Method for quantitatively preparing protein amyloid fiber through pressure dialysis

Abstract

The invention discloses a method for quantitatively preparing protein amyloid fibers through pressure dialysis, and belongs to the technical field of food. The method for quantitatively preparing the protein amyloid fiber through pressure dialysis is provided for the first time, the pressure induction technology and the dialysis technology are combined, the advantages of the pressure induction technology and the dialysis technology are fully exerted, and efficient and controllable preparation of the protein amyloid fiber is achieved. Through a pressure dialysis technology, the pressure and the action time can be accurately controlled, the outflow of small molecular substances is accelerated by utilizing external pressure, the dialysis time is shortened, and the preparation efficiency is improved; fiber loss is reduced, and fiber yield is increased; small molecular impurities are more thoroughly removed, and the fiber purity is enhanced; impurities such as small molecular salt ions and non-fiberized protein molecules are selectively removed through the dialysis bag, interference of the impurities on the fibers is avoided, meanwhile, the stability of the fiber structure is maintained, the technology can be designed in an amplified mode, and large-scale preparation is achieved.

Description

Technical Field

[0001] This invention belongs to the field of food technology, and in particular relates to a method for quantifying the preparation of protein amyloid fibers by pressure dialysis. Background Technology

[0002] Amyloid fibers are a novel, safe, and non-toxic biomaterial prepared from edible plant and animal proteins (such as soy protein and whey protein) through acid-heat induction. When heated under acidic conditions, the protein molecules unfold and self-assemble into nanofibers rich in β-sheet structures. These fibers are fundamentally different from disease-related amyloid aggregates. Since both the raw materials and the preparation process originate from food systems, they possess excellent biocompatibility and food safety.

[0003] In terms of applications, protein amyloid fibers exhibit unique and valuable potential in many aspects: They possess high aspect ratios and excellent interfacial properties, enabling them to act as highly efficient surfactants. For example, whey protein amyloid fibers, even at low protein concentrations, can serve as excellent foaming agents, with foaming performance comparable to traditional foaming agents like egg white. They can also stabilize foam, preventing aggregation and coarsening, effectively improving the texture and mouthfeel of food. As emulsifiers, they can adsorb onto the oil-water interface where small oil droplets form, preventing adjacent droplets from adhering to each other or combining into larger droplets. Compared to natural proteins, they exhibit higher emulsifying activity and stability, significantly improving emulsion quality. Furthermore, protein amyloid fibers possess excellent gelling properties, increasing system viscosity at lower concentrations and improving product texture. For instance, pea globulin amyloid fibers, after pretreatment with a low-frequency magnetic field, show significantly enhanced gelling properties, making them applicable to liquid foods and low-calorie foods. They can also be used to prepare low-fat pork sausages and other foods, bringing more innovative possibilities to the food industry.

[0004] Existing methods for preparing protein amyloid fibers mainly include acid-thermal methods, mechanical induction methods, and multi-component composite methods. Among them, the acid-thermal method can obtain protein amyloid fibers by heating a protein solution at pH=2.0-3.0 and a temperature of 80-90℃, but it has limitations such as low yield and high energy consumption. Mechanical induction methods, such as ultrasonic treatment (20kHz, 200-400W for 10-30 minutes) and shear rheology (5-50r / min shear rate, 90-110℃ oil bath), can significantly improve fiber uniformity and shorten reaction time through cavitation effect or flow field control. Shear rheology technology has achieved large-scale production of 200g per batch. The multi-component composite method introduces components such as chitosan and ferulic acid to construct functional fibers using electrostatic interactions or covalent bonds. For example, the tensile strength of peanut protein-chitosan composite fiber is increased to 25MPa, and the antioxidant activity of lysozyme-ferulic acid composite fiber reaches 85%. However, it still faces challenges such as high cost and insufficient safety assessment in the industrialization process. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for the quantitative preparation of protein amyloid fibers via pressure dialysis. The method provided by this invention offers advantages such as simple operation and low cost, achieving directional alignment and efficient concentration of fibers, thereby improving fiber yield and purity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for quantifying the preparation of protein amyloid fibers by pressure dialysis, comprising the following steps: (1) Preparation of protein amyloid cellulose solution; (2) The protein amyloid fiber solution is placed into a dialysis bag, and pressure is applied to the dialysis bag to perform pressure dialysis to obtain concentrated protein amyloid fiber.

[0007] Further, in step (1), the preparation of the protein amyloid cellulose solution includes the following steps: acid titration, centrifugation and oil bath heating of the protein solution in sequence to obtain the protein amyloid cellulose solution.

[0008] Furthermore, the reagent for the acid titration is 6 mol / L hydrochloric acid, and the titration endpoint is when the solution pH reaches 2.0; The relative centrifugal force for the centrifugation process is 10000×g, and the centrifugation time is 15min; The oil bath heating temperature is 85-90℃, and the oil bath heating time is 10-24h.

[0009] Furthermore, the protein in the protein solution is selected from soy protein, oat protein, lysozyme protein, or whey protein.

[0010] Furthermore, in step (2), the amount of the protein amyloid cellulose solution loaded does not exceed 2 / 3 of the dialysis bag volume.

[0011] Furthermore, in step (2), the molecular weight cutoff of the dialysis bag is 50 kDa.

[0012] Furthermore, in step (2), the pressure of the pressure dialysis is 200g.

[0013] Furthermore, in step (2), the dialysate for pressure dialysis is water with a pH of 2.

[0014] Furthermore, in step (2), the pressure dialysis temperature is 4°C and the pressure dialysis time is 28h.

[0015] The present invention also provides a protein amyloid fiber, which is prepared according to the method described in the above technical solution.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention proposes for the first time a method for the quantitative preparation of protein amyloid fibers via pressure dialysis. It combines pressure induction with dialysis technology, fully leveraging the advantages of both to achieve efficient and controllable preparation of protein amyloid fibers. Pressure dialysis allows for precise control of pressure and treatment time, utilizing external pressure to accelerate the outflow of small molecules, shortening dialysis time and improving preparation efficiency; reducing fiber loss and increasing fiber yield; more thoroughly removing small molecule impurities and enhancing fiber purity; and selectively removing small molecule salt ions, unfibrinated protein molecules, and other impurities through the dialysis bag, avoiding interference with the fibers while maintaining fiber structural stability. Furthermore, this technology is scalable and can be designed for large-scale preparation, providing a novel approach and method for concentrating protein amyloid fibers, with significant scientific value and broad application prospects.

[0017] This invention employs a pressure dialysis quantification method to prepare amyloid fibers. This process does not introduce any chemical reagents. Pressure dialysis increases the yield of protein amyloid fibers, more thoroughly removes small molecule impurities, enhances fiber purity, and avoids interference from impurities on the fibers. This provides a promising method for expanding the application range of protein amyloid fibers in the food industry. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a process flow diagram of the method for quantifying the preparation of protein amyloid fibers by pressure dialysis in Examples 1-4; Figure 2 The diagrams shown are schematic representations of the pressure dialysis apparatus in Examples 1-4, where the left figure is the front view and the right figure is the top view. Figure 3 The image shows an atomic force microscope image of the concentrated soybean protein amyloid fibers obtained in Example 1, where the left image is before dialysis and the right image is after dialysis. Figure 4 The image shows an atomic force microscope image of the concentrated oat protein amyloid fibers obtained in Example 2, where the left image is before dialysis and the right image is after dialysis. Figure 5 The image shows an atomic force microscope image of the concentrated lysozyme protein amyloid fibers obtained in Example 3, where the left image is before dialysis and the right image is after dialysis. Figure 6 The image shows an atomic force microscope image of the concentrated whey protein amyloid fibers obtained in Example 4, where the left image is before dialysis and the right image is after dialysis. Figure 7 This is a graph showing the change in protein concentration in the solute over time in Example 1. Figure 8 The average length of the concentrated protein amyloid fibers obtained in Example 1. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] This invention provides a method for quantifying the preparation of protein amyloid fibers via pressure dialysis, comprising the following steps: (1) Preparation of protein amyloid cellulose solution; (2) The protein amyloid fiber solution is placed into a dialysis bag, and pressure is applied to the dialysis bag to perform pressure dialysis to obtain concentrated protein amyloid fiber.

[0022] In a preferred embodiment, step (1) involves the following steps: sequentially subjecting the protein solution to acid titration, centrifugation, and oil bath heating to obtain the protein amyloid fiber solution.

[0023] In a preferred embodiment, the protein in the protein solution is selected from soy protein, oat protein, lysozyme protein, or whey protein; the concentration of the protein solution is 3% (w / v).

[0024] In a preferred embodiment, the reagent for acid titration is 6 mol / L hydrochloric acid, and the titration endpoint is when the solution pH reaches 2.0.

[0025] In a preferred embodiment, the relative centrifugal force of the centrifugation process is 10000×g, and the centrifugation time is 15min.

[0026] In a preferred embodiment, the oil bath heating temperature is 85-90°C, and the oil bath heating time is 10-24 hours.

[0027] In a preferred embodiment, in step (2), the amount of amyloid cellulose solution loaded does not exceed 2 / 3 of the dialysis bag volume. This invention controls the amount of amyloid cellulose solution loaded to allow sufficient space for the solution to flow and expand under pressure.

[0028] In a preferred embodiment, in step (2), the molecular weight cutoff of the dialysis bag is 50 kDa.

[0029] In a preferred embodiment, in step (2), the pressure of the pressure dialysis is 200g. This invention uses water with pH=2 as the dialysate, and accelerates the transmembrane diffusion of small molecule impurities (such as salt ions and denaturants) through pressure-driven diffusion, while maintaining the supersaturated state of the protein solution, thereby increasing the yield and purity of protein amyloid fibers.

[0030] In a preferred embodiment, in step (2), the dialysate for pressure dialysis is water with a pH of 2.

[0031] In a preferred embodiment, in step (2), the temperature of the pressure dialysis is 4°C and the pressure dialysis time is 28h.

[0032] The present invention also provides a protein amyloid fiber, which is prepared according to the method described in the above technical solution.

[0033] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0034] Example 1 A method for quantifying the preparation of protein amyloid fibers by pressure dialysis is shown in the process flow diagram. Figure 1 The specific steps are as follows: (1) Soy protein isolate was uniformly dispersed in deionized water and stirred for 10 h to obtain a 3% (w / v, i.e., 30 mg / mL) soy protein stock solution. The soy protein stock solution was titrated with 6 mol / L hydrochloric acid until the pH value of the solution reached 2.0. Then, it was centrifuged at 10000×g for 15 min to obtain a uniform soy protein suspension. The suspension did not contain fine insoluble soy protein solid particles. The obtained soy protein suspension was then transferred to a sealed container and heated in an oil bath at 85℃ and 200 rpm for 24 h to induce the formation of amyloid fibers in soy protein to obtain a soy protein amyloid fiber solution. (2) Using a pipette, load the soybean protein amyloid cellulose solution obtained in step (1) into a dialysis bag with a molecular weight cutoff of 50 kDa. Avoid splashing or generating air bubbles. The volume of the soybean protein amyloid cellulose solution should not exceed 2 / 3 of the dialysis bag's volume. After loading, secure both ends of the dialysis bag with a dialysis bag clamp to ensure a good seal and prevent leakage. Before sealing, gently squeeze the dialysis bag to expel air. Then, place the sealed dialysis bag into a pressure dialysis apparatus (see...). Figure 2 In this study, a pressure of 200g was applied to the dialysis bag at a temperature of 4℃, and deionized water with pH=2.0 was used as the dialysis solution for dialysis. Dialysis was performed for 28 hours. After dialysis, the dialysis bag was removed and the concentrated soybean protein starch fiber inside the bag was collected.

[0035] Figure 2 The diagram shows the structure of the pressure dialysis apparatus in Examples 1-4. As can be seen, the pressure dialysis apparatus used in Examples 1-4 mainly consists of a 3L beaker, a cylindrical strainer, a 200g weight, and a partition. The beaker provides space for liquid flow; the cylindrical strainer is used to place the dialysis bag containing the sample; and the weight is used to apply pressure to accelerate the dialysis speed.

[0036] Figure 3 The images show atomic force microscopy (AFM) images of the concentrated soybean protein amyloid fibers obtained in Example 1, with the left image showing the result before dialysis and the right image showing the result after dialysis. Figure 3 It can be seen that before dialysis, soybean protein amyloid fibers are mostly flexible fibers and some macromolecules. After dialysis, background impurities are significantly reduced.

[0037] The purity of the fiber in Example 1 was measured to increase from 28.83% before dialysis to 39.91% after dialysis, and the fiber yield increased from 35.98% before dialysis to 42.81% after dialysis.

[0038] Example 2 A method for quantifying the preparation of protein amyloid fibers by pressure dialysis is shown in the process flow diagram. Figure 1 The specific steps are as follows: (1) Oat protein was uniformly dispersed in deionized water and stirred for 10 h to obtain an oat protein stock solution with a concentration of 3% (w / v, i.e., 30 mg / mL); the above oat protein stock solution was acid-titrated with 6 mol / L hydrochloric acid until the pH value of the solution reached 2.0, and then centrifuged at 10000×g for 15 min to obtain a uniform oat protein suspension. The suspension did not contain fine insoluble oat protein solid particles; the obtained oat protein suspension was then transferred to a sealed container and stirred and heated at 90℃ oil bath and 200 rpm for 10 h to induce oat protein to form amyloid fibers, thus obtaining an oat protein amyloid fiber solution. (2) Using a pipette, load the oat protein amyloid cellulose solution obtained in step (1) into a dialysis bag with a molecular weight cutoff of 50 kDa. Avoid splashing or generating air bubbles. The volume of the oat protein amyloid cellulose solution should not exceed 2 / 3 of the dialysis bag's volume. After loading, secure both ends of the dialysis bag with a dialysis bag clamp to ensure a good seal and prevent leakage. Before sealing, gently squeeze the dialysis bag to expel air. Then, place the sealed dialysis bag into a pressure dialysis apparatus (see...). Figure 2 In this study, a pressure of 200g was applied to the dialysis bag at a temperature of 4℃, and deionized water with pH=2.0 was used as the dialysate for dialysis. Dialysis was performed for 28 hours. After dialysis, the dialysis bag was removed and the concentrated oat protein amyloid fibers in the bag were collected.

[0039] Figure 4 The images shown are atomic force microscopy images of the concentrated oat protein amyloid fibers obtained in Example 2, with the left image showing the result before dialysis and the right image showing the result after dialysis. Figure 4 It can be seen that there are many unassembled particulate aggregates before dialysis, and the short fiber morphology is basically preserved after dialysis.

[0040] The purity of the fiber in Example 2 was measured to increase from 25.56% before dialysis to 30.50% after dialysis, and the fiber yield increased from approximately 28% before dialysis to 32.01% after dialysis.

[0041] Example 3 A method for quantifying the preparation of protein amyloid fibers by pressure dialysis is shown in the process flow diagram. Figure 1 The specific steps are as follows: (1) Disperse lysozyme protein evenly in deionized water and stir for 10 h to prepare a lysozyme protein stock solution with a concentration of 3% (w / v, i.e., 30 mg / mL); titrate the above lysozyme protein stock solution with 6 mol / L hydrochloric acid until the pH value of the solution reaches 2.0, and then centrifuge at 10000×g for 15 min to obtain a uniform lysozyme protein suspension. The suspension does not contain fine insoluble lysozyme protein solid particles; then transfer the obtained lysozyme protein suspension to a sealed container and heat it in a 90℃ oil bath at 200 rpm for 24 h to induce the formation of amyloid fibers from lysozyme protein to obtain a lysozyme protein amyloid fiber solution. (2) Using a pipette, load the lysozyme protein amyloid cellulose solution obtained in step (1) into a dialysis bag with a molecular weight cutoff of 50 kDa. Avoid splashing or generating air bubbles. The volume of the lysozyme protein amyloid cellulose solution should not exceed 2 / 3 of the dialysis bag's volume. After loading, secure both ends of the dialysis bag with a dialysis bag clamp to ensure a good seal and prevent leakage. Before sealing, gently squeeze the dialysis bag to expel air. Then, place the sealed dialysis bag into a pressure dialysis apparatus (see...). Figure 2 In this study, a pressure of 200g was applied to the dialysis bag at a temperature of 4℃, and deionized water with pH=2.0 was used as the dialysis solution for dialysis. Dialysis was performed for 28 hours. After dialysis, the dialysis bag was removed and the concentrated lysozyme protein amyloid fibers in the bag were collected.

[0042] Figure 5 The images shown are atomic force microscopy images of the concentrated lysozyme protein amyloid fibers obtained in Example 3, with the left image showing the result before dialysis and the right image showing the result after dialysis. Figure 5 It can be seen that there are a small number of oligomers in the background before dialysis, while the fibers are densely distributed and highly uniform after dialysis.

[0043] The purity of the fiber in Example 3 was found to have increased from 35.97% before dialysis to 50.23% after dialysis, and the fiber yield was found to have increased from 38.41% before dialysis to 54.93% after dialysis.

[0044] Example 4 A method for quantifying the preparation of protein amyloid fibers by pressure dialysis is shown in the process flow diagram. Figure 1 The specific steps are as follows: (1) Disperse whey protein evenly in deionized water and stir for 10 h to obtain a whey protein stock solution with a concentration of 3% (w / v, i.e., 30 mg / mL); titrate the above whey protein stock solution with 6 mol / L hydrochloric acid until the pH value of the solution reaches 2.0, and then centrifuge at 10000×g for 15 min to obtain a uniform whey protein suspension. The suspension does not contain fine insoluble whey protein solid particles; then transfer the obtained whey protein suspension to a sealed container and stir and heat at 85℃ oil bath and 200 rpm for 10 h to induce whey protein to form amyloid fibers, and obtain a whey protein amyloid fiber solution. (2) Using a pipette, load the whey protein amyloid cellulose solution obtained in step (1) into a dialysis bag with a molecular weight cutoff of 50 kDa. Avoid splashing or generating air bubbles. The volume of the whey protein amyloid cellulose solution should not exceed 2 / 3 of the dialysis bag's volume. After loading, secure both ends of the dialysis bag with a dialysis bag clamp to ensure a good seal and prevent leakage. Before sealing, gently squeeze the dialysis bag to expel air. Then, place the sealed dialysis bag into a pressure dialysis apparatus (see...). Figure 2 In this study, a pressure of 200g was applied to the dialysis bag at a temperature of 4℃, and deionized water with pH=2.0 was used as the dialysis solution for dialysis. Dialysis was performed for 28 hours. After dialysis, the dialysis bag was removed and the concentrated whey protein amyloid fibers in the bag were collected.

[0045] Figure 6 The images shown are atomic force microscopy images of the concentrated whey protein amyloid fibers obtained in Example 4, with the left image showing the result before dialysis and the right image showing the result after dialysis. Figure 6 It can be seen that there are a large number of spherical oligomers before dialysis, and the fibers disperse after dialysis, resulting in a cleaner background.

[0046] The purity of the fiber in Example 4 was found to have increased from 30.12% before dialysis to 38.14% after dialysis, and the fiber yield was found to have increased from 27.31% before dialysis to 33.87% after dialysis.

[0047] Figure 7 This is a graph showing the change in protein concentration in the solute over time in Example 1. The control in the graph refers to the state before dialysis. From... Figure 7 It can be seen that the protein concentration in the solute increases significantly with increasing dialysis time.

[0048] Figure 8 This represents the average length of the concentrated protein amyloid fibers obtained in Example 1. From... Figure 8 It can be seen that the fiber length was 1100nm before dialysis and 2100nm after dialysis, an increase of about 1000nm.

[0049] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for quantifying the preparation of protein amyloid fibers by pressure dialysis, characterized in that, Includes the following steps: (1) Preparation of protein amyloid cellulose solution; (2) The protein amyloid fiber solution is placed into a dialysis bag, and pressure is applied to the dialysis bag to perform pressure dialysis to obtain concentrated protein amyloid fibers.

2. The method for quantitatively preparing protein amyloid fibers by pressure dialysis according to claim 1, characterized in that, In step (1), the preparation of the protein amyloid cellulose solution includes the following steps: acid titration, centrifugation and oil bath heating of the protein solution in sequence to obtain the protein amyloid cellulose solution.

3. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 2, characterized in that, The reagent used for acid titration is 6 mol / L hydrochloric acid, and the titration endpoint is when the solution pH reaches 2.

0. The relative centrifugal force for the centrifugation process is 10000×g, and the centrifugation time is 15min; The oil bath heating temperature is 85-90℃, and the oil bath heating time is 10-24h.

4. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 2, characterized in that, The protein in the protein solution is selected from soy protein, oat protein, lysozyme protein, or whey protein.

5. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 1, characterized in that, In step (2), the amount of the protein amyloid cellulose solution loaded does not exceed 2 / 3 of the dialysis bag volume.

6. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 1, characterized in that, In step (2), the molecular weight cutoff of the dialysis bag is 50 kDa.

7. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 1, characterized in that, In step (2), the pressure of the pressure dialysis is 200g.

8. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 1, characterized in that, In step (2), the dialysate for pressure dialysis is water with a pH of 2.

9. The method for quantifying the preparation of protein amyloid fibers by pressure dialysis according to claim 1, characterized in that, In step (2), the pressure dialysis temperature is 4°C and the pressure dialysis time is 28h.

10. A protein amyloid fiber, characterized in that, Prepared by the method according to any one of claims 1-9.

Images