Preparation method for forming amyloid fiber through microwave induction of oat globulin
The treatment of oat globulin solution by microwave-induced method solves the problem of slow and uneven formation of protein amyloid fibers under traditional heating technology, achieving efficient and uniform protein fibrosis, and has environmental advantages.
Patent Information
- Application Number
- CN202411958207.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-08
AI Technical Summary
Traditional heating technology takes a long time to prepare protein amyloid fibers and the protein fibrosis is uneven.
The oat globulin solution was treated by microwave-induced method, and protein molecules were promoted through microwave heating to promote the denaturation and self-assembly of proteins to form amyloid fibers.
The formation speed and level of oat globulin amyloid fibers is improved, and more uniform protein fibrosis is achieved, and the method is environmentally friendly and efficient.
Smart Images
Figure CN119777014A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for preparing amyloid fibers formed by microwave-induced oat globulin, and belongs to the technical field of food processing. Background Art
[0002] Proteins can form fibrous structures, protein amyloid fibers, when heated to a temperature above their denaturation temperature (80-90°C) and a pH value far below their isoelectric point (~pH 2.0) and stirred for several hours (5-24h). During the fibrillation process, proteins undergo thermal denaturation and unfolding, hydrolyze into polypeptides, and finally self-assemble into ordered fibrous aggregates. Although plant proteins have shown their advantages in sustainability and resource efficiency and have become a preferred source of protein amyloid fibers, they still face some challenges and limitations in practical applications. Most plant proteins are affected by their subunit diversity, which increases the complexity of protein purification and research. The allergenicity of some proteins from plant sources (such as soybeans and peas) may affect biomedical and food applications.
[0003] As an ancient crop, oats have a long history of cultivation and consumption. In recent years, with the in-depth study of the nutritional components of oats, they have been highly praised, especially in the field of healthy diet and nutrition. As a protein-rich cereal, oats have the highest protein content among all cereals, and have a unique amino acid composition and bioactive peptides, making them a hot spot in plant protein research. Oat protein not only provides essential amino acids, but is also widely studied for its hypoallergenicity, high nutritional value and potential health benefits. The content of oat protein ranges from 11.2 to 19.9%, and is composed of globulin, albumin, alcohol-soluble protein and glutenin. Among them, globulin is the main storage protein, accounting for 70-80% of oat protein. Oat globulin can be divided into 12S globulin, 7S globulin and 3S globulin according to the sedimentation coefficient. 12S globulin accounts for about 80% of oat globulin, and is composed of 32kDa α-subunit and 22kDa β-subunit connected by disulfide bonds to form a 54kDa dimer. Overall, oat globulins showed excellent applicability in the preparation of protein amyloid fibers.
[0004] However, at present, traditional heating technology for preparing protein amyloid fibers takes a long time and the protein fibrillation is uneven. Summary of the invention
[0005] Based on the defects and shortcomings in the above-mentioned prior art, the purpose of the present invention is to provide a method for preparing oatmeal globulin amyloid fibers induced by microwaves, so as to improve the speed and level of oatmeal globulin amyloid fiber formation.
[0006] Conventional heating first heats the contact part of the container through heat conduction, and then the heat is transferred to the entire solution through heat conduction and convection. However, microwave heating increases the temperature of the solution by interacting with the polar molecules in the protein solution. Under the electromagnetic field of microwaves, water molecules and other polar molecules in the protein solution rotate rapidly, and the molecules rub and collide with each other to generate heat energy. In addition, microwave radiation excites the torsional vibration of the protein skeleton, thereby affecting the rate of tertiary structure rearrangement, such as denaturation and refolding. Therefore, microwave heating is more efficient in promoting the formation of protein amyloid fibers.
[0007] Based on the above principles, the technical problem to be solved by the present invention is achieved through the following technical solutions:
[0008] One of the technical solutions of the present invention provides a method for preparing amyloid fibers formed by oat globulin induced by microwaves, the steps of which are as follows:
[0009] Step S1: Preparation of oat globulin
[0010] S1.1 Disperse defatted oatmeal powder in deionized water to obtain a defatted oatmeal powder solution, stir evenly, and centrifuge to obtain a precipitate I and a supernatant I;
[0011] S1.2 Add the precipitate I obtained in step S1.1 to the NaCl solution, and repeat the stirring and centrifugation steps described in S1.1 to obtain supernatant II;
[0012] S1.3 adjusting the pH of the supernatant II to 3.5-4.5, centrifuging after standing to obtain precipitate II, redispersing the precipitate II in deionized water, dialyzing and freeze-drying to obtain oat globulin;
[0013] Step S2: Preparation of oatmeal amyloid fibers
[0014] S2.1 Dissolve the oat globulin prepared in step S1 in deionized water with a pH of 2.0, stir, store at 0-4°C overnight, and then adjust the pH to 2.0-2.5 to obtain an oat globulin solution;
[0015] S2.2 The oatmeal protein solution is treated by microwave heating and stirred at the same time; after heating, the solution is treated by ice water bath to obtain oatmeal protein amyloid fibers.
[0016] Furthermore, in step S1.1, the ratio of defatted oatmeal powder to deionized water is (1-2) g:10 mL; preferably 1 g:10 mL.
[0017] Furthermore, the stirring conditions in step S1.1 are: mechanical stirring at a speed of 400 to 800 rpm for 1 to 2 hours at room temperature; the speed is preferably 400 rpm, and the time is preferably 1 hour;
[0018] Furthermore, the centrifugation conditions in step S1.1 are: low-temperature centrifugation at 8000-10000 g for 15-30 min, wherein the low temperature is 0-4° C. The rotation speed is preferably 8000 g, the centrifugation time is preferably 15 min, and the temperature is preferably 4° C.
[0019] Furthermore, the amount of the NaCl solution in step S1.2 is 12 to 15 times, preferably 15 times, the volume of the precipitate I; and the concentration of the NaCl solution is 1 to 2 mol / L.
[0020] Furthermore, the reagent used to adjust the pH of the supernatant II in step S1.3 is preferably a 1-3 M HCl solution; the standing time is 20-30 min, preferably 20 min; the centrifugation condition after standing is 8000-10000g low-temperature centrifugation for 15-30 min, and the low temperature is 0-4°C; preferably 8000g 4°C centrifugation for 15 min.
[0021] Furthermore, in step S1.3, the precipitate II is dialyzed for 20 to 30 hours, and freeze-dried for 36 to 72 hours, preferably dialyzed for 24 hours and freeze-dried for 36 hours.
[0022] Furthermore, the concentration of oat globulin in step S2.1 is 20-30 mg / mL, preferably 20 mg / mL.
[0023] Furthermore, the stirring in step S2.1 is preferably carried out in a magnetic stirrer at a speed of 300 to 600 rpm, preferably 500 rpm; the time is 1 to 3 hours, preferably 2 hours; the reagent used to adjust the pH of the solution is preferably a 1 to 3 M HCl solution;
[0024] Furthermore, the temperature of the heating treatment in step S2.2 is 90-95°C, preferably 90°C, and the time is 0-10h; the stirring speed is 300-600rpm, preferably 300rpm; and the time of the ice water bath is 10-20min, preferably 10min.
[0025] The dielectric heating of microwaves causes the protein molecules to rapidly reach an unfolded state, exposing more positively charged groups, and the enhanced electrostatic interactions promote the formation of oatmeal amyloid fibrils.
[0026] The second technical solution of the present invention provides oat globulin amyloid fibers prepared in the above technical solution.
[0027] Furthermore, the oat globulin amyloid fibers have a linear aggregate structure, with a length of 1 to 10 μm and a diameter of 1 to 10 nm.
[0028] Compared with the prior art, this application has at least the following advantages:
[0029] (1) High efficiency: The present invention provides a new method for modifying oat protein, which uses microwaves to induce oat globulin to form amyloid fibers. Compared with traditional methods, microwave heating, as a feasible and effective protein fibrillation strategy, improves the speed and level of protein amyloid fiber formation, greatly expanding new ways to optimize protein structure and improve its physical and chemical properties.
[0030] (2) Uniformity: The present invention utilizes microwave dielectric heating to cause polar molecules (such as water molecules) to rotate rapidly, generating a large amount of frictional heat, thereby achieving heating. This heating method is internal and uniform. Uniform heating helps protein molecules to begin denaturation, expansion, aggregation and reorganization at the same time in various regions.
[0031] (3) Environmental protection: In addition, microwave heating can effectively utilize energy and reduce heat loss during the transfer process. The microwave heating process usually does not involve the discharge of liquid or gas. Therefore, compared with some traditional heating methods, microwave heating does not produce a large amount of wastewater, waste gas or waste heat, reducing the need to treat and discharge these wastes, which is more environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 Effects of two heating methods on the morphology of oatmeal amyloid fibrils; (a) conventional heating, (b) microwave heating;
[0033] Figure 2 Effects of two heating methods on the Congo red binding spectra of oatmeal amyloid fibrils; (a) conventional heating, (b) microwave heating;
[0034] Figure 3 Effects of two heating methods on the zeta potential of oatmeal amyloid fibrils; CH stands for conventional heating, MH stands for microwave heating;
[0035] Figure 3 Different lowercase letters indicate significant differences (P<0.05);
[0036] Figure 4 Effects of two heating methods on the particle size of oatmeal amyloid fibrils; CH means conventional heating, MH means microwave heating;
[0037] Figure 4 Different lowercase letters indicate significant differences (P<0.05). DETAILED DESCRIPTION
[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0039] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.
[0040] The deionized water with a pH of 2.0 described in the present application is deionized water after the pH is adjusted with hydrochloric acid, and after the adjustment, it is called deionized water (pH=2.0).
[0041] Comparative Example 1 Preparation of oatmeal amyloid fibers by conventional heating
[0042] (1) Preparation of oat globulin: Defatted oatmeal was dispersed in deionized water (1:10), mechanically stirred at 400 rpm for 1 h at room temperature, and then centrifuged at 8000 g for 15 min to obtain precipitate I and supernatant I, and supernatant I was discarded. 15 times the volume of 1.2 mol / L NaCl solution was added to precipitate I, mechanically stirred at 400 rpm for 1 h at room temperature, and then centrifuged at 8000 g for 15 min to obtain supernatant II, and the precipitate was discarded. The pH of supernatant II was adjusted to 4 using 1M HCl solution, and after standing for 20 min, centrifuged at 8000 g for 15 min to obtain precipitate II. Precipitate II was redispersed in deionized water, dialyzed for 24 h, and freeze-dried for 36 h to obtain oat globulin.
[0043] (2) Preparation of oat globulin amyloid fibers: The above oat globulin was dissolved in deionized water at pH 2, and the oat globulin concentration was 20 mg / mL. After magnetic stirring at 500 rpm for 2 h, the solution pH was adjusted to 2 with 1 M HCl solution, and the pH was adjusted to 2 after storage at 4°C overnight. Conventional heating was performed at 90°C and 300 rpm for 0, 2, 4, 6, 8, and 10 h. After heating, ice water bath was performed for 10 min to obtain oat globulin amyloid fibers.
[0044] The conventional heating in step (2) refers to: C-MAG HS7 heating magnetic stirrer, heating at 90°C and 300 rpm for 0, 2, 4, 6, 8, 10 h.
[0045] Example 1 Preparation of oatmeal amyloid fibers using microwave heating
[0046] (1) Preparation of oat globulin: Defatted oatmeal was dispersed in deionized water (1:10), mechanically stirred at 400 rpm for 1 h at room temperature, and then centrifuged at 8000 g for 15 min to obtain precipitate I and supernatant I, and supernatant I was discarded. 15 times the volume of 1.2 mol / L NaCl solution was added to precipitate I, mechanically stirred at 400 rpm for 1 h at room temperature, and then centrifuged at 8000 g for 15 min to obtain supernatant II, and the precipitate was discarded. The pH of supernatant II was adjusted to 4 using 1M HCl solution, and after standing for 20 min, centrifuged at 8000 g for 15 min to obtain precipitate II. Precipitate II was redispersed in deionized water, dialyzed for 24 h, and freeze-dried for 36 h to obtain oat globulin.
[0047] (2) Preparation of oat globulin amyloid fibers: The above oat globulin was dissolved in deionized water at pH 2, and the oat globulin concentration was 20 mg / mL. After magnetic stirring at 500 rpm for 2 h, the solution pH was adjusted to 2 with 1 M HCl solution. After storage at 4°C overnight, the pH was adjusted to 2. Microwave heating (carried out in a microwave solid-liquid phase synthesis extractor, model XH-200A) was performed at 90°C and 300 rpm for 0, 2, 4, 6, 8, and 10 h. After heating, ice water bath was placed for 10 min to obtain oat globulin amyloid fibers.
[0048] Example 2
[0049] The oat globulin amyloid fibers prepared in Example 1 and Comparative Example 1 were structurally characterized:
[0050] The structural characterization method of the prepared oat globulin amyloid fibers is as follows:
[0051] (1) Morphology: Dilute the sample solution to 0.1 mg / mL with deionized water adjusted to pH 2.0. Add 10 μL of the sample onto the copper grid, then add 10 μL of ultrapure water, and finally add 10 μL of 3% uranyl acetate dye solution. Dry overnight at room temperature and observe the morphology using a transmission electron microscope.
[0052] like Figure 1 As shown in the figure, the globular protein particles aggregated to form small plaques when heated for 0 h, and oatmeal amyloid fibrils were formed after 2 h. The number of oatmeal amyloid fibrils increased with the extension of heating time. After 6 h of microwave heating, the entanglement of long fibers increased. Finally, oatmeal amyloid fibrils were assembled into long and straight protein amyloid fibrils with a length of several microns. Compared with traditional heating ( Figure 1 A) compared to microwave heating ( Figure 1 B) Significantly promoted the formation of oatmeal amyloid fibrils.
[0053] (2) Congo red binding spectrum: Prepare 100 μM Congo red working solution with 10 mM phosphate buffer solution (150 mM NaCl, pH 7.4). Take 5 μL of sample and mix with 200 μL Congo red working solution. Incubate in the dark for 15 min and scan with an ELISA reader at 400-700 nm. Use Congo red working solution as blank control ( Figure 2 middle CR curve).
[0054] like Figure 2 As shown in the figure, Congo red has a strong affinity for the ordered β-folding structure and cross-β structure in protein amyloid fibrils. Congo red has a characteristic absorption peak at 486nm. After binding to oatmeal amyloid fibrils, the peak undergoes a red shift, confirming the formation of protein amyloid fibrils. Under microwave heating conditions, the more significant red shift of the characteristic peak reflects that microwaves are more efficient in promoting the formation of oatmeal amyloid fibrils.
[0055] (3) Zeta potential: The sample solution was diluted to 0.5 mg / mL with deionized water adjusted to pH 2.0. The refractive index and absorbance were set to 1.45 and 0.001, respectively, and the zeta potential was measured using a Malvern ZetaSizer Nano ZS after 120 s of equilibration.
[0056] like Figure 3 As shown in Figure 2, the increase in Zeta potential indicates that under acidic conditions, the electrostatic repulsion between particles increases, and the stability and dispersibility of oatmeal amyloid fiber solution are improved. Figure 3 The potential value of the MH group) increased more than that of the traditional heating group ( Figure 3 The results show that the thermal and non-thermal effects of microwaves promote the unfolding of protein structures and expose more positively charged groups. Therefore, microwave treatment can accelerate the formation of protein amyloid fibers through electrostatic interactions.
[0057] (4) Particle size distribution: The sample solution was diluted to 0.5 mg / mL with deionized water adjusted to pH 2.0. After equilibration for 120 s, the particle size was measured using a NanoBrook 173Plus particle size analyzer at a refractive index of 1.450 and a scattering angle of 173°.
[0058] like Figure 4 As shown in Figure 2, oat protein is hydrolyzed into peptides under acidic and heated conditions, which is the first step in the formation of oat protein amyloid fibrils. In addition, it was observed in the transmission electron microscopy images that a small amount of protein amyloid fibrils can be formed by heating for 2 h (see Figure 2). Figure 1 ). Therefore, the particle size decreased significantly after heating for 2 h. As the heating time increased (2 to 10 h), the average particle size gradually increased, indicating that the fibrils were in the growth and elongation stage. In the process of fibrillation, the microwave heating group ( Figure 4The particle size of the samples in the MH group) is larger than that in the conventional heating group ( Figure 4 This may be because the dielectric heating of microwaves causes the protein molecules to quickly reach an unfolded state, which is conducive to the formation of oatmeal amyloid fibrils.
[0059] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.
Claims
1. A method for preparing oatmeal globulin to form amyloid fibers induced by microwaves, characterized in that: The method steps are as follows: Step S1: Preparation of oat globulin S1.1 Disperse defatted oatmeal powder in deionized water to obtain a defatted oatmeal powder solution, stir evenly, and centrifuge to obtain a precipitate I and a supernatant I; S1.2 Add the precipitate I obtained in step S1.1 to the NaCl solution, and repeat the stirring and centrifugation steps described in S1.1 to obtain supernatant II; S1.3 adjusting the pH of the supernatant II to 3.5-4.5, centrifuging after standing to obtain precipitate II, redispersing the precipitate II in deionized water, dialyzing and freeze-drying to obtain oat globulin; Step S2: Preparation of oatmeal amyloid fibers S2.1 Dissolve the oat globulin prepared in step S1 in deionized water with a pH of 2.0, stir, store at 0-4°C overnight, and then adjust the pH to 2.0-2.5 to obtain an oat globulin solution; S2.2 The oatmeal protein solution is treated by microwave heating and stirred at the same time; after heating, the solution is treated by ice water bath to obtain oatmeal protein amyloid fibers.
2. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The ratio of defatted oatmeal powder to deionized water in step S1.1 is (1-2) g:10 mL.
3. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The stirring conditions in step S1.1 are: mechanical stirring at a speed of 400-800 rpm for 1-2 hours at room temperature; the centrifugation conditions are: low-temperature centrifugation at 8000-10000 g for 15-30 minutes, and the low temperature is 0-4°C.
4. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The amount of the NaCl solution used in step S1.2 is 12 to 15 times the volume of the precipitate I, and the concentration of the NaCl solution is 1 to 2 mol / L.
5. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The standing time in step S1.3 is 20 to 30 minutes; the centrifugation condition after standing is 8000 to 10000g low-temperature centrifugation for 15 to 30 minutes, and the low temperature is 0 to 4°C; the dialysis time of the precipitate II is 20 to 30 hours, and the freeze-drying time is 36 to 72 hours.
6. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The concentration of oat globulin described in step S2.1 is 20-30 mg / mL.
7. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The stirring speed in step S2.1 is 300-600 rpm and the time is 1-3 h.
8. The method for preparing microwave-induced oat globulin amyloid fibers according to claim 1, characterized in that: The temperature of the heating treatment in step S2.2 is 90-95° C., and the time is 0-10 h; the stirring speed is 300-600 rpm; and the time of the ice water bath is 10-20 min.
9. An oat globulin amyloid fiber, characterized in that: Prepared according to the method according to any one of claims 1 to 8.
10. The oat globulin amyloid fiber according to claim 9, characterized in that: The oat globulin amyloid fiber has a linear aggregate structure, a length of 1 to 10 μm, and a diameter of 1 to 10 nm.