Wheat gluten protein amyloid fiber and preparation method thereof

Abstract

The invention relates to a wheat gluten protein amyloid fiber and a preparation method thereof, and belongs to the technical field of food biology. According to the difference of amyloid fibers formed by main components (prolamin and glutenin) of gluten protein and a nuclear induced fibrosis theory, breakthrough is realized through the following steps: (1) component compounding regulation: optimizing the mass ratio of prolamin to glutenin, and activating an optimal synergistic self-assembly effect; and (2) nucleus induction: introducing a primary nucleus and a secondary nucleus of the gliadin amyloid fiber, and constructing a double-track graded induction system so as to improve the forming speed and efficiency of the gluten protein amyloid fiber. According to the method, the forming efficiency of the gluten protein amyloid fiber is improved, a new perspective is provided for establishing a gluten protein proliferation technology, and meanwhile, a new strategy is provided for preparation and application development of the amyloid fiber.

Description

Technical Field

[0001] This invention relates to a wheat gluten amyloid fiber and its preparation method, specifically to a method that controls the relative ratio of prolysin and glutenin, belonging to the field of food biotechnology. Background Technology

[0002] Amyloid fibers are highly ordered protein nanostructures formed by the self-assembly of partially denatured or hydrolyzed proteins under specific conditions. Their core characteristic is a unique cross-beta-sheet structure. Due to their extremely high aspect ratio, excellent mechanical strength, abundant surface functional groups, and good interfacial activity, amyloid fibers show broad application prospects in food science and materials science, including but not limited to: emulsifiers, hydrogels, cell culture meat scaffolds, and delivery carriers.

[0003] Wheat gluten protein is one of the main byproducts of wheat processing, with abundant and inexpensive sources. Modifying gluten protein into amyloid fibers is an effective strategy to enhance its functional properties and added value. Although gluten protein has the potential to form amyloid fibers, its current preparation efficiency is generally low. Studies report that even under optimized conditions, the conversion rate of gluten protein to amyloid fibers is typically only around 50%, meaning that nearly half of the raw material fails to be effectively converted into the target fiber structure.

[0004] The two main components of gluten protein (gliadin and glutenin) have a synergistic effect in amyloid fibril formation, but the natural ratio is not the golden ratio for optimal synergistic efficiency. Excessive glutenin may hinder the overall process due to its weak nucleation ability and rich α-helix structure; insufficient gliadin may limit the number of fibril nuclei formed. The nucleation step is a key rate-limiting step in gluten amyloid fibril formation. Low efficiency in primary nucleation is the main reason for low overall conversion rate and long time. Secondary nucleation, on the other hand, is key to exponentially increasing the rate and yield of fibril formation.

[0005] No research has yet systematically combined the strategies of "precisely controlling the gliadin / glutenin ratio" and "actively introducing pre-prepared nuclei (especially distinguishing between primary and secondary nuclei)" to form a dual-track regulatory mechanism to synergistically enhance the formation rate and final conversion efficiency of gluten amyloid fibers. This process aims to fill this gap by providing an ideal combination of fiber building blocks through component blending and overcoming the nucleation bottleneck through a nucleation-inducing strategy, achieving efficient and controllable preparation of wheat gluten amyloid fibers, thus laying the foundation for their large-scale application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a wheat gluten amyloid fiber and its preparation method. This method shortens the formation time, improves the reaction rate, and increases the conversion efficiency by controlling the relative ratio of prolysin and glutenin, and introducing primary and secondary nuclei of prolysin amyloid fibers in different proportions.

[0007] The technical solution of the present invention: a method for preparing wheat gluten amyloid fibers, comprising the following steps: (1) enzymatic hydrolysis of prolamins and glutenins:

[0008] a. Prepare prolysin and glutenin solutions by dispersing them separately in ultrapure water; adjust the pH of the protein solution system to 8.0 using NaOH solution, and then place it in a constant temperature magnetically stirred water bath for full hydration;

[0009] b. Add trypsin to the two protein solutions respectively to carry out enzymatic hydrolysis. During the enzymatic hydrolysis process, add NaOH solution dropwise at intervals to maintain a constant pH of the enzymatic hydrolysis system.

[0010] c. After the enzymatic hydrolysis reaction is completed, immediately place the two protein solutions in a high-temperature water bath to inactivate trypsin; after inactivation, rapidly cool the protein hydrolysate to room temperature in an ice-water bath, centrifuge, and collect the supernatant; pass the supernatant through a microporous membrane under vacuum filtration to remove insoluble impurities; collect the filtrate, and freeze-dry it to obtain alcohol-soluble protein hydrolysate and glutenin hydrolysate.

[0011] (2) Preparation of primary and secondary nuclei of alcohol-soluble amyloid fibrils:

[0012] d. Disperse the freeze-dried alcohol-soluble protein hydrolysate sample obtained in step (1) in ultrapure water to prepare an alcohol-soluble protein hydrolysate solution;

[0013] e. Transfer the alcohol-soluble protein hydrolysate solution to a constant temperature magnetically stirred water bath and heat it for 0.5 h and 10 h under continuous stirring to induce the formation of primary and secondary nuclei of alcohol-soluble protein amyloid fibers; f. After heating, rapidly cool the sample in an ice-water bath for 10 min to terminate the fibrosis reaction, collect the sample and freeze-dry it; (3) Component preparation and nucleus-induced formation of wheat gluten amyloid fibers:

[0014] g. The glutenin hydrolysate prepared in step 1c is combined with the prolysin hydrolysate prepared in step 1c, the prolysin amyloid fibrous primary nucleus prepared in step 2f, or the prolysin amyloid fibrous prolysin amyloid fibrous secondary nucleus prepared in step 2f, and mixed with ultrapure water to form a mixed protein solution.

[0015] h. Place the mixed protein solution in a constant temperature magnetically stirred water bath and heat it under continuous stirring to obtain wheat gluten amyloid fibers. After heating, rapidly cool it in an ice water bath to terminate the fiberization reaction, collect the sample and store it at low temperature to obtain wheat gluten amyloid fibers.

[0016] Furthermore, during the hydration process in step 1a, the concentrations of alcohol-soluble protein and glutenin solutions are 10-50 mg / mL, and the pH of the protein solution system is adjusted to 8.0 using 1 mol / L NaOH solution; the hydration conditions for the protein solution are 30-50℃, 200-500 rpm / min, pH 6.0-9.0, and 1-4 h.

[0017] Further, the trypsin activity described in step 1b is ≥2500 units / mg, and the mass ratio to the protein sample is 1:10-1000; the enzymatic hydrolysis conditions are 30-50℃, 200-500 rpm / min, pH 6.0-9.0, and 2-10 h; during the enzymatic hydrolysis process, 1 mol / L NaOH solution is added dropwise every hour to maintain a constant pH in the enzymatic hydrolysis reaction system.

[0018] Further, immediately after the enzymatic hydrolysis reaction in step 1c, the protein solution is incubated in a 95°C water bath for 5 minutes to inactivate trypsin; the centrifugation conditions are 4-25°C, 8000-15000×g, 10-30 minutes; the microporous membrane is specifically 0.45μm. Further, the concentration of the alcohol-soluble protein hydrolysate solution described in step 2d is 10-50 mg / mL.

[0019] Furthermore, the heating conditions in step 2e are 50-100℃, 200-500rpm / min, and 1-24h.

[0020] Furthermore, in step 2f, rapid cooling in an ice-water bath for 10 minutes terminates the fibrosis reaction.

[0021] Further, in step 3g: the specific compounding ratio is as follows: the mass ratio of glutenin hydrolysate to prolysin hydrolysate is 1:0-10; the mass ratio of glutenin hydrolysate to prolysin amyloid fibrous primary nucleus is 1:0-5; or the mass ratio of prolysin amyloid fibrous secondary nucleus to glutenin hydrolysate is 1:0-5.

[0022] The concentration of the mixed protein solution is 10-50 mg / mL; the heating conditions are: 50-100℃, 200-500 rpm / min, reaction for 12-48 h.

[0023] Furthermore, the heating conditions in step 3h are 50-100℃, 200-500rpm / min, 1-24h; after heating, the fibrosis reaction is terminated by rapid cooling in an ice-water bath for 8-12min, and the collected samples are stored at 4℃.

[0024] Finally, the fluorescence increment, fibrinization hysteresis time, and rate of increase of maximum thioflavone T fluorescence intensity of the formed wheat gluten amyloid fibers were measured using thioflavone T fluorescence analysis. Furthermore, the fiber conversion rate and amount of the formed wheat gluten amyloid fibers were measured using ultrafiltration centrifugation.

[0025] The fluorescence increment of wheat gluten amyloid fibers measured was 600-1000 a.u.; the fibrinization hysteresis time was 0.3-4.5 h; the maximum fluorescence intensity increase rate was 50-150 a.u. / h; the nucleation free energy barrier was 93-100 kJ mol-1; the fiber elongation free energy consumption was 90-95 kJ mol-1; the fiber conversion rate was 50-85%; and the fiber yield was 10-17 mg / mL.

[0026] Another technical solution of the present invention is wheat gluten protein starch-like fiber prepared by the above method.

[0027] The beneficial effects of this invention are as follows: By precisely controlling the ratio of prolysin to glutenin (providing ideal building blocks) and introducing pre-formed nuclei (overcoming nucleation bottlenecks and accelerating growth), this invention effectively solves the core problems of low conversion rate, slow production rate, and long time consumption in the traditional preparation of wheat gluten amyloid fibers. It significantly improves fiber conversion rate (>75%), greatly shortens the production cycle (<24h), and makes the production process more controllable and repeatable. This invention lays a solid foundation for the efficient and large-scale preparation of wheat gluten amyloid fibers and their wide application in food, materials, and biomedicine. Attached Figure Description

[0028] Figure 1 This is a process flow diagram of the present invention.

[0029] Figure 2 This is a comparison graph of the fluorescence intensity increment of thioflavone T in Examples 1-11.

[0030] Figure 3 This is a comparison graph of the fibrinization hysteresis time and the rate of increase in maximum thioflavone T fluorescence intensity in Examples 1-11;

[0031] Figure 4 This is a comparison chart of fiber conversion rate and fiber generation amount for Examples 1 to 11. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings:

[0033] The method for detecting wheat gluten amyloid fibers in this invention

[0034] (1) Fluorescence analysis of thioflavin T

[0035] Thioflavin T is a fluorescent dye that specifically binds to the characteristic cross-beta-sheet structure of amyloid fibers and is commonly used to monitor amyloid fiber formation. 80 mg of thioflavin T was dissolved in 100 mL of phosphate buffer (10 mM, pH 7.0) to prepare a 2.5 mM thioflavin T stock solution. After filtration through a 0.22 μm filter, the stock solution was diluted 50 times to obtain the working solution. Based on the specific binding characteristic of amyloid fibers to thioflavin T, 50 μL of the amyloid fiber sample solution was reacted with 4 mL of the thioflavin T working solution in the dark for 3 min. Subsequently, the maximum thioflavin T fluorescence intensity of the sample was measured using a fluorescence spectrophotometer at excitation and emission wavelengths of 440 and 480 nm, respectively.

[0036] The data on the change of thioflavin T fluorescence intensity over time were fitted to Equation (1) to determine the kinetic parameters of wheat gluten amyloid fiber formation: apparent rate constant k app and halfway time t 1 / 2 (That is, the time required to reach half of the final state from the initial state):

[0037]

[0038] Where F(t) represents the fluorescence intensity at time t; F max F0 and F0 represent the final and initial ThT fluorescence intensities of the sample, respectively.

[0039] Lag time t lag Given by formula (2):

[0040] t lag = t 1 / 2 - 2 / k app (2)

[0041] The maximum rate of increase in fluorescence intensity (df / dt)max is calculated according to formula (3):

[0042]

[0043] Furthermore, the data on the change of thioflavone T fluorescence intensity over time at different temperatures were fitted to the Finke-Watzky two-step nucleation-autocatalytic growth model (Equation 4) to determine the nucleation rate constant k1 and fiber elongation rate constant k2 of wheat gluten amyloid fiber formation under different temperature conditions.

[0044]

[0045] Where [A] represents the initial monomeric protein concentration.

[0046] Based on the transition state theory, the nucleation rate constant k1 and fiber elongation rate constant k2 under different temperature conditions are substituted into the Eyring equation (Equation 5) to derive the activation enthalpy (ΔH) and activation entropy (ΔS) for nucleation and fiber elongation of wheat gluten amyloid fibers:

[0047]

[0048] Where k and T represent the rate constant and thermodynamic temperature, respectively; k B is Boltzmann constant; R is gas constant; h is Planck constant. The activation enthalpy and entropy values ​​for nucleation and fiber extension can be determined by linear fitting according to the Eyring equation (5): activation enthalpy is calculated by the slope of the fitted line; activation entropy is calculated by the intercept of the fitted line.

[0049] Finally, the nucleation energy barrier (ΔG) and fiber elongation free energy consumption (ΔG) of wheat gluten amyloid fiber formation are calculated by substituting ΔH and ΔS into the Gibbs-Helmholtz equation (Equation 6).

[0050] ΔG = ΔH - TΔS (6)

[0051] Among them, the nucleation energy barrier represents the energy barrier that gluten protein monomers need to overcome to form the initial fibrous nucleus, that is, the change in the transition state free energy from the aggregation of disordered peptide segments into a cross-beta-sheet nucleus. The fiber elongation free energy consumption represents the change in the free energy required for gluten protein monomers to be added to the existing fiber end, that is, the transition state energy barrier of monomers binding to the fiber end through diffusion and directional alignment.

[0052] (3) Determination of fiber conversion rate

[0053] Gluten amyloid fibrous and non-aggregated peptides were separated by ultrafiltration centrifugation. The gluten amyloid fibrous sample was diluted to 2 mg / mL with ultrapure water. Then, the diluted fibrous solution was ultrafiltered for 15 min at 3000 g using a centrifuge filter (MWCO 100 kDa). After each centrifugation cycle, the retentate was restored to its original volume with ultrapure water. After three rounds of separation, the final retentate solution was the purified gluten amyloid fibrous. The filtrate from each round of centrifugation was collected, and the protein concentrations of the unfibrinated protein stock solution and the ultrafiltration filtrate were determined by the Coomassie brilliant blue method. The conversion rate of enzymatically hydrolyzed peptides to gluten amyloid fibrous was calculated according to formula (7):

[0054]

[0055] Where P is the initial concentration of the unfibrinated protein stock solution (mg / mL), and F is the total protein concentration of the filtrate after ultrafiltration and centrifugation (mg / mL).

[0056] Example 1

[0057] (1) Enzymatic hydrolysis of prolamins and glutenins:

[0058] a. Disperse the alcohol-soluble protein and glutenin samples separately in ultrapure water to prepare protein solutions with a concentration of 20 mg / mL. Adjust the pH of the protein solution system to 8.0 using 1 mol / L NaOH solution, and place it in a constant temperature magnetically stirred water bath for 1 h at a speed of 300 rpm / min and a temperature of 37°C.

[0059] b. Add trypsin at an enzyme-substrate ratio of 1:100 (w / w), maintain a stirring rate of 300 rpm / min, and hydrolyze for 2 hours at pH 8.0 and 37°C. During the hydrolysis process, add 1 mol / L NaOH solution dropwise every hour to maintain a constant pH in the hydrolysis system.

[0060] c. After the enzymatic hydrolysis reaction is complete, immediately incubate the protein solution in a 95°C water bath for 5 minutes to inactivate trypsin. After inactivation, rapidly cool the hydrolysate to room temperature in an ice-water bath, then centrifuge at 15000×g for 20 minutes and collect the supernatant. Filter the supernatant through a 0.45μm microporous membrane under vacuum filtration to remove insoluble impurities. Collect the filtrate and freeze-dry it to obtain the hydrolyzed peptides of prolysin and glutenin.

[0061] (2) Preparation of primary and secondary nuclei of alcohol-soluble amyloid fibrils:

[0062] d. Disperse the freeze-dried alcohol-soluble protein hydrolysate sample in ultrapure water to prepare a protein solution of 20 mg / mL.

[0063] e. Transfer the solution to a constant temperature magnetically stirred water bath and heat it at 300 rpm / min and 90°C for 0.5 and 10 h respectively to induce the formation of primary and secondary nuclei of alcohol-soluble amyloid fibrils.

[0064] f. After heating, rapidly cool in an ice-water bath for 10 minutes to terminate the fibrosis reaction, collect the sample and freeze-dry it.

[0065] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0066] g. Mix wheat gluten hydrolysate peptides and alcohol-soluble protein hydrolysate peptides in a ratio of 1:0.67, and prepare a mixed protein solution with a concentration of 20 mg / mL using ultrapure water.

[0067] h. Place the mixed protein solution in a thermostatically heated magnetically stirred water bath and heat at 300 rpm / min and 90°C for 24 h to obtain wheat gluten amyloid fibers. After heating, rapidly cool in an ice-water bath for 10 min to terminate the fibrosis reaction, collect the sample and store it at 4°C.

[0068] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 1 was 713.79±23.82au, the hysteresis time was 1.19±0.05h, the maximum fluorescence intensity increase rate was 73.26±7.85au / h, the nucleation free energy barrier was 98.91±0.46kJ mol-1, the fiber elongation free energy consumption was 93.47±0.39kJ mol-1, the fiber conversion rate was 57.29±0.7%, and the fiber yield was 11.46±0.14mg / mL.

[0069] Example 2

[0070] (1) Enzymatic hydrolysis of prolamins and glutenins:

[0071] a. Disperse the alcohol-soluble protein and glutenin samples separately in ultrapure water to prepare protein solutions with a concentration of 20 mg / mL. Adjust the pH of the protein solution system to 8.0 using 1 mol / L NaOH solution, and place it in a constant temperature magnetically stirred water bath for 1 h at a speed of 300 rpm / min and a temperature of 37°C.

[0072] b. Add trypsin at an enzyme-substrate ratio of 1:500 (w / w), maintain a stirring rate of 300 rpm / min, and hydrolyze for 2 hours at pH 8.0 and 37°C.

[0073] The enzymatic hydrolysis steps and conditions are then the same as step 1c in Example 1.

[0074] Steps (2) and (3) are the same as in Example 1.

[0075] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 2 was 691.62±35.9au, the hysteresis time was 1.54±0.05h, the maximum fluorescence intensity increase rate was 62.04±9.79au / h, the nucleation free energy barrier was 98.67±0.34kJ mol-1, the fiber elongation free energy consumption was 93.41±0.36kJ mol-1, the fiber conversion rate was 55.12±1.3%, and the fiber yield was 11.02±0.03mg / mL.

[0076] The combined results of Examples 1 and 2 indicate that an excessively high enzyme-to-substrate ratio is detrimental to the formation of wheat gluten amyloid fibers, possibly because some proteins are not effectively hydrolyzed.

[0077] Example 3

[0078] (1) Enzymatic hydrolysis of prolamins and glutenins:

[0079] a. Disperse the alcohol-soluble protein and glutenin samples separately in ultrapure water to prepare protein solutions with a concentration of 20 mg / mL. Adjust the pH of the protein solution system to 8.0 using 1 mol / L NaOH solution, and place it in a constant temperature magnetically stirred water bath for 1 h at a speed of 300 rpm / min and a temperature of 37°C.

[0080] b. Add trypsin at an enzyme-substrate ratio of 1:100 (w / w), maintain a stirring rate of 300 rpm / min, and hydrolyze for 4 hours at pH 8.0 and 37°C.

[0081] The enzymatic hydrolysis steps and conditions are then the same as step 1c in Example 1.

[0082] Steps (2) and (3) are the same as in Example 1.

[0083] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 3 was 751.83±98.46au, the hysteresis time was 1.08±0.05h, the maximum fluorescence intensity increase rate was 91.16±11.94au / h, the nucleation free energy barrier was 98.03±0.02kJ mol-1, the fiber elongation free energy consumption was 93.16±0.23kJ mol-1, the fiber conversion rate was 63.61±1.14%, and the fiber yield was 12.72±0.23mg / mL.

[0084] The combined results of Examples 1 and 3 indicate that increasing the enzymatic hydrolysis time significantly improved the generation, rate, and conversion rate of wheat gluten amyloid fibers. Furthermore, the extended hydrolysis time reduced the nucleation free energy barrier and the fiber elongation free energy consumption, suggesting that the initial folding state of gluten monomers becomes simpler, thereby reducing the free energy required for the formation of cross-beta-sheet nuclei through conformational changes and for the formation of protofibrils through binding to fiber ends.

[0085] Example 4

[0086] Steps (1) and (2) are the same as in Example 3.

[0087] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0088] g. Mix wheat gluten hydrolysate peptides and alcohol-soluble protein hydrolysate peptides at a ratio of 1:0.25, and prepare a mixed protein solution with a concentration of 20 mg / mL using ultrapure water.

[0089] The subsequent fiberization steps and conditions are the same as step 3h in Example 1.

[0090] The results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 4 was 724.79±12.03au, the hysteresis time was 1.29±0.06h, the maximum fluorescence intensity increase rate was 84.44±1.4au / h, the nucleation free energy barrier was 98.4±0.02kJ mol-1, the fiber elongation free energy consumption was 93.33±0.32kJ mol-1, the fiber conversion rate was 61.08±0.72%, and the fiber yield was 12.22±0.14mg / mL.

[0091] Example 5

[0092] Steps (1) and (2) are the same as in Example 4.

[0093] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0094] g. Mix wheat gluten hydrolysate peptides and alcohol-soluble protein hydrolysate peptides in a 1:4 ratio, and prepare a mixed protein solution with a concentration of 20 mg / mL using ultrapure water.

[0095] The subsequent fiberization steps and conditions are the same as step 3h in Example 1.

[0096] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 5 was 786.99±49.96au, the hysteresis time was 0.85±0.04h, the maximum fluorescence intensity increase rate was 108.6±6.9au / h, the nucleation free energy barrier was 97.23±0.39kJ mol-1, the fiber elongation free energy consumption was 92.67±0.02kJ mol-1, the fiber conversion rate was 76.52±0.49%, and the fiber yield was 15.3±0.04mg / mL.

[0097] The results of Examples 3, 4, and 5 indicate that when the proportion of alcohol-soluble protein hydrolysates in the protein sample is high, the amount of wheat gluten amyloid fibers generated is greater, faster, and with a higher conversion rate. Similarly, as the proportion of alcohol-soluble protein hydrolysates increases, the nucleation free energy barrier and fiber elongation free energy consumption for the formation of wheat gluten amyloid fibers decrease. This may be because (1) alcohol-soluble protein hydrolysates provide more pre-folded β-chain templates, reducing the recombination energy required to form cross-β-sheet nuclei; and (2) a high proportion of alcohol-soluble protein hydrolysates optimizes the hydrophobic interaction with glutenin hydrolysates, increasing the diffusion rate of monomers to the fiber ends.

[0098] Example 6

[0099] Steps (1) and (2) are the same as in Example 5.

[0100] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0101] g. At a ratio of 1:0.67, glutenin hydrolysate peptides and prolysin amyloid fibrous primary nuclei were combined and mixed with ultrapure water to prepare a mixed protein solution with a concentration of 20 mg / mL.

[0102] The subsequent fiberization steps and conditions are the same as step 3h in Example 1.

[0103] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 6 was 831.26±39.53au, the hysteresis time was 0.88±0.05h, the maximum fluorescence intensity increase rate was 113.05±5.38au / h, the nucleation free energy barrier was 96.58±0.21kJ mol-1, the fiber elongation free energy consumption was 91.78±0.46kJ mol-1, the fiber conversion rate was 70.33±2.28%, and the fiber yield was 14.07±0.56mg / mL.

[0104] Example 7

[0105] Steps (1) and (2) are the same as in Example 5.

[0106] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0107] g. At a ratio of 1:0.67, glutenin hydrolysate peptides and prolysin amyloid fibrous secondary nuclei were combined and mixed with ultrapure water to prepare a mixed protein solution with a concentration of 20 mg / mL.

[0108] h. The subsequent fiberization steps and conditions are the same as step (3) in Example 1.

[0109] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 7 was 888.7±49.53au, the hysteresis time was 0.32±0.03h, the maximum fluorescence intensity increase rate was 148.54±6.59au / h, the nucleation free energy barrier was 95.45±0.28kJ mol-1, the fiber elongation free energy consumption was 90.71±0.5kJ mol-1, the fiber conversion rate was 79.19±1.67%, and the fiber yield was 15.84±0.33mg / mL.

[0110] The results of Examples 5, 6, and 7 combined demonstrate that introducing gliadin amyloid fibrillary nuclei can increase the generation, rate, and conversion rate of wheat gluten amyloid fibers. Furthermore, introducing gliadin amyloid fibrillary nuclei can reduce the nucleation free energy barrier and the free energy consumption required for fiber elongation. Specifically, compared to primary nuclei, secondary nuclei have a shorter induction lag period and lower free energy consumption for nucleation and fiber elongation.

[0111] Example 8

[0112] Steps (1) and (2) are the same as in Example 5.

[0113] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0114] g. At a ratio of 1:0.25, glutenin hydrolysate peptides and prolysin amyloid fibrous primary nuclei were combined and mixed with ultrapure water to prepare a mixed protein solution with a concentration of 20 mg / mL.

[0115] h. The subsequent fiberization steps and conditions are the same as step (3) in Example 1.

[0116] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 8 was 749.26±15.09au, the hysteresis time was 1.08±0.02h, the maximum fluorescence intensity increase rate was 95.91±1.93au / h, the nucleation free energy barrier was 96.95±0.03kJ mol-1, the fiber elongation free energy consumption was 91.95±0.38kJ mol-1, the fiber conversion rate was 66.57±2.07%, and the fiber yield was 13.31±0.41mg / mL.

[0117] Example 9

[0118] Steps (1) and (2) are the same as in Example 5.

[0119] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0120] g. Mix wheat gluten hydrolysate with prolyl amyloid fibrous primary nucleus at a ratio of 1:4, and prepare a mixed protein solution with a concentration of 20 mg / mL using ultrapure water.

[0121] The subsequent fiberization steps and conditions are the same as step 3h in Example 1.

[0122] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 9 was 894.88±30.14 au, the hysteresis time was 0.69±0.07 h, the maximum fluorescence intensity increase rate was 140.5±4.73 au / h, the nucleation free energy barrier was 95.79±0.11 kJ mol-1, the fiber elongation free energy consumption was 91.3±0.02 kJ mol-1, the fiber conversion rate was 78.88±0.75%, and the fiber yield was 15.78±0.15 mg / mL.

[0123] The results from Examples 6, 8, and 9 indicate that a higher proportion of primary nuclei of prolyl amyloid fibrils in the protein samples leads to higher generation, rate, and conversion rates of wheat gluten amyloid fibrils; and lower required energy consumption for nucleation and fibril elongation. The primary nucleus, acting as a pre-assembled β-sheet template, results in a higher density of ordered structures within the system. Therefore, monomeric proteins do not need to search the conformational space de novo and can directly attach to the nucleus surface, reducing conformational disorder in the nucleation transition state.

[0124] Example 10

[0125] Steps (1) and (2) are the same as in Example 5.

[0126] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0127] g. At a ratio of 1:0.25, glutenin hydrolysate peptides and prolysin amyloid fibrous secondary nuclei were combined and mixed with ultrapure water to prepare a mixed protein solution with a concentration of 20 mg / mL.

[0128] The subsequent fiberization steps and conditions are the same as step 3h in Example 1.

[0129] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 10 was 872.37±19.39au, the hysteresis time was 0.4±0.06h, the maximum fluorescence intensity increase rate was 135.22±3.04au / h, the nucleation free energy barrier was 96.09±0.46kJ mol-1, the fiber elongation free energy consumption was 91.14±0.28kJ mol-1, the fiber conversion rate was 71.92±2.73%, and the fiber yield was 14.38±0.55mg / mL.

[0130] Example 11 (4:1)

[0131] Steps (1) and (2) are the same as in Example 5.

[0132] (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers:

[0133] g. Mix glutenin hydrolysate with prolyl amyloid fibrous secondary nuclei at a ratio of 1:4, and prepare a mixed protein solution with a concentration of 20 mg / mL using ultrapure water.

[0134] The subsequent fiberization steps and conditions are the same as step 3h in Example 1.

[0135] The fiber nucleation energy barrier and fiber extension free energy consumption of Examples 1-11 are shown in Table 1.

[0136] Table 1

[0137]

[0138]

[0139] The measurement results showed that the thioflavone T fluorescence intensity increment of the wheat gluten amyloid fibers obtained in Example 11 was 332.06±10.22au, the hysteresis time was 3.75±0.04h, the maximum fluorescence intensity increase rate was 88.37±1.35au / h, the nucleation free energy barrier was 95.86±0.07kJ mol-1, the fiber elongation free energy consumption was 91.41±0.15kJ mol-1, the fiber conversion rate was 76.52±0.49%, and the fiber yield was 15.3±0.1mg / mL.

[0140] The results from Examples 7, 10, and 11 indicate that a higher proportion of secondary nuclei in the protein samples, particularly those of gliadin amyloid fibers, leads to increased fiber formation, faster formation rate, and improved conversion rate. However, an excessively high proportion of secondary nuclei results in decreased fiber formation, slower formation rate, and reduced conversion rate, while also increasing the free energy required for nucleation and fiber elongation. This may be because the secondary nuclei may be damaged by high temperatures during heating, thus weakening their inductive ability.

[0141] As demonstrated by the above examples, the enzyme-to-substrate ratio and enzymatic hydrolysis time are the core factors regulating the formation of wheat gluten amyloid fibers. An excessively high enzyme-to-substrate ratio hinders the effective formation of amyloid fibers, while appropriately increasing the hydrolysis time significantly improves the amount, rate, and conversion rate of amyloid fiber formation, and reduces the free energy required for nucleation and fiber elongation. Furthermore, increasing the proportion of prolysin hydrolysates in the protein sample promotes greater amyloid fiber formation, faster formation rate, higher conversion rate, and lower nucleation free energy barriers and fiber elongation free energy consumption. Introducing prolysin amyloid fiber nuclei (including primary and secondary nuclei) can effectively improve the amount, rate, and conversion rate of amyloid fibers, and reduce the nucleation free energy barrier and fiber elongation free energy consumption. Among these, secondary nuclei significantly shorten the lag time compared to primary nuclei, thus reducing the nucleation free energy barrier and fiber elongation free energy consumption. The higher the proportion of primary nuclei in a protein sample, the better the induction effect. Increasing the proportion of secondary nuclei within a reasonable range can also increase yield, speed up the process, and improve conversion rate. However, if the proportion is too high, it will lead to a decrease in fiber production, a slower process, a decrease in energy efficiency, and a reduction in fiber conversion rate.

[0142] The significance of this invention lies in its systematic revelation of these key process variables and their complex synergistic and balancing relationships, and based on this, it proposes an optimized and controllable production scheme. This scheme solves the efficiency problems (such as long preparation cycle, low yield, and low conversion rate) of traditional preparation methods, providing a scientific basis and feasible technical path for the efficient and stable production of wheat gluten protein amyloid fibers.

Claims

1. A method for preparing wheat gluten protein amyloid fibers, characterized in that: The steps are as follows: (1) Enzymatic hydrolysis of prolamins and glutenins: a. Alcohol-soluble protein and glutenin were separately dispersed in ultrapure water to prepare alcohol-soluble protein solution and glutenin solution, respectively; The pH of the protein solution system was adjusted to 8.0 using NaOH solution, and then it was placed in a constant temperature magnetically stirred water bath for full hydration. b. Add trypsin to the two protein solutions respectively to carry out enzymatic hydrolysis. During the enzymatic hydrolysis process, add NaOH solution dropwise at intervals to maintain a constant pH of the enzymatic hydrolysis system. c. After the enzymatic hydrolysis reaction is completed, immediately place the two protein solutions in a high-temperature water bath to inactivate trypsin; after inactivation, rapidly cool the protein hydrolysate to room temperature in an ice-water bath, centrifuge, and collect the supernatant; the supernatant is then filtered through a microporous membrane under vacuum to remove insoluble impurities. Collect the filtrate and freeze-dry it to obtain alcohol-soluble protein hydrolysates and glutenin hydrolysates. (2) Preparation of primary and secondary nuclei of alcohol-soluble amyloid fibrils: d. Disperse the freeze-dried alcohol-soluble protein hydrolysate sample obtained in step (1) in ultrapure water to prepare an alcohol-soluble protein hydrolysate solution; e. Transfer the alcohol-soluble protein hydrolysate solution to a constant temperature magnetically stirred water bath and heat it for 0.5 h and 10 h under continuous stirring to induce the formation of primary and secondary nuclei of alcohol-soluble protein amyloid fibrils. f. After heating, rapidly cool in an ice-water bath for 10 minutes to terminate the fibrosis reaction, collect the sample and freeze-dry it. (3) Component formulation and nuclear-induced formation of wheat gluten amyloid fibers: g. The primary or secondary nuclei of alcohol-soluble amyloid fibrils prepared in step 2f are combined with the glutenin hydrolysate prepared in step 1c, and a mixed protein solution is prepared with ultrapure water. h. Place the mixed protein solution in a constant temperature magnetically stirred water bath and heat it under continuous stirring to obtain wheat gluten protein amyloid fibers. After heating, rapid cooling in an ice-water bath terminates the fibrosis reaction. The sample is collected and stored at low temperature to obtain wheat gluten amyloid fiber.

2. The method for preparing wheat gluten amyloid fibers as described in claim 1, characterized in that: During the hydration process in step 1a, the concentration of alcohol-soluble protein and glutenin solution is 10-50 mg / mL, and the pH of the protein solution system is adjusted to 8.0 using 1 mol / L NaOH solution; the hydration conditions for the protein solution are 30-50℃, 200-500 rpm / min, pH 6.0-9.0, and 1-4 h.

3. The method for preparing wheat gluten amyloid fibers as described in claim 1, characterized in that: The trypsin activity described in step 1b is ≥2500 units / mg, and the mass ratio of trypsin to protein sample is 1:10-1000; the enzymatic hydrolysis conditions are 30-50℃, 200-500 rpm / min, pH 6.0-9.0, and 2-10 h; during the enzymatic hydrolysis process, 1 mol / L NaOH solution is added dropwise every hour to maintain a constant pH in the enzymatic hydrolysis reaction system.

4. The method for preparing wheat gluten amyloid fibers as described in claim 1, characterized in that: step After the 1c enzymatic digestion reaction is completed, the protein solution is immediately incubated in a 95℃ water bath for 5 min to inactivate trypsin; the centrifugation conditions are 4-25℃, 8000-15000×g, 10-30 min; the microporous filter membrane is specifically 0.45μm.

5. The method for preparing wheat gluten amyloid fibers as described in claim 1, characterized in that: The concentration of the alcohol-soluble protein hydrolysate solution in step 2d is 10-50 mg / mL.

6. The method for preparing wheat gluten protein amyloid fibers as described in claim 1, characterized in that: The heating conditions in step 2e are 50-100℃, 200-500rpm / min, and 1-24h.

7. The method for preparing wheat gluten amyloid fibers as described in claim 1, characterized in that: In step 2f, rapid cooling in an ice-water bath for 10 minutes terminates the fibrosis reaction.

8. The method for preparing wheat gluten amyloid fiber as described in claim 1, characterized in that step 3g comprises: a specific compounding ratio of glutenin hydrolysate to prolysin hydrolysate in a mass ratio of 1:0-10; glutenin hydrolysate to prolysin amyloid fiber primary core in a mass ratio of 1:0-5; or prolysin amyloid fiber secondary core to glutenin hydrolysate in a mass ratio of 1:0-5; and the concentration of the mixed protein solution is 10-50 mg / mL; The heating conditions are: 50-100℃, 200-500 rpm / min, reaction time 1-24h.

9. The method for preparing wheat gluten amyloid fibers as described in claim 1, characterized in that: The heating conditions in step 3h are 50-100℃, 200-500rpm / min, 1-24h; after heating, the fiberization reaction is terminated by rapid cooling in an ice-water bath for 8-12min, and the collected samples are stored at 4℃.

10. Wheat gluten amyloid fibers prepared by the method according to any one of claims 1-10.

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