Rice protein with high solubility and emulsibility and preparation method thereof

Abstract

The invention belongs to the technical field of food processing, and particularly relates to rice protein with high solubility and emulsibility and a preparation method of the rice protein. The method comprises the following steps: mixing choline chloride or glucose with citric acid to obtain a citric acid-based eutectic solvent; then mixing with rice protein, carrying out hydration reaction, and after the reaction is finished, carrying out gradient boosting treatment in a high-pressure jet mill; finally, trypsin and glutaminase are added for continuous reaction, centrifugation, dialysis and freeze drying are carried out, and the rice protein with high solubility and emulsibility is obtained. According to the method disclosed by the invention, the structure and functional characteristics of the rice protein are directionally regulated and controlled through a multi-dimensional synergistic effect of chemical controllable deamidation treatment, physical crushing and biological modification by adopting a method of carrying out differential deamidation on two natural deep eutectic solvents, then carrying out high-pressure jet milling treatment and carrying out double-enzyme time sequence concerted catalysis. The protein molecule conformation is accurately regulated and controlled, the limitation of a traditional single deamidation technology is broken through, and the functional characteristics are comprehensively improved while excessive hydrolysis is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a rice protein with high solubility and emulsification and a preparation method thereof. Background Art

[0002] Rice protein (RP) is a recognized protein with low allergenicity and high nutritional value. It also has a balanced amino acid profile and high biological value and is widely used in the food industry. However, due to its high hydrophobicity and cross-linking of intermolecular disulfide bonds, natural rice protein has poor water solubility and emulsification, which is mainly attributed to the formation of intramolecular / intermolecular hydrogen bonds by the amide groups of glutamine and asparagine, leading to protein aggregation, limiting its application in food systems. Traditional deamidation technologies mainly include acid method, alkaline method and enzyme method, but all have limitations. In order to overcome these limitations, researchers have developed a variety of modification technologies in recent years, including physical, biological and chemical methods.

[0003] The acid method and alkaline method used in the prior art utilize strong acid (HCl) or strong base (NaOH) treatment, which easily leads to excessive protein hydrolysis (Degree of Hydrolysis, DH≥15%), destroying the functional structure, and the deamidation and emulsification activity effects are poor. Therefore, there is an urgent need to provide a new method. Summary of the invention

[0004] The object of the invention is to overcome the deficiencies in the prior art, provide a kind of rice protein with high solubility and emulsification and preparation method thereof, specifically adopt the following technical scheme: In a first aspect, the present invention provides a method for preparing rice protein with high solubility and emulsification, comprising the following steps: S1. Mix choline chloride or glucose with citric acid, then dissolve in water, and stir at 75° C.-90° C. until a transparent and uniform liquid is formed to obtain a citric acid-based deep eutectic solvent; S2, dissolving the rice protein powder in the citric acid-based deep eutectic solvent to carry out a hydration reaction to obtain deamidated rice protein; S3, placing the deamidated rice protein in a high-pressure jet mill for gradient boosting treatment; the pressure of the gradient boosting treatment is 200MPa-250MPa-280MPa successively; S4, the rice protein after S3 processing is adjusted to pH 6-8, trypsin is added to react, then glutaminase is added to continue the reaction, centrifugation, dialysis, freeze drying, to obtain rice protein with high solubility and emulsification.

[0005] The present invention adopts a glucose / choline chloride-citric acid binary NADES system (molar ratio of 1:1-1:1.5) to achieve controllable deamidation; combines gradient pressure-increasing jet mill treatment (pressure range of 200-280MPa, increasing in stages) with trypsin-transglutaminase sequential synergistic catalysis (enzyme ratio of 1:2-1:8, added in steps), breaks through the bottleneck of a single technology, and finally obtains a method for controllable deamidation of rice protein with significantly improved solubility (pH3>65%) and emulsification activity (EAI>50m² / g).

[0006] The present invention uses natural deep eutectic solvents (NADESs) composed of two or more natural components derived from biological metabolites, which have lower toxicity and better biocompatibility than traditional low eutectic solvents. The excellent solubility of NADESs is affected by its polarity, which in turn affects the interaction between proteins and solvents. Therefore, the appropriate polarity of NADESs is crucial for protein modification.

[0007] Secondly, the present invention adopts an efficient physical modification technology of high-pressure jet mill, which can significantly change the particle size and structure of protein through mechanical effects such as high-speed shearing, collision and friction. High-pressure jet mill treatment can destroy the aggregation structure of protein, reduce the particle size, increase the surface area of ​​protein, thereby improving its solubility and emulsification. Studies have shown that high-pressure jet mill treatment can effectively open the higher-order structure of protein, expose more hydrophilic groups, enhance the interaction between protein and water, and thus improve its water solubility. In addition, high-pressure jet mill can also improve the emulsification performance of protein, so that it forms a more stable interface film at the oil-water interface.

[0008] Finally, the present invention adopts two forms of enzymatic modification, namely hydrolysis and cross-linking. Enzymatic hydrolysis modification refers to the use of proteases to catalyze the hydrolysis of proteins under mild conditions to achieve the purpose of modifying the composition or structure of proteins; proteins are hydrolyzed by enzymes to become small molecule peptides, the molecular weight is reduced, and the molecular structure and hydrophobicity are also changed, thereby changing the functional properties of proteins. Enzymatic cross-linking modification refers to the covalent cross-linking or polymerization reaction between or within protein molecules after the protein is treated with enzymes, which changes the ratio of hydrophobic groups to hydrophilic groups on the surface of the protein, and ultimately improves the functional properties of the protein.

[0009] In summary, the present invention adopts choline chloride-citric acid natural eutectic solvent controlled deamidation treatment, high pressure jet mill treatment, and dual enzyme sequential synergistic catalysis to have significant synergistic effect. Choline chloride-citric acid natural eutectic solvent controlled deamidation treatment enhances its functional characteristics by changing the charge distribution of protein. High pressure jet mill treatment can destroy the higher structure of protein, increase its surface area, and provide more reaction sites for subsequent dual enzyme sequential synergistic catalysis. Dual enzyme sequential synergistic catalysis further degrades the hydrophobic region of protein through enzymolysis, generates small molecule peptides, and enhances its solubility and emulsification. The three are combined to form a chemical-physical-biological three-dimensional synergistic synergistic technology, which can significantly improve the solubility and emulsification of rice protein and broaden its application in the food industry.

[0010] As a further preferred embodiment, the molar ratio of choline chloride or glucose to citric acid in S1 is 1:1-1:1.5.

[0011] As a further preferred embodiment, it is characterized in that the citric acid-based deep eutectic solvent described in S2 is 4.5%-5.5% of the mass of the rice protein powder, w / v, which means that 4.5-5.5g of rice protein powder is dissolved in 100mL of citric acid-based deep eutectic solvent.

[0012] As a further preferred embodiment, the concentration of the citric acid-based deep eutectic solvent in S2 is 0.8 mol / L-1.5 mol / L.

[0013] As a further preferred embodiment, the temperature of the hydration reaction in S2 is 80°C-100°C, and the time of the hydration reaction is 1.5 h-2.5 h.

[0014] As a further preferred embodiment, the time for the gradient boost treatment in S3 is 15 min-25 min; the time for each gradient treatment is 5 min-8 min.

[0015] As a further preferred embodiment, the jet velocity during the high-pressure jet mill treatment in S3 is 400 m / s-430 m / s.

[0016] As a further preferred embodiment, the usage ratio of trypsin and glutaminase in S4 is 1:2-1:8; the concentration of trypsin is 0.3 U / g-0.8 U / g; and the concentration of glutaminase is 0.6 U / g-1.6 U / g.

[0017] As a further preferred embodiment, the interval between adding trypsin and adding glutaminase is 30 min, and the reaction time is 2 h-4 h; the centrifugal speed is 4500 xg, the centrifugal time is 10 min, the dialysis temperature is 4°C, and the dialysis time is 48 h.

[0018] In a second aspect, the present invention provides a rice protein with high solubility and emulsification prepared by the above preparation method.

[0019] The beneficial effects of the present invention are: (1) The present invention adopts a method based on two natural low eutectic solvents, choline chloride-citric acid and glucose-citric acid, followed by differential deamidation and high-pressure jet milling treatment, and dual-enzyme sequential synergistic catalysis, through the multi-dimensional synergistic effect of chemically controllable deamidation treatment, physical fragmentation, and biological modification, so as to directionally regulate the structure and functional properties of rice protein. The protein molecular conformation is precisely regulated, breaking through the limitations of traditional single deamidation technology, and achieving a comprehensive improvement in functional properties while avoiding excessive hydrolysis. In addition, the functional properties of rice protein such as solubility and emulsification are greatly improved, and rice protein with high solubility and emulsification has better applications in food industry fields such as beverages, baking, meat products, and plant meat.

[0020] (2) According to the data results of the present invention, the solubility of rice protein after deamidation treatment by the method of the present invention is significantly improved in a wide pH range, which can exceed 60%; the enhanced emulsifying activity (EAI) is specifically 40.91-48.43 m² / g, and the emulsification stability (ESI) is 49.9-55.3 minutes, which is significantly better than that of unmodified rice protein; the foaming, water-holding and oil-holding properties of the protein are significantly improved.

[0021] (3) The citric acid-based NADES used in the present invention is composed of natural ingredients, has low toxicity, high biocompatibility and degradability, and meets the requirements of green chemistry; and compared with the traditional single enzyme modification, the present method uses high-pressure jet milling to destroy the higher-order structure of rice protein, increase its surface area and reaction sites, and provide a more efficient substrate for the dual-enzyme sequential synergistic catalysis, thereby reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1Shown is a flow chart of the preparation of a natural eutectic solvent of glucose / choline chloride-citric acid; Figure 2 Shown is the SEM image of rice protein during the preparation process of Example 1; Figure 3 Shown is the foaming diagram of rice protein in the preparation process of Example 1; Figure 4 Shown are the foaming properties and foaming stability of rice protein during the preparation process of Example 1; Figure 5 Shown is the solubility of rice protein at pH 3, 7, and 11 during the preparation process of Example 1; Figure 6 Shown is the surface hydrophobicity of rice protein during the preparation process of Example 1; Figure 7 The emulsifying property (EAI) and emulsion stability (ESI) of rice protein during the preparation process of Example 1 are shown. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0025] The specific model of the high-pressure jet mill used in the present invention is ARONIO high-pressure jet mill XCFJ-3256.

[0026] Example 1 A method for preparing rice protein with high solubility and emulsification (its schematic diagram is as shown in FIG. Figure 1 ), specifically including the following steps: (1) Preparation of NADES: Weigh 62.83 g (0.45 mol) of choline chloride (ChCl, molecular weight 139.62 g / mol) and 86.45 g (0.45 mol) of citric acid (CA, molecular weight 192.12 g / mol) and mix them in a 500 mL glass reactor at a molar ratio of 1:1. Add deionized water to a total solution volume of 500 mL (initial water content 10% v / w). Stir magnetically (300 rpm) in an 80°C constant temperature oil bath for 60 min until a transparent homogeneous liquid is formed. After cooling to room temperature, dilute to 1 L with deionized water to obtain a NADES stock solution with a concentration of 0.9 mol / L. (2) Deamidation treatment: Take 100 mL of the NADES solution (0.9 mol / L) obtained in step (1) above and put it into a 250 mL three-necked flask, add 5.0 g of rice protein powder (protein purity ≥ 85%), and disperse it evenly by magnetic stirring (200 rpm). Hydration reaction was carried out in a constant temperature water bath at 90°C for 2 hours, and samples were taken every 30 minutes to detect the dissolution state until the system became a uniform yellow-brown colloid; (3) High-pressure jet mill: The deamidated colloid was transferred to a high-pressure jet mill system (nozzle diameter 0.1 mm, feed rate 50 mL / min) and treated in three stages: the first stage: 200 MPa pressure, jet velocity 420 m / s, and 7 minutes of circulation treatment; the second stage: increasing the pressure to 250 MPa and treating at the same speed for 7 minutes; the third stage: increasing the pressure to 280 MPa and treating for 7 minutes; each stage was cooled to below 37°C with ice water, and finally a nano-scale protein suspension with a particle size of ≤200 nm was obtained; (4) Dual enzyme modification: adjust the pH of the solution obtained after treatment in step (3) to 7.0 (0.1 mol / L NaOH / HCl), and keep in a constant temperature shaking water bath at 37°C (100 rpm); perform enzymatic hydrolysis in two stages: trypsin treatment: add 0.02 g (0.5 U / g protein) of trypsin (enzyme activity 25,000 U / g) and react for 1 hour; transglutaminase treatment: add 0.05 g (1.0 U / g protein) of transglutaminase (enzyme activity 100 U / g) and continue the reaction for 2 hours; nitrogen protection was used throughout the process, and the pH was monitored every 30 minutes and fine-tuned to 7.0; (5) Post-treatment: The reaction solution after treatment in step (4) was centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant was collected; the supernatant was injected into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed against 10 volumes of deionized water at 4°C for 36 hours, with the water being changed every 6 hours; freeze-drying: the dialysate was precooled to -80°C and then vacuum freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a white powdery highly soluble rice protein.

[0027] Its SEM picture is as follows Figure 2 As shown, a in the figure is completely untreated rice protein; b in the figure is rice protein after deamidation (step 2); c in the figure is rice protein after high-pressure jet milling (step 3); d in the figure is rice protein after double enzyme modification (step 4).

[0028] Example 2 A method for preparing rice protein with high solubility and emulsification, specifically comprising the following steps: (1) Preparation of NADES: Weigh glucose (C 6 H 12 O 681.07 g (0.45 mol) of NADES (molecular weight 180.16 g / mol) and 86.45 g (0.45 mol) of citric acid (CA, molecular weight 192.12 g / mol) were mixed in a 500 mL glass reactor at a molar ratio of 1:1, and deionized water was added to a total solution volume of 500 mL (initial water content 10% v / w); magnetic stirring (300 rpm) was carried out in an 80°C constant temperature oil bath for 60 minutes until a transparent homogeneous liquid was formed. After cooling to room temperature, the volume was adjusted to 1 L with deionized water to obtain a NADES stock solution with a concentration of 0.9 mol / L; (2) Deamidation treatment: Take 100 mL of the NADES solution (0.9 mol / L) obtained in step (1) above and put it into a 250 mL three-necked flask, add 5.0 g of rice protein powder (protein purity ≥ 85%), and disperse it evenly by magnetic stirring (200 rpm). Hydration reaction was carried out in a constant temperature water bath at 90°C for 2 hours, and samples were taken every 30 minutes to detect the dissolution state until the system became a uniform yellow-brown colloid; (3) High-pressure jet mill: The deamidated colloid was transferred to a high-pressure jet mill system (nozzle diameter 0.1 mm, feed rate 50 mL / min) and treated in three stages: the first stage: 200 MPa pressure, jet velocity 420 m / s, and 7 minutes of circulation treatment; the second stage: increasing the pressure to 250 MPa and treating at the same speed for 7 minutes; the third stage: increasing the pressure to 280 MPa and treating for 7 minutes; each stage was cooled to below 37°C with ice water, and finally a nano-scale protein suspension with a particle size of ≤200 nm was obtained; (4) Dual enzyme modification: The solution obtained after treatment in step (3) was adjusted to pH 7.0 (0.1 mol / L NaOH / HCl) and kept in a constant temperature shaking water bath at 37°C (100 rpm). The enzymatic hydrolysis was carried out in two stages: trypsin treatment: 0.02 g trypsin (enzyme activity 25,000 U / g) (0.5 U / g protein) was added and the reaction was continued for 1 hour; transglutaminase treatment: 0.05 g transglutaminase (enzyme activity 100 U / g) (1.0 U / g protein) was added and the reaction was continued for 2 hours; nitrogen protection was passed throughout the process, and the pH was monitored every 30 minutes and fine-tuned to 7.0.

[0029] (5) Post-treatment: The reaction solution after treatment in step (4) was centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant was collected; the supernatant was injected into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed against 10 volumes of deionized water at 4°C for 36 hours, with the water being changed every 6 hours; freeze-drying: the dialysate was precooled to -80°C and then vacuum freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a white powdery highly soluble rice protein.

[0030] Comparative Example 1 A method for preparing rice protein with high solubility and emulsification, specifically comprising the following steps: (1) Preparation of NADES: Weigh 62.83 g (0.45 mol) of choline chloride (ChCl, molecular weight 139.62 g / mol) and 86.45 g (0.45 mol) of citric acid (CA, molecular weight 192.12 g / mol) and mix them in a 500 mL glass reactor at a molar ratio of 1:1. Add deionized water to a total solution volume of 500 mL (initial water content 10% v / w). Stir magnetically (300 rpm) in an 80°C constant temperature oil bath for 60 min until a transparent homogeneous liquid is formed. After cooling to room temperature, dilute to 1 L with deionized water to obtain a NADES stock solution with a concentration of 0.9 mol / L. (2) Deamidation treatment: Take 100 mL of the NADES solution (0.9 mol / L) obtained in step (1) above and put it into a 250 mL three-necked flask, add 5.0 g of rice protein powder (protein purity ≥ 85%), and disperse it evenly by magnetic stirring (200 rpm); hydrate the solution in a 90°C constant temperature water bath for 2 hours, and take samples every 30 minutes to detect the dissolution state until the system becomes a uniform yellow-brown colloid; (3) High-pressure jet mill: The deamidated colloid was transferred to a high-pressure jet mill system (nozzle diameter 0.1 mm, feed rate 50 mL / min) and treated in one stage: 250 MPa pressure, jet velocity 420 m / s, and cycle treatment for 21 minutes; ice water was passed through every 7 minutes to cool to below 37°C, and finally a nano-scale protein suspension with a particle size of ≤200 nm was obtained; (4) Dual enzyme modification: adjust the pH of the solution obtained after treatment in step (3) to 7.0 (0.1 mol / L NaOH / HCl), and keep in a constant temperature shaking water bath at 37°C (100 rpm); perform enzymatic hydrolysis in two stages: trypsin treatment: add 0.02 g (0.5 U / g protein) of trypsin (enzyme activity 25,000 U / g) and react for 1 hour; transglutaminase treatment: add 0.05 g (1.0 U / g protein) of transglutaminase (enzyme activity 100 U / g) and continue the reaction for 2 hours; nitrogen protection was used throughout the process, and the pH was monitored every 30 minutes and fine-tuned to 7.0; (5) Post-treatment: The reaction solution after treatment in step (4) was centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant was collected; the supernatant was injected into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed against 10 volumes of deionized water at 4°C for 36 hours, with the water being changed every 6 hours; freeze-drying: the dialysate was precooled to -80°C and then vacuum freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a white powdery highly soluble rice protein.

[0031] Comparative Example 2 A method for preparing rice protein with high solubility and emulsification, specifically comprising the following steps: (1) Preparation of NADES: Weigh 62.83 g (0.45 mol) of choline chloride (ChCl, molecular weight 139.62 g / mol) and 86.45 g (0.45 mol) of citric acid (CA, molecular weight 192.12 g / mol) and mix them in a 500-mL glass reactor in a 1:1 molar ratio. Add deionized water to a total solution volume of 500 mL (initial water content 10% v / w). Stir magnetically (300 rpm) in an 80°C constant temperature oil bath for 60 min until a transparent homogeneous liquid is formed. After cooling to room temperature, dilute to 1 L with deionized water to obtain a NADES stock solution with a concentration of 0.9 mol / L.

[0032] (2) Deamidation treatment: Take 100 mL of the NADES solution (0.9 mol / L) obtained in step (1) above and put it into a 250 mL three-necked flask, add 5.0 g of rice protein powder (protein purity ≥ 85%), and disperse it evenly by magnetic stirring (200 rpm); hydrate the solution in a 90°C constant temperature water bath for 2 hours, and take samples every 30 minutes to detect the dissolution state until the system becomes a uniform yellow-brown colloid; (3) High-pressure jet mill: The deamidated colloid was transferred to a high-pressure jet mill system (nozzle diameter 0.1 mm, feed rate 50 mL / min) and treated in one stage: 250 MPa pressure, jet velocity 420 m / s, and cycle treatment for 21 minutes; ice water was passed through the mill to cool to below 37°C every 7 minutes, and a nanoscale protein suspension with a particle size of ≤200 nm was finally obtained.

[0033] (4) Dual enzyme modification: The pH of the solution obtained after the treatment in step (3) was adjusted to 7.0 (0.1 mol / L NaOH or HCl was added to adjust the pH), and the solution was kept in a constant temperature shaking water bath at 37°C (100 rpm). Enzymatic hydrolysis was performed by adding 0.02 g (0.5 U / g protein) of trypsin (enzyme activity 25,000 U / g) and 0.05 g (1.0 U / g protein) of transglutaminase (enzyme activity 100 U / g) at the same time. The reaction was carried out for 3 hours. Nitrogen protection was applied throughout the process, and the pH was monitored every 30 minutes and fine-tuned to 7.0. (5) Post-treatment: The reaction solution after treatment in step (4) was centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant was collected; the supernatant was injected into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed against 10 volumes of deionized water at 4°C for 36 hours, with the water being changed every 6 hours; freeze-drying: the dialysate was precooled to -80°C and then vacuum freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a white powdery highly soluble rice protein.

[0034] Comparative Example 3 A method for preparing rice protein with high solubility and emulsification, specifically comprising the following steps: (1) Preparation of NADES: Weigh 172.908 g (0.9 mol) of citric acid (CA, molecular weight 192.12 g / mol), add deionized water to a total solution volume of 500 mL (initial water content 10% v / w), and stir magnetically (300 rpm) in an 80°C constant temperature oil bath for 60 minutes until a transparent homogeneous liquid is formed. After cooling to room temperature, dilute to 1 L with deionized water to obtain a NADES stock solution with a concentration of 0.9 mol / L.

[0035] (2) Deamidation treatment: Take 100 mL of the NADES solution (0.9 mol / L) obtained in step (1) above and put it into a 250 mL three-necked flask, add 5.0 g of rice protein powder (protein purity ≥85%), and disperse it evenly by magnetic stirring (200 rpm); hydrate it in a constant temperature water bath at 90°C for 2 hours, and take samples every 30 minutes to check the dissolution state until the system becomes a uniform yellow-brown colloid.

[0036] (3) High-pressure jet mill: The deamidated colloid was transferred to a high-pressure jet mill system (nozzle diameter 0.1 mm, feed rate 50 mL / min) and treated in one stage: 250 MPa pressure, jet velocity 420 m / s, and cycle treatment for 21 minutes; ice water was passed through every 7 minutes to cool to below 37°C, and finally a nano-scale protein suspension with a particle size of ≤200 nm was obtained; (4) Dual enzyme modification: adjust the pH of the solution obtained after treatment in step (3) to 7.0 (0.1 mol / L NaOH / HCl), and keep in a constant temperature shaking water bath at 37°C (100 rpm); perform enzymatic hydrolysis in two stages: trypsin treatment: add 0.02 g (0.5 U / g protein) of trypsin (enzyme activity 25,000 U / g) and react for 1 hour; transglutaminase treatment: add 0.05 g (1.0 U / g protein) of transglutaminase (enzyme activity 100 U / g) and continue the reaction for 2 hours; nitrogen protection was used throughout the process, and the pH was monitored every 30 minutes and fine-tuned to 7.0; (5) Post-treatment: The reaction solution after treatment in step (4) was centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant was collected; the supernatant was injected into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed against 10 volumes of deionized water at 4°C for 36 hours, with the water being changed every 6 hours; freeze-drying: the dialysate was precooled to -80°C and then vacuum freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a white powdery highly soluble rice protein.

[0037] Comparative Example 4 A method for preparing rice protein with high solubility and emulsification, specifically comprising the following steps: (1) Preparation of NADES: Weigh 62.83 g (0.45 mol) of choline chloride (ChCl, molecular weight 139.62 g / mol) and 86.45 g (0.45 mol) of citric acid (CA, molecular weight 192.12 g / mol), mix them in a 500 mL glass reactor at a molar ratio of 1:1, add deionized water to a total solution volume of 500 mL (initial water content 10% v / w), and stir magnetically (300 rpm) in an 80°C constant temperature oil bath for 60 minutes until a transparent homogeneous liquid is formed. After cooling to room temperature, dilute to 1 L with deionized water to obtain a NADES stock solution with a concentration of 0.9 mol / L; (2) Deamidation treatment: Take 100 mL of the NADES solution (0.9 mol / L) obtained in step (1) above and put it into a 250 mL three-necked flask, add 5.0 g of rice protein powder (protein purity ≥ 85%), and disperse it evenly by magnetic stirring (200 rpm); hydrate the solution in a 90°C constant temperature water bath for 2 hours, and take samples every 30 minutes to detect the dissolution state until the system becomes a uniform yellow-brown colloid; (3) High-pressure jet mill: The deamidated colloid was transferred to a high-pressure jet mill system (nozzle diameter 0.1 mm, feed rate 50 mL / min) and treated in three stages: the first stage: 200 MPa pressure, jet velocity 420 m / s, and 7 minutes of circulation treatment; the second stage: increasing the pressure to 250 MPa and treating at the same speed for 7 minutes; the third stage: increasing the pressure to 280 MPa and treating for 7 minutes; each stage was cooled to below 37°C with ice water, and finally a nano-scale protein suspension with a particle size of ≤200 nm was obtained; (4) Post-treatment: The reaction solution after treatment in step (3) was centrifuged at 4°C and 5000×g for 15 minutes, and the supernatant was collected; the supernatant was injected into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed against 10 volumes of deionized water at 4°C for 36 hours, with the water being changed every 6 hours; freeze-drying: the dialysate was precooled to -80°C and then vacuum freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a white powdery highly soluble rice protein.

[0038] Example 3 The rice protein prepared in Example 1 was subjected to a performance test, and the rice protein prepared in Example 1 was subjected to a performance test comparison with Comparative Examples 1-4.

[0039] (1) Solubility Dispersions of deamidated RP samples (0.1%) were adjusted to pH 3, 7, and 11 using 0.1 mol / L NaOH and HCl and hydrated for 2 h by magnetic stirring. The supernatant was collected after centrifugation (4000 × g, 10 min). The soluble protein fraction was measured using the BCA assay kit. The solubility was calculated using the following formula: (2) Emulsification properties After adjusting the protein solution (16 mL, 1 mg / mL) to pH 7.0 with HCl or NaOH (0.1 mol / L), it was mixed with 4 mL of corn oil and then homogenized at 12,000 r / min for 2 min to form an emulsion. Subsequently, 50 μL of the emulsion at 0 min and 10 min in the centrifuge tube was mixed with 5 mL of SDS solution (1 mg / mL). The mixture was used to measure the absorbance at 500 nm. EAI and ESI were calculated by different formulas as shown below: Among them, A 0 and A 10 are the absorbance of different diluted emulsions at 0 min and 10 min, respectively; D refers to the dilution factor; C is the initial concentration of the deamidated RP sample (g / mL); φ is the volume fraction.

[0040] This example uses completely untreated rice protein (RP) as a control, wherein STEP1 represents the rice protein after the first step of binary NADES controlled deamidation; STEP2 represents the rice protein after the second step of high-pressure jet milling treatment; STEP3 represents the rice protein after the third step of dual enzyme sequential synergistic catalysis treatment.

[0041] The above test results are as follows Figure 2-7 As shown in the results, it can be seen that the particle size and structure of rice protein were significantly changed after controlled deamidation treatment with glucose / choline chloride-citric acid natural eutectic solvent, high-pressure jet milling treatment, and dual enzyme sequential synergistic catalysis ( Figure 2 At the same time, as the three steps proceed, the foaming properties of rice protein are significantly improved ( Figure 3 , 4), the foaming stability is also improved to a certain extent ( Figure 4 ). It can be seen that the deacetylation of citric acid and NADESs significantly reduces the surface hydrophobicity ( Figure 6 ), which greatly improved the solubility of RP. The solubility of untreated rice protein is about 8%, while the solubility of rice protein prepared by this process is more than 60%, 20%, and 55% at pH 3, 7, and 11, respectively ( Figure 5 ). The emulsifying activity (EAI) was significantly improved, and the emulsifying stability was also improved to a certain extent ( Figure 7 ). The test results show that the solubility of the deamidated rice protein in Example 1 exceeds 60%, 20%, and 55% at pH 3, 7, and 11, respectively, the emulsification activity (EAI) is 40.91–48.43 m² / g, and the emulsification stability (ESI) is 49.9-55.3 minutes.

[0042] The rice protein prepared in Example 1 was tested for performance with Comparative Examples 1-4, and the comparative data are shown in Table 1.

[0043] The single-stage high-pressure treatment of Comparative Example 1 may result in uneven distribution of protein particles, partial aggregation, reduced solubility and emulsification; foaming and stability decreased due to the larger particle size, and the surface hydrophobicity increased slightly (the hydrophilic groups were not fully exposed) (Table 1).

[0044] In comparative example 2, the simultaneous action of trypsin and transglutaminase may induce a competitive reaction, resulting in reduced enzymatic efficiency and insufficient cross-linking; the emulsification stability is reduced (insufficient cross-linking leads to weak interfacial film strength), and the surface hydrophobicity is further increased (some hydrophobic areas are not masked) (Table 1).

[0045] In comparative example 3, citric acid alone could not effectively form a hydrogen bond network, the deamidation efficiency was low, and the protein solubility was significantly reduced; the surface hydrophobicity was greatly increased (the protein structure was not fully opened), and the emulsification and foaming abilities were limited (Table 1).

[0046] Comparative Example 4 was not subjected to enzymatic hydrolysis and cross-linking, the protein molecular weight was too large and the structure was highly rigid, and the solubility and emulsification were the lowest; The surface hydrophobicity was the highest (the unmodified hydrophobic groups were completely exposed), and the foaming and stability were the worst (Table 1).

[0047] Table 1 The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of ​​the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the inspiration of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. A method for preparing rice protein with high solubility and emulsification, characterized in that, The following steps are involved: S1. Mix choline chloride or glucose with citric acid, then dissolve in water, and stir at 75° C.-90° C. until a transparent and uniform liquid is formed to obtain a citric acid-based deep eutectic solvent; S2, dissolving the rice protein powder in the citric acid-based deep eutectic solvent to carry out a hydration reaction to obtain deamidated rice protein; S3, placing the deamidated rice protein in a high pressure jet mill for gradient boost treatment; The pressure of the gradient pressure boosting treatment is 200MPa-250MPa-280MPa in sequence; S4, the rice protein after S3 processing is adjusted to pH 6-8, trypsin is added to react, then glutaminase is added to continue the reaction, centrifugation, dialysis, freeze drying, to obtain rice protein with high solubility and emulsification.

2. The preparation method according to claim 1, characterized in that: The molar ratio of choline chloride or glucose to citric acid in S1 is 1:1-1:1.

5.

3. The preparation method according to claim 1, characterized in that: The mass volume ratio of the rice protein powder to the citric acid-based deep eutectic solvent in S2 is 4.5% w / v-5.5% w / v.

4. The preparation method according to claim 1, characterized in that: The concentration of the citric acid-based deep eutectic solvent in S2 is 0.8 mol / L-1.5 mol / L.

5. The preparation method according to claim 4, characterized in that: The temperature of the hydration reaction in S2 is 80°C-100°C, and the time of the hydration reaction is 1.5 h-2.5 h.

6. The preparation method according to claim 1, characterized in that: The time for the gradient boost treatment in S3 is 15 min-25 min; the time for each gradient treatment is 5 min-8 min.

7. The preparation method according to claim 6, characterized in that: The jet velocity during the high-pressure jet mill treatment in S3 is 400 m / s-430 m / s.

8. The preparation method according to claim 1, characterized in that: The usage ratio of trypsin and glutaminase in the S4 is 1:2-1:8; the concentration of trypsin is 0.3 U / g-0.8 U / g; and the concentration of glutaminase is 0.6 U / g-1.6 U / g.

9. The preparation method according to claim 1, characterized in that: The interval between adding trypsin and adding glutaminase reaction is 30 min, and the reaction time is 2 h-4 h; the centrifugal speed is 4500 xg, the centrifugal time is 10 min, the dialysis temperature is 4°C, and the dialysis time is 48 h.

10. A rice protein with high solubility and emulsification, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 9.

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