Preparation method of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles

Abstract

The present invention provides a method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, and relates to the technical field of nanoparticle preparation. The specific raw materials and preparation method are as follows: rice bran protein is extracted from defatted rice bran by an alkali dissolution and acid precipitation method; rice bran protein-epigallocatechin gallate-carboxymethyl chitosan (R-E-C) ternary nanoparticles are prepared by a pH cycle method; and freeze-dried to obtain the R-E-C nanoparticle product. The nanoparticles prepared by the present invention have a short preparation cycle, good dispersibility, and high yield; they protect epigallocatechin gallate, reduce its activity loss due to environmental factors, and improve its bioavailability; and the nanoparticles have the ability to control release, which can prolong the antioxidant effect of epigallocatechin gallate; no chemical reagents are added during the preparation process, which is green and safe, and further expands the application potential of the prepared nanoparticles.

Description

Technical Field

[0001] The invention relates to a preparation method of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, belonging to the technical field of nanoparticle preparation. Background Art

[0002] As the world's largest producer of rice bran resources, my country produces tens of millions of tons of rice bran annually, making it a vast resource. Rice bran contains a variety of nutrients and physiologically active substances, including rice bran protein, considered an emerging high-quality plant protein. However, rice bran is currently primarily used as boiler fuel, animal feed, or simply discarded as agricultural waste. The added value of this agricultural byproduct remains largely untapped, causing significant damage and waste to the environment and resources. Therefore, comprehensive processing and utilization of rice bran are essential to promote its application in the food industry.

[0003] There is an existing patent, "A method for preparing soy protein nanoparticles for encapsulating and delivering proanthocyanidins" (CN202410240169.8). By ultrasonically and enzymatically treating soy protein, the nanoparticles can achieve better encapsulation effects on proanthocyanidins. This patent uses a pH cycle method to prepare rice bran protein-epigallocatechin gallate-carboxymethyl chitosan (REC) ternary nanoparticles, which protect epigallocatechin gallate, reduce its activity loss caused by environmental factors, improve its bioavailability, and prolong the antioxidant effect of epigallocatechin gallate. At the same time, EGCG contains abundant hydroxyl groups, which enables it to form hydrogen bonds with other compounds such as proteins, thus having a good modification effect on protein structure. However, single protein nanoparticle carriers are easily affected by the external environment. Therefore, adding biosafe, high-performance carboxymethyl chitosan to the protein-polyphenol system can give the nanoparticles better structural and functional properties, providing new methods and new ideas for the future development of new carrier materials. Summary of the Invention

[0004] The present invention provides a method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, which not only ensures the biological activity of epigallocatechin gallate in the nanoparticles, but also improves the stability of the protein nanoparticles and imparts them with better functional properties.

[0005] The present invention adopts the following technical solution: a method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, comprising the following steps:

[0006] 1) Rice bran protein extraction: Defatted rice bran is mixed with deionized water, homogenized and stirred at room temperature, the pH value of the mixture is adjusted to alkaline, stirred, filtered to remove rice bran residue and centrifuged, the pH value of the supernatant obtained after centrifugation is adjusted to acidic, allowed to stand and then centrifuged, the precipitate obtained after centrifugation is collected and diluted, the pH value thereof is adjusted to neutral, and freeze-dried to obtain rice bran protein RBP;

[0007] 2) Preparation of stock solution: Dissolve the rice bran protein RBP prepared in step 1) in deionized water, adjust the pH of the solution with 1 M NaOH, and stir at room temperature. Then, hydrate the rice bran protein RBP solution at 4° C. overnight to prepare the RBP stock solution.

[0008] 3) Preparation of Rice Bran Protein-Epigallocatechin Gallate (RE) Nanoparticles: The RBP stock solution from step 2) was mixed with epigallocatechin gallate (EGCG), and deionized water was added to control the total solids concentration of the solution. The pH of the mixture was adjusted to alkaline with 1 M NaOH solution, and the mixture was stirred continuously for 24 h. The mixture was dialyzed in an 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 6-8 h, to obtain a RE nanoparticle solution, which was freeze-dried to produce RE nanoparticles.

[0009] 4) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan REC nanoparticles: Using a pH cycle method, freeze-dried RE nanoparticle powder was dissolved in deionized water, carboxymethyl chitosan CMCS was added, and deionized water was added to control the total solid concentration of the solution. After magnetic stirring, the mixture was uniformly mixed, and the pH value of the mixture was slowly adjusted to acidic with 0.1 M HCl solution. Magnetic stirring was continued, and finally the pH value of the mixture was adjusted to neutral. The mixture was centrifuged, and the supernatant was collected to obtain a REC nanoparticle solution, which was freeze-dried to produce REC nanoparticles.

[0010] The preparation method of the rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles comprises the following steps: step 1) specifically comprising: mixing defatted rice bran and deionized water in a ratio of 1:5, homogenizing for 4-6 minutes, and then magnetically stirring at room temperature for 1-2 hours; adjusting the pH value of the mixture to 9.5 using 1M NaOH, and magnetically stirring at 55°C for 1-2 hours; filtering to remove rice bran residue, and then centrifuging at 10,000 r / min for 30 minutes; adjusting the pH value of the supernatant obtained after centrifugation to 4.4 using 1M HCl; standing for 30-40 minutes, and then centrifuging at 10,000 r / min for 30 minutes; collecting the precipitate obtained after centrifugation, diluting it, adjusting its pH value to neutral, and freeze-drying it to obtain rice bran protein.

[0011] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 2), the concentration of the rice bran protein RBP stock solution is 10 mg / mL and the pH value is 12.0.

[0012] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 3), the amount of epigallocatechin gallate EGCG added is 1.5% to 3.5% of the mass of rice bran protein RBP.

[0013] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 3), the total solid concentration of the solution is controlled to be 0.1% and the system pH is 9.0.

[0014] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 4), the freeze-dried RE nanoparticles are dissolved in deionized water to a concentration of 10 mg / mL.

[0015] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 4), the mass ratio of the added amount of carboxymethyl chitosan CMCS to the RE nanoparticles is 2:1 to 6:1.

[0016] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 4), the total solid concentration of the REC nanoparticle solution is 0.2% to 0.5%.

[0017] In the above-mentioned method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, in step 4), the pH value of the mixed system is adjusted to an acidic pH of 2.5 to 4.5.

[0018] In the preparation method of the rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, the freeze-drying conditions in steps 3) and 4) are as follows: pre-freeze at -20°C for 24 hours, place in a freeze dryer, and freeze-dry at 0.1-0.5 mbar and -60--50°C for 48 hours.

[0019] The present invention provides a method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles. Compared with the prior art, the method of the present invention has the following beneficial effects:

[0020] 1. The present invention utilizes a pH cycle method to modify carboxymethyl chitosan onto the surface of RE nanoparticles. Carboxymethyl chitosan has unique biological activity and rich physiological functions. The entire nanoparticle preparation method and preparation process are simple, fast, and have a high yield.

[0021] 2. The present invention utilizes carboxymethyl chitosan and rice bran protein to reduce the activity loss of epigallocatechin gallate due to environmental factors, thereby improving its bioavailability. In addition, the nanoparticles have the ability of controlled release, which can prolong the antioxidant activity of epigallocatechin gallate.

[0022] 3. The present invention makes full use of the pH cycle method to prepare protein-polyphenol-polysaccharide ternary nanoparticles, effectively enhancing the functional properties of protein nanoparticles. In addition, no chemical emulsifier is added during the preparation process, which is green and safe, further expanding the application potential of the prepared nanoparticles.

[0023] 4. The present invention utilizes rice bran, a by-product of grain and oil processing, and extracts rice bran protein as a nanoparticle carrier, thereby saving crop resources and improving the application value of rice bran.

[0024] 5. The rice bran protein-epigallocatechin gallate-carboxymethyl chitosan ternary nanoparticles prepared in the present invention have a particle size of 112.1-566.3 nm, a PDI of 0.141-0.696, a Zeta potential of -29.4--14.0 mV, and a turbidity of 0.0576-1.2466. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a flow chart for the preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan ternary nanoparticles.

[0026] Figure 2 The particle size and turbidity diagram of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan ternary nanoparticles prepared in Examples 1-5 of the present invention.

[0027] Figure 3 The PDI and Zeta potential diagrams of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan ternary nanoparticles prepared in Examples 1-5 of the present invention. DETAILED DESCRIPTION

[0028] Example 1

[0029] A method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, comprising the following specific steps:

[0030] (1) Protein extraction: 1 kg of defatted rice bran was mixed with 5000 mL of deionized water, homogenized for 5 minutes, and then magnetically stirred at room temperature for 1 hour. The pH value of the mixture was adjusted to 9.5 using 1 M NaOH, and magnetically stirred at 55°C for 1 hour. After filtering to remove the rice bran residue, the mixture was centrifuged at 10,000 rpm for 30 minutes. The pH value of the supernatant obtained after centrifugation was adjusted to 4.4 using 1 M HCl. After standing for 40 minutes, the mixture was centrifuged at 10,000 rpm for 30 minutes. The precipitate obtained after centrifugation was collected and diluted, and its pH value was adjusted to neutral. The mixture was freeze-dried to obtain rice bran protein (RBP).

[0031] (2) Preparation of stock solution: 1 g of rice bran protein (RBP) from step (1) was dissolved in 100 ml of deionized water, the pH was adjusted to 12.0 with 1 M NaOH solution, and the mixture was magnetically stirred at 600 rpm at room temperature for 5 h. The RBP solution was then hydrated at 4°C overnight to prepare a 10 mg / mL RBP stock solution.

[0032] (3) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan REC ternary nanoparticles: 0.03 g of epigallocatechin gallate EGCG was added to the 10 mg / mL RPB stock solution in step (2), and 3 mL of deionized water was added to the system to control the total solid concentration to 0.1%. The pH value of the mixed solution was then adjusted to 9.0 with 1 M NaOH, and magnetic stirring was continued for 24 h. The mixture was dialyzed in an 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 8 h to obtain RE nanoparticle solution, which was freeze-dried to obtain RE nanoparticles. Using the pH cycling method, 0.03 g of the aforementioned RE nanoparticle powder was dissolved in 10 ml of deionized water, to which 0.06 g of CMCS was added. 8 mL of deionized water was then added to control the total solids concentration to 0.5%. The mixture was magnetically stirred for 1 hour. After mixing, the pH of the mixture was slowly adjusted to 3.0 with 0.1 M HCl solution. Magnetic stirring was continued for 1 hour. Finally, the pH of the mixture was adjusted to neutral, and the mixture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected to obtain the REC nanoparticle solution.

[0033] (4) Drying: The REC nanoparticle solution prepared in step (3) was frozen at -20°C for 24 h, and then placed in a freeze dryer and freeze-dried at 0.1-0.5 mbar and -60--50°C for 48 h to obtain the REC nanoparticle product.

[0034] The prepared REC had a particle size of 156.3 nm, a turbidity of 0.2066, a PDI of 0.289, and a potential of -18.3 mV.

[0035] Example 2

[0036] A method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, comprising the following specific steps:

[0037] (1) Protein extraction: 1 kg of defatted rice bran was mixed with 5000 mL of deionized water, homogenized for 5 minutes, and then magnetically stirred at room temperature for 1 hour. The pH value of the mixture was adjusted to 9.5 using 1 M NaOH, and magnetically stirred at 55 ° C for 1 hour. After filtering to remove the rice bran residue, the mixture was centrifuged at 10,000 r / min for 30 minutes. The pH value of the supernatant obtained after centrifugation was adjusted to 4.4 using 1 M HCl. After standing for 40 minutes, the mixture was centrifuged at 10,000 r / min for 30 minutes. The precipitate obtained after centrifugation was collected and diluted, and its pH value was adjusted to neutral. The rice bran protein was obtained after freeze-drying.

[0038] (2) Preparation of stock solution: 1 g of rice bran protein (RBP) from step (1) was dissolved in 100 ml of deionized water, the pH was adjusted to 12.0 with 1 M NaOH solution, and the mixture was magnetically stirred at 600 rpm at room temperature for 5 h. The RBP solution was then hydrated at 4°C overnight to prepare a 10 mg / mL RBP stock solution.

[0039] (3) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan REC ternary nanoparticles: 0.03 g of epigallocatechin gallate EGCG was added to the 10 mg / mL RPB stock solution in step (2), and 2.5 mL of deionized water was added to the system to control the total solid concentration to 0.1%. The pH value of the mixed solution was then adjusted to 9.0 with 1 M NaOH, and magnetic stirring was continued for 24 h. The solution was dialyzed in a 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 8 h to obtain RE nanoparticle solution, which was freeze-dried to obtain RE nanoparticles. Using the pH cycle method, 0.03 g of the above RE nanoparticle powder was dissolved in 10 ml of deionized water, and 0.09 g of CMCS, and add 50 mL of deionized water to the system to control the total solid concentration to 0.2%, magnetically stir for 1 hour, and after mixing evenly, slowly adjust the pH value of the mixed system to 2.5 with 0.1 M HCl solution, continue magnetic stirring for 1 hour, and finally adjust the pH value of the mixed system to neutral, centrifuge at 10000 r / min for 10 minutes, and collect the supernatant to obtain REC nanoparticle solution;

[0040] (4) Drying: The REC nanoparticle solution prepared in step (3) was frozen at -20°C for 24 h, and then placed in a freeze dryer and freeze-dried at 0.1-0.5 mbar and -60--50°C for 48 h to obtain the REC nanoparticle product.

[0041] The prepared REC had a particle size of 148 nm, a turbidity of 0.0866, a PDI of 0.246, and a potential of -20.6 mV.

[0042] Example 3

[0043] A method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, comprising the following specific steps:

[0044] (1) Protein extraction: 1 kg of defatted rice bran was mixed with 5000 mL of deionized water, homogenized for 5 minutes, and then magnetically stirred at room temperature for 1 hour. The pH value of the mixture was adjusted to 9.5 using 1 M NaOH, and magnetically stirred at 55°C for 1 hour. After filtering to remove the rice bran residue, the mixture was centrifuged at 10,000 rpm for 30 minutes. The pH value of the supernatant obtained after centrifugation was adjusted to 4.4 using 1 M HCl. After standing for 40 minutes, the mixture was centrifuged at 10,000 rpm for 30 minutes. The precipitate obtained after centrifugation was collected and diluted, and its pH value was adjusted to neutral. The mixture was freeze-dried to obtain rice bran protein (RBP).

[0045] (2) Preparation of stock solution: 1 g of rice bran protein (RBP) from step (1) was dissolved in 100 ml of deionized water, the pH was adjusted to 12.0 with 1 M NaOH solution, and the mixture was magnetically stirred at 600 rpm at room temperature for 5 h. The RBP solution was then hydrated at 4°C overnight to prepare a 10 mg / mL RBP stock solution.

[0046] (3) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan REC ternary nanoparticles: 0.03 g of epigallocatechin gallate EGCG was added to the 10 mg / mL RPB stock solution in step (2), and 3 mL of deionized water was added to the system to control the total solid concentration to 0.1%. The pH value of the mixed solution was then adjusted to 9.0 with 1 M NaOH, and magnetic stirring was continued for 24 h. The solution was dialyzed in a 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 8 h to obtain RE nanoparticle solution, which was freeze-dried to obtain RE nanoparticles. Using the pH cycle method, 0.03 g of the above RE nanoparticle powder was dissolved in 10 ml of deionized water, and 0.12 g of CMCS, and add 40 mL of deionized water to the system to control the total solid concentration to 0.3%, magnetically stir for 1 hour, and after mixing evenly, slowly adjust the pH value of the mixed system to 2.5 with 0.1 M HCl solution, continue magnetic stirring for 1 hour, and finally adjust the pH value of the mixed system to neutral, centrifuge at 10000 r / min for 10 minutes, and collect the supernatant to obtain REC nanoparticle solution;

[0047] (4) Drying: The REC nanoparticle solution prepared in step (3) was frozen at -20°C for 24 h, and then placed in a freeze dryer and freeze-dried at 0.1-0.5 mbar and -60--50°C for 48 h to obtain the REC nanoparticle product.

[0048] The prepared REC had a particle size of 114.8 nm, a turbidity of 0.0576, a PDI of 0.141, and a potential of -24.1 mV.

[0049] Example 4

[0050] A method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, comprising the following specific steps:

[0051] (1) Protein extraction: 1 kg of defatted rice bran was mixed with 5000 mL of deionized water, homogenized for 5 minutes, and then magnetically stirred at room temperature for 1 hour. The pH value of the mixture was adjusted to 9.5 using 1 M NaOH, and magnetically stirred at 55°C for 1 hour. After filtering to remove the rice bran residue, the mixture was centrifuged at 10,000 rpm for 30 minutes. The pH value of the supernatant obtained after centrifugation was adjusted to 4.4 using 1 M HCl. After standing for 40 minutes, the mixture was centrifuged at 10,000 rpm for 30 minutes. The precipitate obtained after centrifugation was collected and diluted, and its pH value was adjusted to neutral. The mixture was freeze-dried to obtain rice bran protein (RBP).

[0052] (2) Preparation of stock solution: 1 g of rice bran protein (RBP) prepared in step (1) was dissolved in 100 ml of deionized water, the pH was adjusted to 12.0 with 1 M NaOH solution, and the mixture was stirred magnetically at 600 rpm for 5 h at room temperature. The RBP solution was then hydrated overnight at 4°C to prepare a 10 mg / mL RBP stock solution.

[0053] (3) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan (REC) ternary nanoparticles: 0.03 g of epigallocatechin gallate EGCG was added to the 10 mg / mL RPB stock solution in step (2), and 3 mL of deionized water was added to the system to control the total solid concentration to 0.1%. The pH value of the mixed solution was then adjusted to 9.0 with 1 M NaOH, and magnetic stirring was continued for 24 h. The solution was dialyzed in a 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 8 h to obtain RE nanoparticle solution, which was freeze-dried to obtain RE nanoparticles. Using the pH cycle method, 0.03 g of the above RE nanoparticle powder was dissolved in 10 ml of deionized water, and 0.15 g of CMCS, and add 35 mL of deionized water to the system to control the total solid concentration to 0.4%, magnetically stir for 1 hour, and after mixing evenly, slowly adjust the pH value of the mixed system to 3.5 with 0.1 M HCl solution, continue magnetic stirring for 1 hour, and finally adjust the pH value of the mixed system to neutral, centrifuge at 10000 r / min for 10 minutes, and collect the supernatant to obtain REC nanoparticle solution;

[0054] (4) Drying: The REC nanoparticle solution prepared in step (3) was frozen at -20°C for 24 h, and then placed in a freeze dryer and freeze-dried at 0.1-0.5 mbar and -60--50°C for 48 h to obtain the REC nanoparticle product.

[0055] The prepared REC had a particle size of 290.3 nm, a turbidity of 0.9566, a PDI of 0.571, and a potential of -17.8 mV.

[0056] Example 5

[0057] A method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, comprising the following specific steps:

[0058] (1) Protein extraction: 1 kg of defatted rice bran was mixed with 5000 mL of deionized water, homogenized for 5 minutes, and then magnetically stirred at room temperature for 1 hour. The pH value of the mixture was adjusted to 9.5 using 1 M NaOH, and magnetically stirred at 55°C for 1 hour. After filtering to remove the rice bran residue, the mixture was centrifuged at 10,000 rpm for 30 minutes. The pH value of the supernatant obtained after centrifugation was adjusted to 4.4 using 1 M HCl. After standing for 40 minutes, the mixture was centrifuged at 10,000 rpm for 30 minutes. The precipitate obtained after centrifugation was collected and diluted, and its pH value was adjusted to neutral. The mixture was freeze-dried to obtain rice bran protein (RBP).

[0059] (2) Preparation of stock solution: 1 g of rice bran protein (RBP) from step (1) was dissolved in 100 ml of deionized water, the pH was adjusted to 12.0 with 1 M NaOH solution, and the mixture was magnetically stirred at 600 rpm at room temperature for 5 h. The RBP solution was then hydrated at 4°C overnight to prepare a 10 mg / mL RBP stock solution.

[0060] (3) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan REC ternary nanoparticles: 0.03 g of epigallocatechin gallate EGCG was added to the 10 mg / mL RPB stock solution in step (2), and 3 mL of deionized water was added to the system to control the total solid concentration to 0.1%. The pH value of the mixed solution was then adjusted to 9.0 with 1 M NaOH, and magnetic stirring was continued for 24 h. The solution was dialyzed in a 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 8 h to obtain RE nanoparticle solution, which was freeze-dried to obtain RE nanoparticles. Using the pH cycle method, 0.03 g of the above RE nanoparticle powder was dissolved in 10 ml of deionized water, and 0.18 g of CMCS, and add 60 mL of deionized water to the system to control the total solid concentration to 0.3%, magnetically stir for 1 hour, and after mixing evenly, slowly adjust the pH value of the mixed system to 3.5 with 0.1 M HCl solution, continue magnetic stirring for 1 hour, and finally adjust the pH value of the mixed system to neutral, centrifuge at 10000 r / min for 10 minutes, and collect the supernatant to obtain REC nanoparticle solution;

[0061] (4) Drying: The REC nanoparticle solution prepared in step (3) was frozen at -20°C for 24 h, and then placed in a freeze dryer and freeze-dried at 0.1-0.5 mbar and -60--50°C for 48 h to obtain the REC nanoparticle product.

[0062] The prepared REC had a particle size of 300 nm, a turbidity of 1.04, a PDI of 0.645, and a potential of -15 mV.

[0063] Figure 2 and Figure 3 The particle size, turbidity, particle size distribution (PDI) and Zeta-potential were used as research indicators to explore the effects of different polysaccharide addition amounts, pH and total polymer concentration on the REC nanoparticle formation process in Examples 1-5.

[0064] like Figure 2As shown, the mass ratio of the polysaccharide CMCS to RE nanoparticles in the REC nanoparticles prepared in Examples 1-5 ranged from 2:1 to 6:1, resulting in turbidity values ​​ranging from 0.0675 to 1.04 and particle sizes ranging from 114.8 to 300 nm. These results demonstrate that an appropriate amount of CMCS can tightly bind to RE through electrostatic interactions, minimizing the particle size and turbidity of the REC nanoparticles. Subsequently, as the polysaccharide CMCS ratio increases, the binding rate of CMCS to RBP increases with the amount of CMCS added, leading to increases in the particle size and turbidity of the REC nanoparticles. In Example 3, the particle size and turbidity of the REC nanoparticles were minimized when the mass ratio was 4:1.

[0065] The pH of the mixed system prepared in Examples 1-5 is 2.5-3.5. The closer the pH is to the isoelectric point of RBP (4.4), the lower the solubility of RBP, resulting in a large amount of aggregation of proteins and polysaccharides and an increase in particle size. The total polymer concentration in Examples 1-5 is 0.2%-0.5%. When the total polymer concentration is too high, the number of REC nanoparticles increases, and aggregation forms between the nanoparticles, resulting in an increase in the particle size of the nanoparticles. The particle size and turbidity of the REC nanoparticles prepared under the conditions of pH 2.5 and total polymer concentration of 0.3% in Example 3 are the smallest.

[0066] like Figure 3 As shown in the figure, it can be seen that the PDI of the REC nanoparticles in Examples 1-5 is between 0.141 and 0.645, and the potential is between -15 and -24.1 mv, indicating that the nanoparticles prepared in all examples have uniform particle size distribution and good stability. The REC nanoparticles prepared in Example 3 have the smallest particle size potential value, smaller PDI, and good REC dispersion, indicating that the system is relatively stable at this time and is suitable for preparing REC nanoparticles.

[0067] In summary, by adjusting the amount of polysaccharide CMCS added, the system pH and the total polymer concentration, the various indicators of REC nanoparticles can be optimized. In Example 3, when the mass ratio of CMCS to RE was 4:1, the pH was 2.5, and the total polymer concentration was 0.3%, the REC nanoparticles prepared showed the best PDI value, good particle size distribution and low turbidity. Under these conditions, the interaction between RE and CMCS reached the best state.

Claims

1. A method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles, characterized in that: The steps include: 1) Rice bran protein extraction: Defatted rice bran is mixed with deionized water, homogenized, and stirred at room temperature. The pH of the mixture is adjusted to alkaline, stirred, filtered to remove rice bran residue, and centrifuged. The pH of the supernatant obtained after centrifugation is adjusted to acidic, allowed to stand, and then centrifuged. The precipitate obtained after centrifugation is collected and diluted, and its pH is adjusted to neutral. The mixture is freeze-dried to obtain rice bran protein (RBP). 2) Preparation of stock solution: Dissolve the rice bran protein (RBP) prepared in step 1) in deionized water, adjust the pH of the solution with 1 M NaOH, and stir at room temperature. Then, hydrate the RBP solution at 4°C overnight to prepare a 10 mg / mL RBP stock solution with a pH of 12.

0. 3) Preparation of Rice Bran Protein-Epigallocatechin Gallate (RE) Nanoparticles: The RBP stock solution from step 2) was mixed with epigallocatechin gallate (EGCG) at an amount of 1.5% to 3.5% of the mass of rice bran protein (RBP). Deionized water was added to control the total solids concentration of the solution. The pH of the mixture was adjusted to alkaline with 1 M NaOH solution. The mixture was stirred continuously for 24 h and dialyzed in an 8000-14000 kDa dialysis bag at 4°C for 48 h, with the water changed every 6-8 h to obtain a RE nanoparticle solution. The RE nanoparticles were freeze-dried. 4) Preparation of rice bran protein-epigallocatechin gallate-carboxymethyl chitosan REC nanoparticles: The freeze-dried RE nanoparticle powder was dissolved in deionized water using the pH cycle method, and carboxymethyl chitosan CMCS was added. The mass ratio of carboxymethyl chitosan CMCS to RE nanoparticles was 4:

1. Deionized water was added to control the total solid concentration of the solution to 0.3%. After magnetic stirring, the mixture was evenly mixed and the pH value of the mixture was slowly adjusted to 2.5 with 0.1 M HCl solution. The magnetic stirring was continued and the pH value of the mixture was finally adjusted to neutral. The mixture was centrifuged and the supernatant was collected to obtain the REC nanoparticle solution, which was freeze-dried to obtain REC nanoparticles.

2. The method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles according to claim 1, wherein: Step 1) is specifically as follows: defatted rice bran and deionized water are mixed in a ratio of 1:5, homogenized for 4 to 6 minutes, and then magnetically stirred at room temperature for 1 to 2 hours. The pH value of the mixture is adjusted to 9.5 using 1 M NaOH, and magnetically stirred at 55° C. for 1 to 2 hours. After filtering to remove rice bran residue, the mixture is centrifuged at 10,000 r / min for 30 minutes. The pH value of the supernatant obtained after centrifugation is adjusted to 4.4 using 1 M HCl. After standing for 30 to 40 minutes, the mixture is centrifuged at 10,000 r / min for 30 minutes. The precipitate obtained after centrifugation is collected and diluted, and its pH value is adjusted to neutral. The rice bran protein is obtained after freeze-drying.

3. The method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles according to claim 1, wherein: In the step 3), the total solid concentration of the solution is controlled to be 0.1% and the pH of the system is 9.

0.

4. The method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles according to claim 1, wherein: In the step 4), the freeze-dried RE nanoparticles are dissolved in deionized water to a concentration of 10 mg / mL.

5. The method for preparing rice bran protein-epigallocatechin gallate-carboxymethyl chitosan nanoparticles according to claim 1, wherein: The freeze-drying conditions in steps 3) and 4) are as follows: pre-freeze at -20°C for 24 hours, place in a freeze dryer, and freeze-dry at 0.1-0.5 mbar and -60--50°C for 48 hours.

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