a new way for the high-value utilization of rice protein.

[0004] In a first aspect, the present invention provides a method for preparing rice protein amyloid fibers, comprising: reducing a rice protein dispersion to obtain a rice protein solution under a food-grade reducing agent; hydrating the rice protein solution at pH=1.0-3.0 and 0℃-6℃ for 8h-16h and then separating the hydrated solution; stirring and dissolving a composite calcium source in the hydrated solution, and then heat-treating it at 80℃-90℃ to obtain rice protein amyloid fibers; wherein the composite calcium source comprises organic calcium and / or inorganic calcium.

[0005] Optionally, the food-grade reducing agent includes one of cysteine, glutathione, and sodium sulfite; and / or, the mass ratio of the food-grade reducing agent to rice protein is 1:(5-20); and / or, the concentration of rice protein in the rice protein dispersion is 0.5wt%-2wt%; and / or, the reduction is carried out in a dispersion at pH=7.5-8.5; and / or, the reduction is carried out at 20℃-40℃; and / or, the reduction is carried out for 30min-120min to obtain a rice protein solution.

[0006] Optionally, the rice protein solution is hydrated under intermittent ultrasound; and / or, the rice protein solution is subjected to restriction enzyme digestion and enzyme inactivation at pH 1.0-3.0 and 0℃-6℃ under the action of acidic protease; and / or, the rice protein solution is hydrated under the action of a hydration promoter, wherein the hydration promoter includes one of ethanol, sodium citrate, and sodium lactate; and / or, after hydration, the supernatant is collected by centrifugation to obtain a hydrated solution.

[0007] Optionally, the total calcium concentration after dissolving the composite calcium source in the hydrated solution is 10 mmol / L-200 mmol / L; and / or, heat treatment is performed in a non-oxidizing atmosphere, which includes either a nitrogen atmosphere or an argon atmosphere; and / or, the organic calcium includes either calcium citrate or calcium gluconate; and / or, the inorganic calcium includes calcium chloride; and / or, the molar ratio of the organic calcium to the inorganic calcium is 1:(2-5).

[0008] Secondly, the present invention also provides rice protein amyloid fiber prepared by any of the above-mentioned optional preparation methods, wherein the functionality of rice protein amyloid fiber is improved compared with that of rice protein, and the functionality includes at least one of solubility, foaming property and emulsifying property.

[0009] Thirdly, the present invention also provides the application of rice protein amyloid fibers prepared by any of the above-mentioned optional preparation methods in emulsions, foams, gels, and delivery systems.

[0010] The method for preparing rice protein amyloid fibers provided by this invention has at least one of the following beneficial technical effects compared with the prior art: 1. By reducing the rice protein dispersion under the action of food-grade reducing agent, it can react with the disulfide bonds in the rice protein molecules and promote the breakage of the disulfide bonds, making the densely aggregated rice protein structure loose, thereby fully exposing the hydrophobic regions and calcium ion binding sites embedded in the rice protein, and increasing the number of nucleation sites during the assembly of amyloid fibers. 2. By breaking the disulfide bonds in rice protein molecules with a reducing agent and hydrating them in an acidic environment, the formation of amyloid fibers in rice protein is promoted under the action of calcium ions. This effectively improves the solubility, emulsification, foaming properties, and rheology of rice protein. The operation is simple, green, and safe, and does not require the use of complex mechanical equipment, which is conducive to industrial production and provides a new way for the high-value utilization of rice protein. Attached Figure Description

[0011] Figure 1 A flowchart illustrating a method for preparing rice protein amyloid fibers provided by the present invention; Figure 2 This is a comparison graph showing the changes in ThT fluorescence intensity under different heat treatment times in Examples 1 to 5 of this invention; Figure 3 This is a comparison graph showing the changes in ANS fluorescence intensity under different heat treatment times in Examples 1 to 5 of this invention; Figure 4 This is a comparison chart of the soluble protein concentrations in the amyloid cellulose solution systems prepared in Examples 1 to 5 of this invention. Figure 5 This is a comparison graph showing the viscosity changes of the amyloid fiber solution systems prepared in Examples 1 to 5 of this invention. Figure 6 A comparative diagram showing the emulsification characteristics of the amyloid fiber solution systems prepared in Examples 1 to 5 of this invention; Figure 7 A comparative diagram showing the foaming characteristics of the amyloid fiber solution systems prepared in Examples 1 to 5 of this invention; Figure 8 The images show a comparison of the morphology of the amyloid fiber solution systems prepared in Examples 1 to 5 of this invention at 0 min and 30 min after foaming. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0013] See Figure 1 This invention provides a method for preparing rice protein amyloid fibers, comprising the following steps: S1. Rice protein dispersion is reduced with a food-grade reducing agent to obtain rice protein solution; S2. After hydrating the rice protein solution at pH=1.0-3.0 and 0℃-6℃ for 8h-16h, separate the hydrated solution. S3. The composite calcium source was stirred and dissolved in the hydrated solution, and then separated into rice protein starch-like fibers after heat treatment at 80℃-90℃.

[0014] In fact, this invention reduces the rice protein dispersion with a reducing agent in step S1, specifically breaking the disulfide bonds in the rice protein and promoting the unfolding of the rice protein's folded structure. This not only improves the solubility of the rice protein but also fully exposes the active sites inside the rice protein. Then, in step S2, the rice protein is fully extended and forms a hydrated solution by low-temperature hydration in an acidic environment using electrostatic repulsion. Finally, in step S3, calcium ions are used to generate electrostatic shielding and ion bridging, and in conjunction with a high-temperature acidic environment, the peptides undergo conformational rearrangement, directional self-assembly, and form β-sheet-rich amyloid fibers.

[0015] In some embodiments, the food-grade reducing agent used in step S1 is a disulfide bond reducing agent. This agent specifically breaks the disulfide bonds in rice protein. Using food-grade raw materials improves the safety of the resulting amyloid fibers, allowing them to be directly used as raw materials in the food industry. For example, rice protein amyloid fibers can be used as food-grade thickeners, emulsifiers, or to make food gels, or combined with small molecule active substances to form encapsulation carriers. Specifically, the food-grade reducing agent used includes one of cysteine, glutathione, and sodium sulfite, and the mass ratio of the food-grade reducing agent to rice protein is 1:(5-20).

[0016] Example 1: A method for preparing a rice protein amyloid cellulose solution, comprising the following steps: T1. At 25℃ and 500rpm, 5g of rice protein was mixed with 500mL of ultrapure water to obtain a rice protein dispersion. T2. After adjusting the pH of the rice protein dispersion to 2.0 with hydrochloric acid and hydrating it at 4℃ for 12 h, centrifuge it at 4800 rpm for 10 min and separate the supernatant to obtain the hydrated solution (RP). T3. Dissolve calcium chloride in the hydrated solution by stirring, adjust the calcium ion concentration to 50 mmol / L, and then heat-treat in a water bath at 85℃ for 12 h to obtain rice protein amyloid fiber solution (RPF-50).

[0017] Exploratory Examples 2 to 5: Each provides a method for preparing rice protein amyloid fibers. The difference from Exploratory Example 1 is that the concentration of calcium ions in the system is different in step T3, as shown in Table 1 below.

[0018] Table 1. Calcium ion concentrations during hydration in Examples 1 to 5

[0019] Exploration, characterization, and analysis: 1. Fiber Formation Amount: Solutions from Experimental Examples 1 to 5, heat-treated for 0 min, 30 min, 60 min, 360 min, and 720 min respectively in step T3, were used as test samples. A 0.8 mg / mL thioflavin T (ThT) solution (solvent consisting of 150 mmol / L sodium chloride and 10 mmol / L phosphate buffer, pH=7.4) was diluted 50 times and mixed with the test samples at a volume ratio of 25:1. After reacting at 25°C in the dark for 5 min, the fluorescence intensity was measured using a microplate reader (excitation wavelength 440 nm, emission wavelength 482 nm) to characterize the degree of amyloid fiber formation in the test samples. The results are as follows: Figure 2 As shown.

[0020] from Figure 2 As can be seen, the ThT fluorescence intensity of rice protein gradually increases during the formation of amyloid fibers from Experiment 1 to Experiment 5. This indicates that rice protein is gradually forming amyloid fibers rich in β-sheet structures. Among them, the sample tested in Experiment 1 showed the highest ThT fluorescence intensity when the calcium ion concentration was 50 mmol / L. This shows that adding an appropriate amount of calcium ions can promote the formation of amyloid fibers by reducing the electrostatic repulsion between protein molecules, promoting the orderly arrangement of molecules, and enhancing the intermolecular interaction.

[0021] Furthermore, when the calcium ion concentration reached 150 mmol / L and 200 mmol / L, the ThT fluorescence intensity decreased instead, indicating that excessively high calcium ion concentrations are not conducive to the formation of ordered amyloid fibers. This may be because high concentrations of calcium ions can damage the protein hydration layer, induce disordered aggregation, and enhance ion bridging, thereby inhibiting the formation of ordered β-sheet structures. In addition, except for Experimental Example 5, the ThT fluorescence intensity of Experimental Examples 1 to 4, which had calcium ions added, all showed a decreasing trend after a long treatment of 720 min, indicating that the fibers may undergo local gelation under the action of calcium ions in the later stages.

[0022] 2. Fiber Hydrophobicity: Solutions from Experimental Examples 1 to 5, heat-treated for 0 min, 30 min, 60 min, 360 min, and 720 min respectively in step T3, were used as test samples. A 2 mmol / L ammonium 8-anilino-1-naphthalenesulfonate (ANS) solution (solvent: 10 mmol / L phosphate buffer, pH=7.4) was used. After diluting the test samples with phosphate buffer, 2 μL of ANS solution was thoroughly mixed with 1 mL of the diluted test sample and allowed to react at 25℃ in the dark for 15 min. The fluorescence intensity was then measured using a microplate reader (excitation wavelength: 390 nm, emission wavelength: 48 nm). The ANS working solution was used as a blank fluorescence value for analysis to characterize the changes in hydrophobicity of the test samples during fiber formation. The results are shown below. Figure 3 As shown.

[0023] from Figure 3 As can be seen, the ANS fluorescence intensity of Experimental Examples 1 to 5 gradually increased with the increase of fibrillation time. This indicates that rice protein undergoes structural unfolding during fibrillation, exposing more hydrophobic groups on the molecular surface. In addition, the ANS fluorescence intensity continued to increase with the increase of calcium ion concentration. This indicates that the addition of calcium ions can promote the interaction and conformational changes between protein molecules and help improve the hydrophobicity of the protein surface. This is because calcium ions can accelerate the unfolding of protein structure by reducing the electrostatic repulsion between protein molecules, enhancing hydrophobic interactions, and promoting protein aggregation.

[0024] 3. Soluble protein concentration: The hydrated solution (RP) prepared in step T2 of Preparation Example 1 was used as a blank control. The blank control and the amyloid cellulose solutions (heat-treated for 720 min) prepared in Experimental Examples 1 to 5 were used as test samples. The test samples were centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The protein concentration in the supernatant was determined using a BCA kit to characterize the soluble protein concentration in the amyloid cellulose solution system. The results are as follows: Figure 4 As shown.

[0025] from Figure 4 As can be seen, compared with rice protein (RP) that has not undergone amyloid fibrosis treatment, the soluble protein content in Experimental Examples 1 to 5 is significantly increased. In Experimental Example 5, without the addition of calcium ions as a precursor, the soluble protein concentration increased from 0.37 mg / mL of RP to 0.67 mg / mL. This indicates that fibrosis treatment of rice protein can promote the unfolding of protein structure and improve its stable existence in the aqueous phase.

[0026] Furthermore, in Experimental Examples 1 to 4, the addition of calcium ions resulted in a trend of first increasing and then decreasing soluble protein content. In Experimental Example 1, the soluble protein content reached 0.86 mg / mL, an increase of 131.21% compared to the RP (Recovery Rate). This is because an appropriate amount of calcium ions can reduce the electrostatic repulsion between rice protein molecules, promote protein chain unfolding and fiber structure formation, thereby enhancing the interaction between rice protein and water molecules, and thus improving the solubility of rice protein in the aqueous phase. However, as the calcium ion concentration increased, the soluble protein content gradually decreased, indicating that excessively high calcium ion concentrations can lead to excessive aggregation of rice protein, thereby reducing the stability of the system.

[0027] 4. System Viscosity: The hydrated solution (RP) prepared in step T2 of Preparation Example 1 was used as a blank control (RP). The blank control and the amyloid cellulose solutions (heat-treated for 720 min) prepared in Investigation Examples 1 to 5 were used as test samples. The viscosity of the system was measured using a rheometer. A parallel plate clamp with a diameter of 40 mm was used during the measurement process, the test gap was set to 0.5 mm, the measurement temperature was 25 °C, and the measurement was performed in steady-state shear mode at 0.01 s⁻¹. -1 -100s -1 At a shear rate of [value missing], the apparent viscosity of the system at different shear rates was recorded to characterize the relationship between the apparent viscosity and the shear rate. The results are as follows: Figure 5 As shown.

[0028] from Figure 5 As can be seen, the apparent viscosity of RP and the sample systems of Experimental Examples 1 to 5 gradually decreased with increasing shear rate, exhibiting typical shear-thinning behavior, indicating non-Newtonian fluid characteristics. Compared with untreated RP, the viscosity of Experimental Examples 1 to 5 increased significantly after amyloid fibrillation, indicating that the formation of amyloid fibers can enhance the internal interactions and structural stability of the system. In addition, the viscosity of the system showed an upward trend when the calcium ion concentration increased. The highest apparent viscosity was observed in Experimental Example 4 at a calcium ion concentration of 200 mmol / L. This is because the high concentration of calcium ions greatly enhanced the bridging effect between protein molecules and fibers, promoting the formation of a denser aggregate network.

[0029] 5. Emulsifying Properties: The hydrated solution (RP) prepared in step T2 of Preparation Example 1 was used as a blank control (RP). The blank control and the amyloid cellulose solutions (heat-treated for 720 min) prepared in Investigation Examples 1 to 5 were used as test samples. 3 mL of corn oil was mixed with 1 mL of the test samples (RP, RPF, RPF-50, RPF-100, RPF-150, RPF-200) and homogenized at 10000 rpm for 2 min to prepare a composite emulsion. At min 0 and min 20, 0.01 mL of the composite emulsion was taken from the bottom, diluted 40 times with 1 mg / mL SDS solution, and the absorbance at 500 nm was measured. Using 1 mg / mL SDS solution as a blank sample, the emulsifying activity and emulsifying stability of the test samples were calculated according to the following formulas. The results are as follows: Figure 6 As shown in A and B in the diagram.

[0030]

[0031]

[0032] in: The absorbance value of the composite emulsion at time 0 min is given. The absorbance value of the composite emulsion at 20 minutes is given. This refers to the concentration of rice protein (g / mL). This represents the volume fraction of corn oil in the system.

[0033] from Figure 6 As can be seen, compared with the RP group without amyloid fibrillation, the emulsifying activity and emulsifying stability of rice protein were significantly improved after amyloid fibrillation in Examples 1 to 5. This indicates that the formation of amyloid fibers helps improve the interfacial properties of rice protein. Specifically, the emulsifying activity in Example 5 increased from 53.75 mg / L in the RP group. 2 / g increased to 69.74 m 2 / g, the emulsification stability increased from 69.40% in the RP group to 76.46%, which indicates that the structure of rice protein unfolds during the rice protein fiberization process, enhancing its adsorption capacity and interfacial film formation capacity at the oil-water interface.

[0034] In Experimental Examples 1 through 4, the emulsification characteristics of the amyloid cellulose solution initially increased and then decreased with increasing calcium ion concentration, reaching a peak emulsification activity of 88.16 mg / L in Experimental Example 2 when 100 mmol / L calcium ions were added. 2 / g, which is 64.03% higher than the RP group. In Experiment 1, the addition of 50 mmol / L calcium ions showed the highest emulsifying stability at 84.98%. However, with increasing calcium ion concentration, the emulsifying stability decreased significantly. This indicates that while high concentrations of calcium ions can enhance the hydrophobicity of the protein surface, excessive calcium ions can promote protein aggregation through ion bridging, leading to decreased system stability. Furthermore, it weakens the electrostatic repulsion between emulsion droplets, making the emulsion more prone to flocculation and aggregation. Therefore, it can be concluded that an appropriate amount of calcium ions is beneficial for improving the emulsifying properties of rice protein, while excessive calcium ions lead to decreased emulsion stability and are detrimental to the long-term stability of the emulsion system.

[0035] 6. Foaming Characteristics: The hydrated solution (RP) prepared in step T2 of Preparation Example 1 was used as a blank control (RP). The amyloid cellulose solutions (heat-treated for 720 min) prepared in Experimental Examples 1 to 5 were used as test samples. 20 mL of the test samples (RP, RPF, RPF-50, RPF-100, RPF-150, RPF-200) were transferred to a 50 mL beaker and homogenized at 8000 rpm for 60 s to form foam. The resulting foam was then transferred to a graduated cylinder, and the total volume was recorded at 0 min and 30 min. The foaming properties and foam stability of the test samples were calculated using the following formula. The results are as follows: Figure 7 and Figure 8As shown.

[0036]

[0037]

[0038] in: This represents the total volume of foam at minute 0 after homogenization. This represents the total volume of foam after 30 minutes of homogenization.

[0039] from Figure 7 and Figure 8 It can be seen that, compared with RP without amyloid fibrillation, the amyloid fibrillation of rice protein in Exploratory Examples 1 to 5 significantly enhanced the foaming properties and foam stability of rice protein, indicating that the formation of amyloid fibers helps to improve the adsorption capacity of rice protein at the gas-liquid interface.

[0040] In Experimental Examples 1 to 4, the emulsifying properties of the amyloid cellulose solution, after further addition of calcium ions, showed a trend of first increasing and then decreasing with increasing calcium ion concentration. Experimental Example 1, with the addition of 50 mmol / L calcium ions, exhibited the highest foaming property at 64.17%, an increase of 169.23% compared to the RP group. Combined with... Figure 3 It is known that an appropriate amount of calcium ions can promote the unfolding of rice protein structure and expose more hydrophobic groups, thereby enhancing the protein's ability to rapidly adsorb and oriented at the gas-liquid interface. In addition, the increase in soluble protein content is also conducive to more rice protein migrating to the gas-liquid interface and participating in foam formation.

[0041] Therefore, regarding the foam stability results, all tested samples (RP, RPF, RPF-50, RPF-100, RPF-150, RPF-200) maintained high stability. The highest foam stability (94.41%) was observed in Experimental Example 1 after adding 50 mmol / L calcium ions. Simultaneously, combined with... Figure 5 It was found that the viscosity of the system increased significantly after rice protein underwent amyloid fibrillation, indicating that the formed fiber network can enhance the liquid film strength and slow down liquid discharge, thereby improving foam stability. However, when the calcium ion concentration further increased, both foaming properties and foam stability showed different decreasing trends. This indicates that excessively high calcium ion concentrations can lead to excessive aggregation of rice protein, reducing the flexibility and rearrangement ability of protein molecules at the gas-liquid interface.

[0042] As demonstrated in Examples 1 to 5 above, adding different concentrations of calcium ions to an acidic environment combined with heat treatment can produce rice protein amyloid fibers. The analysis results show that appropriate amounts of calcium ions help rice protein form an ordered β-sheet structure, increase the hydrophobicity of the protein surface and the content of soluble protein, and further improve the emulsifying properties, emulsion stability, foaming properties, and foam stability of the protein. However, when the calcium ion concentration is too high, the protein is prone to excessive aggregation, which is detrimental to the formation of an ordered fiber structure and the maintenance of system stability.

[0043] Example 1: A method for preparing rice amyloid protein fiber, comprising the following steps: S1. At 25℃ and 500rpm, 5g of rice protein and 500mL of ultrapure water were stirred and mixed to obtain a rice protein dispersion. 0.5g of L-cysteine ​​(CAS: 52-90-4) was stirred and dissolved in the rice protein dispersion. The pH was adjusted to 8.0 and the mixture was stirred and reacted at 35℃ for 60min to obtain a rice protein solution. S2. Adjust the pH of the rice protein solution to 2.0 with hydrochloric acid and hydrate it overnight at 4℃ for 12 hours. Then, centrifuge it at 4800 rpm for 10 minutes and separate the supernatant to obtain the hydrated solution. S3. Dissolve calcium chloride in the hydrated solution by stirring, adjust the calcium ion concentration to 50 mmol / L, and then heat-treat in a water bath at 85℃ for 12 h to obtain rice protein amyloid fiber solution.

[0044] Example 2: A method for preparing rice amyloid protein fiber, which differs from Example 1 in that, in step S3, calcium citrate is dissolved in an hydrated solution by stirring, the calcium concentration is adjusted to 50 mmol / L, and then heat-treated in a water bath at 85°C for 12 h to obtain a rice amyloid protein fiber solution.

[0045] Example 3: A method for preparing rice amyloid protein fiber, which differs from Example 1 in that, in step S3, calcium chloride and calcium citrate are stirred and dissolved in an hydrated solution at a molar ratio of 3:1, the calcium concentration is adjusted to 50 mmol / L, and then heat-treated in a water bath at 85°C for 12 h to obtain a rice amyloid protein fiber solution.

[0046] The amyloid cellulose solutions prepared in Examples 1 to 3 were tested for emulsification and foaming properties, and the results are shown in Table 2 below.

[0047] Table 2. Emulsification and foaming properties of the amyloid cellulose solutions prepared in Examples 1 to 3

[0048] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing amyloid fibrils of rice protein, characterized by, include: A rice protein dispersion was reduced using a food-grade reducing agent to obtain a rice protein solution. The rice protein solution was hydrated for 8-16 hours at pH 1.0-3.0 and 0℃-6℃, and then the hydrated solution was separated. The composite calcium source was stirred and dissolved in an hydrated solution, and then separated into rice protein starch-like fibers after heat treatment at 80℃-90℃; wherein the composite calcium source includes organic calcium and / or inorganic calcium.

2. The method of claim 1, wherein: The food-grade reducing agent includes one of cysteine, glutathione, and sodium sulfite; and / or, the mass ratio of the food-grade reducing agent to rice protein is 1:(5-20); and / or, the concentration of rice protein in the rice protein dispersion is 0.5wt%-2wt%; and / or, the reduction is carried out in a dispersion with pH=7.5-8.5; and / or, the reduction is carried out at 20℃-40℃; and / or, the reduction is carried out for 30min-120min to obtain a rice protein solution.

3. The method of claim 1, wherein: Hydration under intermittent ultrasound; and / or, restriction enzyme digestion of rice protein solution under acidic protease at pH=1.0-3.0 and 0℃-6℃ followed by enzyme inactivation; and / or, hydration of rice protein solution under the action of a hydration promoter, said hydration promoter including one of ethanol, sodium citrate, and sodium lactate; and / or, centrifugation after hydration to obtain the hydrated solution.

4. The method of claim 1, wherein: The total calcium concentration after dissolving the composite calcium source in an hydrated solution is 10 mmol / L-200 mmol / L; and / or, heat treatment is performed in a non-oxidizing atmosphere, which includes either a nitrogen atmosphere or an argon atmosphere; and / or, the organic calcium includes either calcium citrate or calcium gluconate; and / or, the inorganic calcium includes calcium chloride; and / or, the molar ratio of the organic calcium to the inorganic calcium is 1:(2-5).

5. The amyloid fibril of rice protein prepared by the production method according to any one of claims 1 to 4, characterized in that, Rice protein amyloid fiber has improved functionality compared to rice protein, including at least one of solubility, foaming properties, and emulsifying properties.

6. The application of rice protein amyloid fibers prepared by any one of claims 1 to 4 in emulsions, foams, gels, and delivery systems.

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