Method for inducing rice bran protein self-assembled nanoparticles to embed fat-soluble polyphenols through cooperation of pH driving and arginine

Abstract

The invention belongs to the field of embedding bioactive substances in vegetable proteins, and provides a method for embedding fat-soluble polyphenol in rice bran protein self-assembled nanoparticles through pH driving and arginine induction. The rice bran protein is promoted to be self-assembled to form nano-particles by adjusting the pH value, and fat-soluble polyphenols such as rutin are embedded; amino, carboxyl and guanidyl in arginine are combined with polyphenol through electrostatic, hydrogen bond and hydrophobic effects, the secondary / tertiary structure of protein is regulated and controlled, the interfacial affinity between the protein and polyphenol is enhanced, and the solubility and stability of nanoparticles are improved. The method does not need an organic solvent and is green and safe, the embedding rate of the obtained rice bran protein-rutin nanoparticles exceeds 90%, the loading capacity reaches 23.4%, rutin is good in stability under ultraviolet irradiation, and the method is suitable for green and efficient embedding of bioactive substances.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant protein embedding bioactive substances, and particularly relates to a method for embedding fat-soluble polyphenols by using pH-driven and arginine-induced rice bran protein self-assembly nanoparticles. BACKGROUND

[0002] Fat-soluble plant polyphenol bioactive substances such as rutin and curcumin are natural antioxidants with excellent free radical capture and scavenging (antioxidant) ability, and can also be used for antibacterial and anti-inflammatory, preventing blood vessel rupture and hemostasis in clinical practice. However, such bioactive substances generally have poor water solubility and poor environmental tolerance, resulting in reduced bioavailability and difficulty in fully exerting the biological activity functions such as anticancer and antioxidant, thereby greatly limiting their application in functional foods and drugs.

[0003] At present, the main technologies for embedding fat-soluble polyphenol bioactive nutrients include liposome embedding technology, nanoemulsion technology, and protein nanoparticle embedding technology. Among them, the protein nanoparticle embedding technology mainly utilizes the interaction between protein molecules and polyphenol bioactive substances to form a complex or microcapsule to achieve embedding. Although this embedding technology has shown good potential in practical application, it still faces technical bottlenecks such as limited encapsulation efficiency and drug loading capacity, limited selection of embedding materials, high cost, and the need to use organic solvents. Therefore, developing a green and environmentally friendly, efficient and controllable new protein nanoparticle embedding technology has important practical significance for improving the bioavailability and stability of fat-soluble polyphenol bioactive substances. SUMMARY

[0004] To solve the above technical problems, the present application provides a method for embedding fat-soluble polyphenols by using pH-driven and arginine-induced rice bran protein self-assembly nanoparticles. By using pH-driven method and alkaline amino acids to enhance the non-covalent bond interactions such as hydrogen bond and hydrophobic interaction between rice bran protein and plant polyphenols, rice bran protein-arginine-polyphenol nanoparticles are constructed, thereby improving the solubility and bioavailability of fat-soluble plant polyphenols. At the same time, the use of polyphenol antioxidant properties can reduce the use of chemical synthetic antioxidants in food and improve the biological safety of food.

[0005] The technical scheme of the present application is as follows: A method for embedding fat-soluble polyphenols by using pH-driven and arginine-induced rice bran protein self-assembly nanoparticles, comprising the following steps: (1) Preparing a rice bran protein solution: taking rice bran protein raw materials, stirring and refrigerating with deionized water, and then centrifuging to remove insoluble large particles, and adjusting the pH to 12.0 with NaOH solution to obtain a rice bran protein solution with a concentration of 35-45 mg / mL; (2) Preparation of fat-soluble plant polyphenol solution: any one of rutin, curcumin is dissolved in an aqueous solution with a pH value of 12.0 to obtain a fat-soluble plant polyphenol stock solution with a concentration of 8-12 mg / mL, and then solid arginine is added to make the final concentration 3 mg / mL, and the pH value of the solution system is kept constant during the whole process.

[0006] (3) Preparation of rice bran protein-fat-soluble polyphenol plant nanoparticles: the rice bran protein solution in (1) and the fat-soluble plant polyphenol solution in (2) are mixed according to a volume ratio of 1:0.8-1.5, then the mixed solution is adjusted to pH 12.0 with NaOH solution, and after stirring, the pH is adjusted to 7.0 with HCl solution to obtain a rice bran protein-fat-soluble polyphenol plant nanoparticle solution, and rice bran protein-fat-soluble polyphenol plant nanoparticles are obtained after freeze-drying.

[0007] Preferably, in (1), the concentration of the rice bran protein stock solution is 20-60 mg / mL; the refrigeration temperature is 2-8℃; and the centrifugation conditions are 5000-10000 rpm for 5-20 min.

[0008] Preferably, in (2), the concentration of the arginine solution is 1-5 mg / mL.

[0009] Another aspect provided by the present application is the use of the above-mentioned rice bran protein-fat-soluble polyphenol plant nanoparticles in the preparation of functional plant protein-based food and plant-based emulsion products.

[0010] The present application has the following advantages and effects relative to the prior art: (1) In the present application, by adjusting the pH, the rice bran protein self-assembles in the solution to form nanoparticles, which have a certain size and structure, and can encapsulate polyphenolic substances inside; the reason is mainly that under different pH conditions, the functional groups on the rice bran protein and the polyphenolic substance molecules will be protonated or deprotonated, which is conducive to the formation of hydrogen bonds between the carboxyl, amino and other groups in the rice bran protein and the hydroxyl, carbonyl and other groups in the polyphenolic substance molecules, at the same time, electrostatic interactions between charged groups may also occur, and the aromatic ring groups of polyphenols can also interact with the hydrophobic regions of the protein, thereby further enhancing the combination of polyphenols and rice bran protein; (2) Arginine is a polar amino acid with good water solubility, and its amino, carboxyl and guanidino groups can combine with polyphenolic substances through electrostatic interactions and hydrogen bonds and other non-covalent bonds to improve their solubility (as shown in Figure 4 , at the same time, arginine can also promote the interaction between rice bran protein and polyphenolic substances by changing the molecular structure of rice bran protein, thereby improving the embedding rate, drug loading rate and stability of polyphenolic substances in rice bran protein nanoparticles; (3) The rice bran protein-rutin nanoparticles prepared by the method have excellent embedding rate and loading capacity, wherein the embedding rate is up to 90% or more, and the loading capacity can reach 23.4%; at the same time, the rutin in the rice bran protein-rutin nanoparticles has good ultraviolet irradiation stability, and the rice bran protein-arginine-rutin composite nanoparticles can improve the solubility of rutin, which is beneficial to the digestion and absorption in vivo and improves the bioavailability thereof; (4) The method for embedding fat-soluble polyphenols by the pH-driven and arginine-induced self-assembly of rice bran protein nanoparticles has the advantages of green safety and simple preparation without using organic solvents throughout. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 The embedding rates of the pH-driven method and the pH-arginine synergistic method under different rutin concentrations; Figure 2 The loading rates of the pH-driven method and the pH-arginine synergistic method under different rutin concentrations; Figure 3 The light stability of rutin, wherein (A) is the retention rate of the rice bran protein-rutin composite nanoparticles under ultraviolet irradiation, and (B) is the retention rate comparison of the nanoparticles and free rutin under ultraviolet irradiation; note: P4, P6, P8, P10, and P12 respectively represent the rice bran protein-rutin composite nanoparticles prepared by the pH-driven method under the rutin concentrations of 4, 6, 8, 10, and 12 mg / mL; A8, A10, and A12 respectively represent the rice bran protein-rutin composite nanoparticles prepared by the pH-driven and arginine-induced method under the rutin concentrations of 8, 10, and 12 mg / mL; Figure 4 The solubility diagram of the composite nanoparticles. DETAILED DESCRIPTION

[0012] In order for those skilled in the art to better understand the present application, the present application will be further described in conjunction with specific embodiments.

[0013] Example 1 A method for embedding fat-soluble polyphenols by pH and alkaline amino acid-induced self-assembly of rice bran protein nanoparticles, comprising the following steps: (1) Preparation of rice bran protein solution: 40 mg / mL rice bran protein raw material is added to deionized water and stirred on a magnetic stirrer for 2 hours and overnight in a 4℃ refrigerator, so as to be completely hydrated. After centrifugation at 8000 rpm for 10 min, the insoluble large particles are removed, and the hydrated rice bran protein (RBP) original solution is adjusted to pH 12.0 with 2 M NaOH solution to obtain a rice bran protein solution with a final concentration of 32.6 mg / mL; (2) Preparation of rutin solution: The pH of the aqueous solution was adjusted to 12.0, and then different amounts of rutin sample were added to prepare rutin stock solutions with concentrations of 8 mg / mL, 10 mg / mL, and 12 mg / mL, respectively. Solid arginine was added to the above rutin stock solutions, with a final arginine concentration of 3 mg / mL. The pH of the solution system was kept constant throughout the process to obtain rutin solutions with concentrations of 8 mg / mL, 10 mg / mL, and 12 mg / mL, respectively.

[0014] (3) Preparation of rice bran protein-lipid-soluble polyphenol plant nanoparticles: Mix rice bran protein solution and rutin solution at a volume ratio of 1:1, and then adjust the pH of the mixed solution to 12.0 with 1 M NaOH solution to avoid pH decrease caused by adding rutin solution. After stirring magnetically for 10 min, adjust the pH to 7.0 with 2 M HCl solution to obtain rice bran protein-rutin nanoparticle solution. After freeze drying, obtain rice bran protein-rutin nanoparticles.

[0015] Based on the different concentrations of the rutin stock solution, the prepared rice bran protein-rutin nanoparticles were named A8, A10, and A12, respectively, for their performance characterization and analysis.

[0016] Comparative Example The preparation of rutin-loaded rice bran protein nanoparticles (using pH-driven method only) includes the following steps: (1) Preparation of rice bran protein solution: Same as the preparation steps in Example 1; (2) Preparation of non-arginine-driven rutin solution: The steps differed from those in Example 1: no arginine solution was added; instead, different amounts of rutin were added to an aqueous solution with a pH of 12.0 to prepare non-arginine-driven rutin solutions with concentrations of 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, and 12 mg / mL, respectively, while maintaining a constant pH value throughout the process.

[0017] (3) Mix rice bran protein solution and non-arginine-driven rutin solution at a volume ratio of 1:1, stir magnetically for 10 min, and adjust the pH of the solution to 7.0 with 2 M HCl to obtain rice bran protein-non-arginine-driven rutin nanoparticle solution. After freeze drying, rice bran protein-non-arginine-driven rutin nanoparticles are obtained.

[0018] The nanoparticles prepared according to different concentrations of rutin were named P4, P6, P8, P10, and P12 for their performance characterization analysis.

[0019] Example 2: Performance determination of nanoparticles The performance indicators of the A8, A10, A12, P4, P6, P8, P10, and P12 nanoparticles prepared in Example 1 and Comparative Example 1 were detected and analyzed.

[0020] 2.1 Determination of Encapsulation Efficiency and Loading Rate: 100 mg of rutin-loaded nanoparticles were mixed with 10 mL of ethanol. The suspension was centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The content of free rutin was analyzed by spectrophotometry at 360 nm. Alternatively, 100 mg of rutin-loaded nanoparticles were mixed with 10 mL of ethanol and then sonicated (40 kHz) in a 60°C water bath for 30 min to extract the encapsulated rutin from the nanoparticles. The sonicated suspension was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to determine the total rutin content.

[0021]

[0022] The embedding rate test results are shown in Table 1: Table 1. Encapsulation efficiency of pH-driven and pH-arginine synergistic methods at different rutin concentrations.

[0023] Table 2 Loading rates of pH-driven and pH-arginine synergistic methods at different rutin concentrations

[0024] The data in Tables 1 and 2 above show that the encapsulation efficiency (EE) and loading rate (LC) reflect the rutin content that can be loaded into rice bran protein and the efficiency of rutin loading per unit carrier system, respectively. Combining Tables 1 and 2... Figure 1 and Figure 2 It was found that under the pH-driven method, the encapsulation rate and loading rate of rutin in rice bran protein-rutin nanoparticles reached their highest values ​​of 96.8% and 19.3%, respectively, when the rutin concentration was 6 mg / mL and 10 mg / mL. For the pH-arginine synergistic method, at higher rutin concentrations of 8-12 mg / mL, the encapsulation rate and loading rate of rutin in rice bran protein-rutin nanoparticles were significantly improved, reaching a maximum of 93.2% and 23.4%, respectively. This indicates that introducing arginine as a functional adjuvant can significantly improve the rutin loading capacity of rice bran protein, which is beneficial for solving problems such as poor rutin solubility and low digestibility and absorption.

[0025] Figure 4 It also shows that, at the same rutin concentration, the transmittance of the bran protein-rutin nanoparticle solution prepared by the pH-arginine synergistic method is significantly higher than that of the sample solution prepared by the pH-driven method. This indicates that arginine, as an adjuvant, can effectively enhance the interaction between bran protein and rutin, thereby significantly improving the solubility of rutin.

[0026] 2.2 Determination of particle size potential: The nanoparticle solution sample was diluted to 1 mg / mL before measurement. The particle size distribution and zeta potential of the sample were determined using a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcestershire, UK). All measurements were performed at 25 ± 1 °C.

[0027] Table 3. Particle size of rice bran protein and its rutin complex nanoparticle solution Sample Particle size (nm) RBP 113.1 ± 1.15 f ]] P4 110.2 ± 1.5 d e ]] P6 108.6 ± 1.0 d ]] P8 105.2 ± 0.4 c <!-- 4 -->]]> P10 111.4 ± 1.1 ef ]] P12 108.6 ± 0.8 d ]] A8 98.8 ± 0.5 a ]] A10 102.5 ±1.5 b ]] A12 102.1 ± 0.8 b ]]

[0028] Table 4. Zeta potential of bran protein and its rutin complex nanoparticle solutions Sample Zeta potential (mV) RBP -28.9 ± 1.3 a ]] P4 -28.2 ± 2.3 ab ]] P6 -28.4 ± 0.3 ab ]] P8 -25.5 ± 0.5 d ]] P10 -27.6 ± 0.6 abc ]] P12 -27.5 ± 0.4 abcd ]] A8 -26.6 ± 0.4 bcd ]] A10 -25.7 ± 1.2 cd ]] A12 -25.8 ± 1.1 b ]]

[0029] Table 3 shows that the particle size of all complex particles is smaller than that of rice bran protein. Furthermore, at the same rutin concentration, the particle size of the complex prepared by the pH-driven synergistic arginine method is significantly reduced. This indicates that both arginine and rutin can interact with rice bran protein to inhibit its aggregation in aqueous solution. In particular, the addition of arginine promotes the binding of rice bran protein to rutin, leading to a smaller particle size of the complex. Additionally, Table 4 shows that the absolute value of the surface potential of the nanoparticles gradually decreases with increasing rutin concentration. This indicates that the interaction between rutin and rice bran protein alters its surface charge distribution, and the addition of arginine promotes this interaction, thus increasing the degree of reduction in the surface charge of the complex.

[0030] 2.3 Stability analysis of rutin in nanoparticles (1) UV stability The optical stability of rutin in nanoparticle dispersions was determined using a controlled ultraviolet lamp. The prepared rice bran protein nanoparticles were placed in a transparent glass bottle and irradiated with ultraviolet light. The samples were treated under ultraviolet light for 30, 60, 90, 120, 240, 360, and 720 minutes, respectively. The amount of rutin remaining in the treated samples was then measured and compared with that of the untreated samples.

[0031] Table 5 Retention rate of rice bran protein-rutin composite nanoparticles under UV irradiation Time P4 P6 P8 P10 P12 A8 A10 A12 30 min 99.1%±0.30 97.0%±1.05 92.2%±2.71 73.6%±2.71 89.4%±2.48 98.1%±1.48 98.7%±1.17 92.9%±2.38 60 min 94.0%±1.49 91.2%±0.69 86.9%±1.56 61.8%±2.21 70.3%±2.71 97.5%±1.73 97.5%±2.02 68.7%±2.78 90 min 89.5%±0.93 88.9%±2.1 85.2%±1.61 55.9%±2.23 57.3%±2.39 96.4%±1.26 93.5%±1.36 58.6%±1.62 120 min 86.6%±0.71 87.9%±1.42 72.6%±1.69 39.7%±1.38 53.0%±2.50 80.4%±1.22 68.2%±1.72 56.9%±2.1 240 min 86.1%±1.19 85.5%±2.53 70.8%±2.57 37.9%±1.06 47.4%±1.36 77.7%±1.91 59.0%±2.38 54.2%±2.07 360 min 82.5%±0.85 83.9%±2.19 68.0%±1.26 35.8%±2.44 44.3%±1.45 74.9%±1.91 55.2%±2.69 51.3%±1.04 720 min 79.3%±1.95 76.4%±1.31 62.1%±2.97 33.0%±2.14 42.2%±2.05 71.2%±0.62 51.6%±2.73 49.2%±1.25

[0032] Table 6. Comparison of retention rates of rice bran protein-rutin composite nanoparticles and free rutin under UV irradiation. Time P6 A8 Ctral6 Ctral8 30 min 97.0%±1.05 98.1%±1.48 97.2%±2.35 92.9%±1.02 60 min 91.2%±0.69 97.5%±1.56 88.8%±0.68 82.3%±2.58 90 min 88.9%±2.10 96.4%±1.26 84.1%±1.60 74.0%±1.25 120 min 87.9%±1.42 80.4%±1.22 75.9%±1.83 69.5%±1.23 240 min 85.5%±2.53 77.7%±1.91 67.3%±2.82 58.1%±2.69 360 min 83.9%±2.19 74.9%±1.91 62.1%±2.63 52.2%±1.22 720 min 76.4%±1.31 71.2%±0.62 55.2%±2.22 43.2%±1.56

[0033] Tables 5 and 6 show that, except for the lack of significant difference in rutin stability between P12 and A12 samples after 60 min of UV irradiation, at other UV irradiation times, when the rutin concentration was the same, the retention rate of rutin embedded in the composite nanoparticles prepared by the pH-arginine synergistic method was higher than that of rutin embedded by the pH-driven method. A comparative study of the UV stability of the P6 and A8 composite nanoparticles with better stability with that of free rutin at the same concentration revealed (see...) Figure 3 Except for the initial 30 min of UV radiation, where there was no significant difference in stability between the embedded rutin and the control, the retention rate of rutin after embedding was higher than that of free rutin at all other treatment times.

[0034] The above UV radiation stability results demonstrate that the composite nanoparticles prepared by the pH-arginine synergistic method not only significantly improve the loading efficiency of rutin but also effectively enhance its photostability. This technology provides an innovative strategy for developing rutin delivery systems with high stability and high bioavailability, and has significant application value in the fields of functional foods and pharmaceutical preparations. The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. All equivalent variations and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for embedding fat-soluble polyphenols with pH-driven synergistic arginine-induced rice bran protein self-assembly nanoparticles, characterized by, The steps include the following: (1) Preparation of rice bran protein solution: Take the rice bran protein raw material, add deionized water, stir, and refrigerate to make it completely hydrated, then centrifuge to remove insoluble large particles, adjust the pH to 12.0 with NaOH solution, and obtain a rice bran protein solution with a concentration of 35-45 mg / mL; (2) Preparation of fat-soluble plant polyphenol solution: Dissolve any one of rutin and curcumin in a water solution with a pH of 12.0 to obtain a fat-soluble plant polyphenol stock solution with a concentration of 8-12 mg / mL, then add solid arginine to make the final concentration 3 mg / mL, and maintain the pH of the solution system constant throughout the process; (3) Preparation of rice bran protein-fat-soluble polyphenol plant nanoparticles: Mix the rice bran protein solution in (1) with the fat-soluble plant polyphenol solution in (2) according to the volume ratio of 1:0.8-1.5, then adjust the mixed solution to pH 12.0 with NaOH solution, stir, adjust the pH to 7.0 with HCl solution, and obtain a rice bran protein-fat-soluble polyphenol plant nanoparticle solution. Freeze-drying obtains rice bran protein-fat-soluble polyphenol plant nanoparticles.

2. A method for pH-driven synergistic arginine-induced self-assembly of rice bran protein nanoparticles for encapsulating fat-soluble polyphenols as claimed in claim 1, wherein, In (1), the concentration of the rice bran protein stock solution is 20-60 mg / mL; the refrigeration temperature is 2-8℃; and the centrifugation conditions are 5000-10000 rpm for 5-20 min.

3. A method for pH-driven synergistic arginine-induced self-assembly of rice bran protein nanoparticles for encapsulating fat-soluble polyphenols as claimed in claim 1, wherein, In (2), the concentration of the arginine solution is 1-5 mg / mL.

4. Use of the rice bran protein-fat-soluble polyphenol plant nanoparticles obtained by the method of any one of claims 1-3 in the preparation of functional plant protein-based food and plant-based emulsion products.

Images