Plant amyloid fiber, preparation method thereof, and composite film and composite coating film preservative

Abstract

The application provides a plant amyloid protein fiber and a preparation method and a composite film and a composite coating film preservative thereof, and belongs to the technical field of protein fibers. Plant protein isolate is mixed with water, and after the pH value is adjusted to 1.5-2.5, solid-liquid separation is carried out, the obtained plant protein-containing liquid material is subjected to fiber growth treatment, and a plant amyloid protein fiber is obtained. According to the growth kinetics curve, the fiber growth treatment comprises sequentially performing first incubation treatment, ultrasonic treatment and second incubation treatment, the time of the first incubation treatment is 0 h-end of the logarithmic growth phase, and the first incubation treatment and the second incubation treatment are performed under non-ultrasonic conditions. When the time of the first incubation treatment is 0 h, the ultrasonic power density of the ultrasonic treatment is greater than or equal to 2.5 W / mL. The method is convenient for simultaneously regulating the growth process and morphology of the fiber, and can reduce the proportion of rigid fibers.

Description

Technical Field

[0001] This invention relates to the field of protein fiber technology, and in particular to a plant amyloid protein fiber, its preparation method, composite film, and composite coating preservative. Background Technology

[0002] In recent years, with limited animal protein supply, plant protein has gained widespread attention as an alternative due to its wide availability. Pea protein isolate, a byproduct of pea starch extraction, is currently the second largest source of plant protein after soybeans, boasting high nutritional value and low allergenicity. Pea protein isolate is primarily composed of globulins; however, the self-assembly of these globular structures into high aspect ratio fibrous structures, forming pea amyloid protein fibers, can expand its application scenarios.

[0003] Currently, the common technique involves directly heating plant proteins to transform them into fibrous structures from spherical structures. However, the protein fibers prepared using this method have a relatively high proportion of rigid fibers. Summary of the Invention

[0004] The purpose of this invention is to provide a plant amyloid protein fiber, its preparation method, a composite film, and a composite coating preservative. The method of this invention for preparing plant amyloid protein fiber allows for simultaneous control of the growth process and morphology of the plant amyloid protein fiber, reducing the proportion of rigid fibers. The composite film prepared using the plant amyloid protein fiber has good mechanical properties, and the composite coating preservative prepared using the plant amyloid protein fiber has good preservation performance.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing plant amyloid protein fibers, comprising the following steps:

[0007] Plant protein isolate is mixed with water to obtain an aqueous solution of plant protein isolate;

[0008] The pH of the plant protein isolate aqueous solution was adjusted to 1.5-2.5, followed by solid-liquid separation to obtain a liquid material containing plant protein.

[0009] The plant protein-containing liquid material is subjected to fiber growth treatment to obtain the plant amyloid protein fiber;

[0010] According to the growth kinetics curve of the plant amyloid fibers, the fiber growth treatment includes a lag phase, a logarithmic growth phase, and a plateau phase in sequence.

[0011] The fiber growth treatment includes sequentially performing a first incubation treatment, an ultrasonic treatment, and a second incubation treatment. The first incubation treatment lasts from 0 hours to the end of the logarithmic growth phase. The first and second incubation treatments are performed under non-ultrasonic conditions. When the first incubation treatment lasts for 0 hours, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL.

[0012] Preferably, the plant protein isolate includes pea protein isolate; the ratio of the plant protein isolate to water is 1-3 g: 100 mL.

[0013] Preferably, the conditions for the ultrasonic treatment include: a temperature of 0–4°C, a total time of 1–3 min, with a pause of 1–3 s after every 1–3 s of ultrasonic treatment; when the first incubation treatment time is 0 h, the ultrasonic power density of the ultrasonic treatment is 2.5–4 W / mL; when the first incubation treatment time is not 0 h, the ultrasonic power density of the ultrasonic treatment is 2–4 W / mL.

[0014] Preferably, the temperature of the first incubation treatment and the second incubation treatment are independently 75-85°C, and the total time of the first incubation treatment and the second incubation treatment is 45-50 hours; the first incubation treatment and the second incubation treatment are carried out under stirring conditions.

[0015] Preferably, the incubation period is from 0 hours to the end of the lag period.

[0016] Preferably, the lag period ranges from 0 to 5 hours, and the logarithmic growth period ranges from 5 to 26.5 hours, with the start of fiber growth treatment as the time point.

[0017] Preferably, the fiber growth treatment includes any one of the following three cases:

[0018] Case 1: The incubation time for the first incubation treatment is 0 hours, and the ultrasonic power density for the ultrasonic treatment is 2.6–4 W / mL;

[0019] Scenario 2: The incubation time for the first incubation treatment is 4-5 hours, and the ultrasonic power density for the ultrasonic treatment is 3-4 W / mL;

[0020] Case 3: The incubation time for the first incubation treatment is 25 to 26.5 hours, and the ultrasonic power density for the ultrasonic treatment is 2 to 2.2 W / mL.

[0021] The present invention provides plant amyloid protein fibers prepared by the preparation method described above, comprising flexible fibers, semi-flexible fibers and rigid fibers, wherein the percentage of flexible fibers in the plant amyloid protein fibers is 4-50%, the percentage of semi-flexible fibers is 10-45%, and the percentage of rigid fibers is 35-65.8%.

[0022] The present invention provides a composite film comprising a chitosan matrix and plant amyloid protein fibers distributed in the chitosan matrix, wherein the plant amyloid protein fibers are the plant amyloid protein fibers described in the above technical solution.

[0023] This invention provides a composite coating preservative, comprising chitosan, plant amyloid protein fiber, epigallocatechin gallate, and a solvent, wherein the plant amyloid protein fiber is the plant amyloid protein fiber described in the above technical solution.

[0024] This invention provides a method for preparing plant amyloid protein fibers, comprising the following steps: mixing plant protein isolate with water to obtain a plant protein isolate aqueous solution; adjusting the pH of the plant protein isolate aqueous solution to 1.5–2.5, followed by solid-liquid separation to obtain a liquid material containing plant protein; subjecting the liquid material containing plant protein to fiber growth treatment to obtain the plant amyloid protein fibers; according to the growth kinetics curve of the plant amyloid protein fibers, the fiber growth treatment sequentially includes a lag phase, a logarithmic growth phase, and a plateau phase; the fiber growth treatment includes sequentially performing a first incubation treatment, ultrasonic treatment, and a second incubation treatment, wherein the first incubation treatment lasts from 0 h to the end of the logarithmic growth phase, and the first and second incubation treatments are performed under non-ultrasonic conditions; when the first incubation treatment lasts for 0 h, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL. The method provided by this invention facilitates the simultaneous control of the growth process and morphology of plant amyloid protein fibers. Specifically, in this invention, the ultrasonic treatment accelerates the growth of plant amyloid fibers, which is significant for the energy-intensive production process of preparing plant amyloid fibers, improving production efficiency and reducing energy consumption. Furthermore, the ultrasonic treatment can regulate the morphology of plant amyloid fibers and reduce the proportion of rigid fibers. Test results show that ultrasonic treatment increases the proportion of flexible and semi-flexible fibers in pea amyloid fibers. The composite film prepared using the plant amyloid fibers of this invention has good mechanical properties; the composite coating preservative prepared using the plant amyloid fibers of this invention has good preservation performance. Test results show that the composite coating preservative has a better inhibitory effect on browning of fresh-cut apples, with a better browning inhibition effect when the content of flexible and semi-flexible fibers is higher. Attached Figure Description

[0025] Figure 1 A comparative diagram showing the preparation process of pea amyloid protein fibers;

[0026] Figure 2 Figure 1 shows the results of the kinetic test of fibrosis kinetics of pea protein isolate treated with ultrasound at t0.

[0027] Figure 3 The figure shows the kinetics of pea protein isolate fiberization after ultrasonic treatment at t0.1.

[0028] Figure 4 Figure 1 shows the kinetics of fibrosis in pea protein isolate after ultrasonic treatment at t0.9.

[0029] Figure 5 TEM image of a pea amyloid protein fiber sample;

[0030] Figure 6 The image shows the outline length distribution of pea amyloid protein fibers obtained by ultrasonic treatment.

[0031] Figure 7 A statistical chart showing the proportions of flexible, semi-flexible, and rigid fibers in pea amyloid protein fibers obtained by ultrasonic treatment.

[0032] Figure 8 The figure shows the test results of tensile strength and elongation at break of the thin film.

[0033] Figure 9 The graph shows the correlation analysis results between the mechanical properties of the film and the stiffness and flexibility of the fiber.

[0034] Figure 10 Figure 1 shows the DPPH free radical scavenging rate test results of pea protein isolate and pea amyloid fibrils.

[0035] Figure 11 This is a diagram showing the storage results of fresh-cut apples. Detailed Implementation

[0036] This invention provides a method for preparing plant amyloid protein fibers, comprising the following steps:

[0037] Plant protein isolate is mixed with water to obtain an aqueous solution of plant protein isolate;

[0038] The pH of the plant protein isolate aqueous solution was adjusted to 1.5-2.5, followed by solid-liquid separation to obtain a liquid material containing plant protein.

[0039] The plant protein-containing liquid material is subjected to fiber growth treatment to obtain the plant amyloid protein fiber;

[0040] According to the growth kinetics curve of the plant amyloid fibers, the fiber growth treatment includes a lag phase, a logarithmic growth phase, and a plateau phase in sequence.

[0041] The fiber growth treatment includes sequentially performing a first incubation treatment, an ultrasonic treatment, and a second incubation treatment. The first incubation treatment lasts from 0 hours to the end of the logarithmic growth phase. The first and second incubation treatments are performed under non-ultrasonic conditions. When the first incubation treatment lasts for 0 hours, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL.

[0042] This invention utilizes ultrasonic processing to simultaneously regulate the growth process and morphology of plant amyloid fibers. Specifically, ultrasound is a highly efficient, low-energy, and environmentally friendly physical processing technology that can alter the structure of proteins at various stages and improve their functional properties without introducing exogenous substances, through external energy input. This invention uses ultrasound to regulate the growth of plant amyloid fibers at different stages (e.g., initial t0, end of lag phase t0.1, end of growth phase t0.9), allowing for flexible timing. Furthermore, it quantifies the rigidity and flexibility indices of plant amyloid fiber morphology, establishing a correlation between morphology and function. Based on this, it further explores the performance differences of plant amyloid fibers with different morphologies in protein-chitosan composite film systems and composite coating preservatives. Samples of plant amyloid fibers with higher contents of flexible and semi-flexible fibers exhibit better mechanical properties, antioxidant properties, and preservation properties. The preparation method of the plant amyloid fibers described in this invention is described in detail below.

[0043] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0044] This invention involves mixing plant protein isolate with water to obtain an aqueous solution of plant protein isolate. As one embodiment of this invention, the plant protein isolate may include pea protein isolate. The preparation method of the pea protein isolate of this invention may include the following steps: mixing pea protein powder with water and performing a first stirring treatment; adjusting the pH value to 7.5–8.5 and then performing a second stirring treatment; performing a first solid-liquid separation; adjusting the pH value of the resulting liquid material to 4–5 and then performing a third treatment; performing a second solid-liquid separation; mixing the resulting solid material with water; adjusting the pH value to 6.5–7.5 under stirring conditions; after the solid material is completely dissolved, performing dialysis; and freeze-drying the resulting purified material to obtain the pea protein isolate.

[0045] In one embodiment of the present invention, the ratio of pea protein powder to water in the mixture can be 0.5–1.5 g: 10 mL, specifically 1 g: 10 mL; the first stirring time can be 1.5–2.5 h, specifically 2 h; the reagent used to adjust the pH value to 7.5–8.5 (specifically 8.0) can be NaOH solution, and the concentration of the NaOH solution can be 3 M; the second stirring time can be 1.5–2.5 h, specifically 2 h. In another embodiment of the present invention, the first and second solid-liquid separations can be performed by centrifugation; the centrifugation temperature can be 3–6 °C, specifically 4 °C; the rotation speed can be 6000–10000 rpm, specifically 8000 rpm; and the time can be 15–25 min, specifically 20 min. In one embodiment of the present invention, the pH value of the liquid material is adjusted to 4-5 (specifically 4.5), and the reagent can be hydrochloric acid, the concentration of which can be 3M; the first stirring treatment time can be 0.5-1.5h, specifically 1h; when the solid material obtained after the second solid-liquid separation is mixed with water, the material-to-liquid ratio can be 0.5-1.5g:5-15mL, specifically 1g:10mL; the reagent used to adjust the pH value to 6.5-7.5 (specifically 7.0) can be NaOH solution, the concentration of which can be 3M. In another embodiment of the present invention, the dialysis conditions include: the molecular weight cutoff of the dialysis bag can be 8-14kDa; the dialysate can be water, specifically deionized water; the temperature is 3-6℃, specifically 4℃; the time is 42-54h, specifically 48h; preferably, the dialysate is replaced every 6h during the dialysis process. The present invention does not specifically limit the freeze-drying conditions; conditions well known to those skilled in the art can be used.

[0046] After obtaining plant protein isolate (such as pea protein isolate), the present invention mixes the plant protein isolate with water to obtain an aqueous solution of plant protein isolate. As one embodiment of the present invention, the ratio of plant protein isolate to water can be 1-3 g:100 mL, specifically 2 g:100 mL. The present invention does not have specific limitations on the method of mixing the plant protein isolate with water, as long as it ensures that the plant protein isolate is fully dissolved in the water.

[0047] After obtaining the aqueous solution of plant protein isolate, the present invention adjusts the pH value of the aqueous solution to 1.5-2.5, followed by solid-liquid separation to obtain a liquid material containing plant protein. In one embodiment of the present invention, the reagent used to adjust the pH value of the aqueous solution of plant protein isolate to 1.5-2.5 (specifically 2.0) can be hydrochloric acid, and the concentration of the hydrochloric acid can be 3M. Adjusting the pH value of the aqueous solution of plant protein isolate to the above range allows for acid hydrolysis of the plant protein isolate during subsequent incubation, which is beneficial for the subsequent preparation of plant amyloid protein fibers. In one embodiment of the present invention, the solid-liquid separation method can be centrifugation; the centrifugation temperature can be 3-6℃, specifically 4℃; the rotation speed can be 6000-10000 rpm, specifically 8000 rpm; and the time can be 15-25 min, specifically 20 min.

[0048] After obtaining the liquid material containing plant protein, the present invention performs fiber growth treatment on the liquid material containing plant protein to obtain the plant amyloid protein fiber. As one embodiment of the present invention, the plant amyloid protein fiber may include pea amyloid protein fiber; specifically, in this embodiment, pea amyloid protein isolate is used as an example to ultimately prepare pea amyloid protein fiber. In this invention, according to the growth kinetic curve of the plant amyloid protein fiber, the fiber growth treatment sequentially includes a lag phase, a logarithmic growth phase, and a plateau phase. The lag phase specifically refers to the initial stage of amyloid protein fiber growth, at which time the native conformation of the protein hydrolyzes into protein monomers or short peptides under high temperature and high acidity. These protein monomers can polymerize through non-covalent interactions to form oligomers rich in cross-β-sheet structures, which is the nucleation process during fiber growth. During the process of protein monomers self-assembling into oligomers, the thioflavone T fluorescent dye, which can specifically bind to the cross-β-sheet structure of amyloid fibers, is used for characterization. It is generally considered that 0-10% of the maximum fluorescence intensity corresponds to the lag phase of amyloid protein fiber growth. The logarithmic growth phase described in this invention specifically refers to the rapid growth stage of amyloid fibrils. During this phase, oligomers grow to a certain size, and using them as templates, protein monomers self-assemble at the oligomer ends through π-π interactions and hydrophobic interactions, forming protofibrils with a high aspect ratio. It is generally considered that 10-90% of the maximum fluorescence intensity corresponds to the logarithmic growth phase. The plateau phase described in this invention specifically refers to the maturation stage of amyloid fibril growth. At this point, the concentration of protein monomers in the system can no longer support the rapid self-assembly of amyloid fibrils, and the growth rate slows down. During this stage, protofibrils begin to influence each other, self-assembling into mature amyloid fibrils through entanglement, orderly arrangement, and other methods. It is generally considered that above 90% of the maximum fluorescence intensity corresponds to the plateau phase. The method for obtaining the growth kinetic curve of pea amyloid fibrils will be described in detail later in this invention and will not be repeated here. In this embodiment of the invention, taking pea amyloid protein fiber as an example, according to its growth kinetic curve, the time range of the lag phase is 0-5h, the time range of the logarithmic growth phase is 5-26.5h, and the plateau phase is after 26.5h.

[0049] In this invention, the fiber growth treatment includes sequentially performing a first incubation treatment, an ultrasonic treatment, and a second incubation treatment. The first incubation treatment lasts from 0 hours to the end of the logarithmic growth phase, and the first and second incubation treatments are performed under non-ultrasonic conditions.

[0050] The incubation period of the first incubation treatment in this invention is 0 hours until the end of the logarithmic growth phase. In one embodiment of this invention, the incubation period can further be 0 hours until the end of the lag phase, specifically 0 hours (i.e., the first incubation treatment is not performed at this time, and the subsequent ultrasonic treatment is performed directly). In another embodiment of this invention, the temperature of the first incubation treatment can be 75-85°C, specifically 80°C; the first incubation treatment can be carried out under stirring conditions, and the stirring speed can be 350-450 rpm, specifically 400 rpm.

[0051] In one embodiment of the present invention, the conditions for ultrasonic treatment include: a temperature of 0–4°C, more specifically 0–2°C; a total time of 1–3 min, specifically 2 min; a pause of 1–3 s after every 1–3 s of ultrasonic treatment, specifically a pause of 2 s after every 2 s of ultrasonic treatment; when the first incubation treatment time is 0 h, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL, more specifically 2.5–4 W / mL, specifically 2.5 W / mL, 2.6 W / mL, 2.7 W / mL, 3 W / mL, 3.5 W / mL, or 4 W / mL; when the first incubation treatment time is not 0 h, the ultrasonic power density of the ultrasonic treatment is 2–4 W / mL, specifically 2 W / mL, 2.2 W / mL, 2.4 W / mL, 2.5 W / mL, 2.6 W / mL, 2.7 W / mL, 3 W / mL, 3.5 W / mL, or 4 W / mL. In this embodiment of the present invention, the diameter of the amplitude transformer used for ultrasonic treatment is specifically 10 mm. In this embodiment of the invention, the effects of different ultrasonic intensities on the growth of plant amyloid fibers were studied. The ultrasonic intensities can range from 1% to 30%, specifically 1%, 5%, 10%, 20%, and 30%, corresponding to actual ultrasonic power densities of 2.1766 W / mL, 2.3909 W / mL, 2.6588 W / mL, 3.1946 W / mL, and 3.7304 W / mL, respectively. In this embodiment of the invention, the rated power of the ultrasonic equipment is 900 W, with the ultrasonic intensity set at 100%.

[0052] In one embodiment of the present invention, the total time for the first incubation treatment and the second incubation treatment can be 45 to 50 hours, specifically 48 hours; the temperature of the second incubation treatment can be 75 to 85°C, specifically 80°C; the second incubation treatment can be carried out under stirring conditions, and the stirring speed can be 350 to 450 rpm, specifically 400 rpm.

[0053] As one embodiment of the present invention, the fiber growth treatment includes any one of the following three cases:

[0054] Scenario 1: The first incubation treatment lasts for 0 hours, and the ultrasonic power density of the ultrasonic treatment is 2.6–4 W / mL. In this embodiment of the invention, the liquid material containing plant protein is subjected to ultrasonic treatment and a second incubation treatment sequentially. During the ultrasonic treatment, the ultrasonic intensity can be 10%, 20%, or 30% (corresponding to actual ultrasonic power densities of 2.6588 W / mL, 3.1946 W / mL, and 3.7304 W / mL, respectively). The temperature of the ultrasonic treatment is provided by ice water, and the total duration of the ultrasonic treatment is 2 minutes, with a 2-second pause after every 2 seconds of treatment. The temperature of the second incubation treatment is 80°C, and the time is 48 hours. The second incubation treatment is carried out under stirring conditions, and the stirring speed is 400 rpm.

[0055] Scenario 2: The first incubation treatment lasts for 4-5 hours, and the ultrasonic power density of the ultrasonic treatment is 3-4 W / mL. In this embodiment of the invention, the liquid material containing plant protein is subjected to a first incubation treatment, an ultrasonic treatment, and a second incubation treatment sequentially; the temperature of the first incubation treatment is 80°C, and the time is 5 hours; the first incubation treatment is carried out under stirring conditions, and the stirring speed is 400 rpm; the ultrasonic intensity during the ultrasonic treatment can be 20% or 30% (corresponding to actual ultrasonic power densities of 3.1946 W / mL and 3.7304 W / mL, respectively), the temperature of the ultrasonic treatment is provided by ice water, the total duration of the ultrasonic treatment is 2 minutes, and there is a 2-second pause every 2 seconds of treatment; the temperature of the second incubation treatment is 80°C, and the time is 43 hours; the second incubation treatment is carried out under stirring conditions, and the stirring speed is 400 rpm.

[0056] Scenario 3: The first incubation treatment lasts for 25–26.5 hours, and the ultrasonic power density of the ultrasonic treatment is 2–2.2 W / mL. In this embodiment of the invention, the liquid material containing plant protein is subjected to a first incubation treatment, an ultrasonic treatment, and a second incubation treatment sequentially; the temperature of the first incubation treatment is 80°C, and the time is 26.5 hours; the first incubation treatment is carried out under stirring conditions, and the stirring speed is 400 rpm; the ultrasonic intensity during the ultrasonic treatment can be 1% (corresponding to an actual ultrasonic power density of 2.1766 W / mL), the temperature of the ultrasonic treatment is provided by ice water, the total duration of the ultrasonic treatment is 2 minutes, and there is a 2-second pause every 2 seconds of treatment; the temperature of the second incubation treatment is 80°C, and the time is 21.5 hours; the second incubation treatment is carried out under stirring conditions, and the stirring speed is 400 rpm.

[0057] In this invention, ultrasonic treatment (with a rated power of 900W and ultrasonic intensities of 1%, 5%, 10%, 20%, and 30%) is applied at different stages of plant amyloid fiber growth (initial t0, end of lag phase t0.1, and end of growth phase t0.9) to regulate its growth process and morphology. Specifically, regarding the fiber growth process, high-intensity ultrasonic treatment at t0 (10%, 20%, and 30%) significantly accelerates the fiberization process without significantly reducing fiber yield; high-intensity ultrasonic treatment at t0.1 (20% and 30%) significantly increases the growth rate of plant amyloid fibers during the logarithmic growth phase; and low-intensity ultrasonic treatment at t0.9 (1%) enhances the growth rate of plant amyloid fibers. In terms of fiber morphology, high-intensity ultrasound (10%, 20%, and 30%) at t0 can significantly reduce the proportion of rigid fibers while increasing the proportion of flexible and semi-flexible fibers; at t0.1, all intensities of ultrasound can significantly reduce the proportion of rigid fibers, and at t0.9, all intensities of ultrasound can reduce the proportion of rigid fibers, while the proportion of flexible fibers increases with the increase of ultrasound intensity.

[0058] After the second incubation treatment, the present invention preferably performs dialysis and freeze-drying sequentially on the obtained product system to obtain the plant amyloid protein fiber. As one embodiment of the present invention, the dialysis conditions include: the molecular weight cutoff of the dialysis bag can be 8-14 kDa; the dialysis solution can be water (pH value specifically 2.0), specifically deionized water; the temperature is 3-6℃, specifically 4℃; the time is 42-54 h, specifically 48 h; the dialysis solution is preferably changed every 6 h during the dialysis process. The present invention does not specifically limit the freeze-drying conditions; conditions well known to those skilled in the art can be used.

[0059] This invention provides plant amyloid protein fibers prepared by the preparation method described above, comprising flexible fibers, semi-flexible fibers, and rigid fibers. The percentage of flexible fibers in the plant amyloid protein fibers is 4-50%, more specifically 10-45%, and even more specifically 20-40%. In the embodiments, the percentages are 4.76%, 4.95%, 6.48%, 5.00%, 10.89%, 13.00%, 15.84%, 16.83%, 25.00%, 31.00%, 36.00%, 40.00%, or 47.00%. The percentage of semi-flexible fibers is 10-45%, more specifically 20-40%, and even more specifically 30%. The percentage of rigid fibers is 35% to 35%, specifically 10.00%, 18.00%, 19.00%, 27.78%, 30.00%, 33.00%, 33.66%, 34.65%, 35.00%, 39.60%, 40.00%, 40.59%, or 43.00% in the examples; the percentage of rigid fibers is 35% to 65.8%, further 40% to 56%, and even further 45% to 50%, specifically 35.00%, 39.00%, 42.00%, 43.56%, 45.00%, 49.51%, 50.00%, 52.00%, 54.46%, 55.24%, 55.45%, or 65.74% in the examples.

[0060] This invention provides a composite film comprising a chitosan matrix and plant amyloid fibers distributed within the chitosan matrix, wherein the plant amyloid fibers are those described in the aforementioned technical solution. In one embodiment of this invention, the mass ratio of chitosan matrix to plant amyloid fibers in the composite film can be 2–4:1, specifically 3:1; the thickness of the composite film can be 0.140–0.160 mm. The composite film provided by this invention can be used for preserving fresh-cut fruits and vegetables.

[0061] As one embodiment of the present invention, the method for preparing the composite film may include the following steps: mixing chitosan solution, plant amyloid protein fiber dispersion and glycerol to obtain a membrane solution; placing the membrane solution in a mold and drying it to remove the solvent to obtain the composite film.

[0062] In one embodiment of the present invention, the preparation method of the chitosan solution may include the following steps: mixing chitosan with an acetic acid solution (volume concentration may be 2%) at a ratio of 2-4 g: 100 mL (specifically 3 g: 100 mL), stirring until the chitosan is completely dissolved, and adjusting the pH of the system to 1.8-2.0 using hydrochloric acid (concentration may be 3M) to obtain the chitosan solution. In another embodiment of the present invention, the plant amyloid cellulose dispersion may be obtained by mixing plant amyloid cellulose with water at a ratio of 0.8-1.2 g: 100 mL (specifically 1 g: 100 mL). In another embodiment of the present invention, the volume ratio of the chitosan solution, the plant amyloid cellulose dispersion, and glycerol may be 15:13-17:0.3-0.7, specifically 15:15:0.5. In this embodiment of the present invention, the mold may specifically be a petri dish with a diameter of 9 mm; the drying temperature may be 45-55℃, specifically 50℃; and the time may be 15-20 h, specifically 18 h. In this embodiment of the invention, the drying process specifically involves placing the obtained membrane material in a desiccator for 24 hours to equilibrate, thereby obtaining the composite film.

[0063] This invention provides a composite coating preservative, comprising chitosan, plant amyloid protein fiber, epigallocatechin gallate (EGCG), and a solvent, wherein the plant amyloid protein fiber is the plant amyloid protein fiber described in the above technical solution.

[0064] In one embodiment of the present invention, the mass ratio of chitosan, plant amyloid protein fiber, and EGCG can be 2-4:0.5-1.5:1-3, specifically 3:1:2. In another embodiment, the concentration of plant amyloid protein fiber in the composite coating preservative can be 8-12 mg / mL, specifically 10 mg / mL. In yet another embodiment, the pH value of the composite coating preservative can be 1.8-2.0; the solvent can specifically be water. The composite coating preservative provided by the present invention can be used for the preservation of fresh-cut fruits and vegetables, such as fresh-cut apples. Test results of the present invention show that the fiber groups with higher contents of flexible and semi-flexible fibers (t0.110%; t0.930%) have the best browning inhibition effect, and their b* values ​​are significantly lower than those of the original isolated protein and the protein fiber groups obtained without ultrasound.

[0065] This invention does not specifically limit the preparation method of the composite coating preservative; simply mixing the components evenly is sufficient. In this embodiment, the membrane solution can be prepared according to the above technical solution, and then the membrane solution is mixed with an EGCG solution to obtain the composite coating preservative. The pH value of the EGCG solution can be 1.8–2.0, and the concentration and volume of the EGCG solution are sufficient to ensure that the concentrations of each component in the obtained composite coating preservative meet the above requirements.

[0066] In this embodiment of the invention, pea protein isolate is specifically used as an example to study the influence of ultrasonic power density and ultrasonic treatment time points during the fibrosis process on the fibrosis kinetics and final product morphology of pea protein isolate. The physicochemical properties and structural differences of the original isolated protein and amyloid protein fibers before and after ultrasonic treatment are analyzed, revealing the ultrasonic regulation mechanism of pea protein isolate fibrosis. Furthermore, the performance differences of pea amyloid protein fibers with different morphologies in protein-chitosan composite membrane systems and coating preservatives are explored.

[0067] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0068] The preparation method of pea protein isolate used in the following experiments includes the following steps:

[0069] Pea protein powder (purchased from Xi'an Virgin Biotechnology Co., Ltd., purity 80wt%) was mixed with deionized water at a ratio of 1g:10mL and stirred for 2 hours. The pH of the system was then adjusted to 8.0 using a 3M NaOH solution, and stirred for another 2 hours to ensure complete dissolution of the pea protein powder. The mixture was centrifuged at 8000rpm for 20 minutes at 4°C, and the supernatant was collected. The pH of the supernatant was adjusted to 4.5 using a 3M HCl solution, stirred for 1 hour, and then centrifuged at 8000rpm for 10 minutes at 4°C. n. Collect the precipitate; mix the precipitate with deionized water at a ratio of 1g:10mL, and adjust the pH of the system to 7.0 with a 3M NaOH solution under continuous stirring. After the precipitate is completely dissolved, dialyze the resulting solution at 4℃ for 48h. The molecular weight cutoff of the dialysis bag used for dialysis is 8-14kDa, and the dialysis solution used for dialysis is deionized water. The dialysis solution is changed every 6h during the dialysis process. Finally, the purified product obtained by dialysis is freeze-dried to obtain the pea protein isolate.

[0070] The following steps are included in the method for obtaining the growth kinetic curve of pea amyloid protein fibers and determining t0.1 and t0.9 in the experiment:

[0071] Pea protein isolate was mixed with deionized water at a ratio of 2 g: 100 mL and magnetically stirred at 25 °C for 2 h to fully dissolve the pea protein isolate, yielding a pea protein isolate solution (2%, w / v). The pH of the pea protein isolate solution was adjusted to 2.0 using 3 M HCl, and the solution was centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was collected. The supernatant was placed in an 80 °C water bath and incubated at 400 rpm for 48 h. During the incubation process, samples were taken at regular intervals, with 15 μL of each sample used for subsequent ThT fluorescence intensity determination.

[0072] 8.0 mg of thioflavone T (ThT) powder was dissolved in 10 mL of phosphate buffer (PBS, pH 7.0) containing 150 mM NaCl and stirred for 20 min. The resulting solution was then filtered through a 0.22 μm microporous membrane to obtain a ThT stock solution (0.08%, w / v). The ThT stock solution was stored at 4 °C protected from light to prevent degradation. The ThT stock solution was then diluted 50 times with PBS to obtain a ThT working solution.

[0073] 15 μL of sample (2%, w / v) was collected at regular intervals during the incubation process and mixed with 135 μL of ultrapure water at pH 2.0. Then, 2850 μL of ThT working solution was added, and the mixture was incubated at 25°C in the dark for 10 min. Afterwards, the ThT values ​​of the samples were measured using a microplate reader at an excitation wavelength of 460 nm and an emission wavelength of 490 nm. The ThT fluorescence intensity data (incubation time and fluorescence intensity) were analyzed using the AmyloFit website (http: / / www.amylofit.ch.cam.ac.uk) to obtain the kinetic curve of pea amyloid fiber growth. Based on the kinetic curve, the time points corresponding to the three stages of pea amyloid fiber growth were determined: the lag phase from 0 to 10% of the maximum fluorescence intensity, the logarithmic growth phase from 10% to 90% of the maximum fluorescence intensity, and the plateau phase from 90% to the end of incubation. The time point corresponding to 10% of the maximum fluorescence intensity was denoted as t0.1, and the time point corresponding to 90% of the maximum fluorescence intensity was denoted as t0.9. After determining these time points, the intermediate product of pea amyloid fiber was subjected to ultrasonic treatment according to the relevant time points in subsequent Example 1.

[0074] Example 1

[0075] The preparation of pea starch-like protein fibers includes the following steps:

[0076] Pea protein isolate was mixed with deionized water at a ratio of 2 g: 100 mL and magnetically stirred at 25 °C for 2 h to fully dissolve the pea protein isolate, yielding a pea protein isolate solution (2%, w / v). The pH of the pea protein isolate solution was adjusted to 2.0 using 3 M HCl, and the solution was centrifuged at 8000 rpm for 10 min at 4 °C, collecting the supernatant. The supernatant was placed in an 80 °C water bath and incubated for 0 h (t0), 5 h (end of lag phase, t0.1), and 26.5 h (end of logarithmic growth phase, t0.9) at a stirring speed of 400 rpm. Afterward, 10 mL of each sample was immersed in ice water and subjected to ultrasonic treatment using a 10 mm diameter amplitude transformer. The ultrasonic intensity for each sample was as follows: The ultrasonic treatments were performed at concentrations of 1%, 5%, 10%, 20%, and 30% (with the ultrasonic intensity set at 100% and the rated power of the ultrasonic equipment at 900W; the actual ultrasonic power density corresponding to each ultrasonic intensity is shown in Table 1). The total ultrasonic treatment time was 2 minutes, with a 2-second pause after every 2 seconds of treatment. The samples were designated as t01–30%, t0.11–30%, and t0.91–30%, respectively. After ultrasonic treatment, the samples were placed back into an 80°C water bath and subjected to a second incubation treatment at a stirring speed of 400 rpm to obtain the pea amyloid protein fibers. The total time for the first and second incubation treatments was 48 hours. During the second incubation treatment, samples were taken at regular intervals to measure the ThT fluorescence intensity using the aforementioned method, and the pea amyloid protein fiber growth kinetic curve was obtained based on the ThT fluorescence intensity data.

[0077] Table 1. Correspondence between ultrasonic intensity and actual ultrasonic power density (measured by isothermal calorimetry)

[0078] Ultrasonic intensity (%) Actual ultrasonic power density (W / mL) 1 2.1766 5 2.3909 10 2.6588 20 3.1946 30 3.7304

[0079] Figure 1 This is a comparative diagram showing the preparation process of pea amyloid protein fibers. Figure 1The lower middle section shows a flowchart of the preparation of pea amyloid protein fibers using ultrasonic treatment, while the upper section shows a flowchart omitting ultrasonic treatment; PPI represents pea protein isolate; control t0, control t0.1, control t0.9 and control48h (t0.9F) represent the PPI samples obtained after incubation at 80℃ for 0h, 5h, 26.5h, and 48h, respectively, with the time points determined based on the kinetic curve of pea amyloid protein fiber growth. t0(1-30%)U, t0.1(1-30%)U, and t0.9(1-30%)U represent the PPI samples obtained after ultrasonic treatment following incubation at 80℃ for 0h, 5h, and 26.5h, with ultrasonic intensities of 1%, 5%, 10%, 20%, and 30%, respectively. t0(1-30%)F, t0.1(1-30%)F, and t0.9(1-30%)F represent the pea amyloid protein fiber samples ultimately formed from t0(1-30%)U, t0.1(1-30%)U, and t0.9(1-30%)U, respectively. To better reflect actual production practices, such as... Figure 1 As shown in the upper middle section, without omitting the ultrasonic treatment, the pea amyloid protein fiber products finally selected for groups t0 and t0.1 were the samples corresponding to 80% of the maximum ThT fluorescence intensity, while the pea amyloid protein fiber products finally selected for group t0.9 were the samples incubated for 48 hours.

[0080] Test Example 1

[0081] 1. Growth kinetics of pea amyloid fibrils treated with ultrasound

[0082] Figure 2 The figure shows the kinetics of pea protein isolate fiberization after t0 ultrasonic treatment. A represents the normalized t0 ultrasonic treatment kinetics curve of pea protein isolate fiberization, and B represents the hysteresis duration of samples at different ultrasonic intensities at t0. 1 / 2 and t growth The test results are shown in Figure C, which represents the maximum ThT fluorescence intensity of pea protein isolate fibers treated with different ultrasonic intensities at time t0; t 1 / 2 t represents the time required to reach half of the maximum ThT fluorescence intensity. growth The duration of the logarithmic growth phase in the fibrosis process is indicated. Results showed that the fibrosis process of pea protein isolate treated with ultrasound at t0 exhibited significant differences. Specifically, when the ultrasound intensity was ≥10% (t0 10%, 20%, and 30%), the fibrosis process was significantly accelerated compared to the control group, which is related to… Figure 2 The shortened hysteresis and half-maximum fluorescence time (t) in B 1 / 2Conversely, the fibrosis process at t01% and t05% ultrasonic intensities was slowed down, indicating that high-power-density ultrasonic treatment at t0 effectively promoted the fibrosis process. This phenomenon may be attributed to the fact that high-power ultrasound promoted the conformational unfolding of pea protein molecules before heating, lowered the energy threshold required for subsequent protein acid hydrolysis, and thus promoted the self-assembly of protein molecules to form a "nucleation" process, ultimately accelerating the fibrosis process. Figure 2 As shown in Figure C, when the ultrasonic power increased, the maximum ThT fluorescence intensity of the t0 group samples decreased slightly, but there was no significant difference compared with the control group. This indicates that ultrasound has little effect on the degree of fibrosis of pea amyloid protein. The maximum fluorescence intensity of t01% was slightly higher than that of the control group (P>0.05), indicating that more fibers were generated. In summary, ultrasound with different powers at t0 can regulate the fibrosis process of pea protein. High-intensity ultrasound accelerates the fibrosis process without significantly reducing fiber yield.

[0083] Figure 3 The figure shows the kinetics of pea protein isolate fiberization after t0.1 ultrasonic treatment. A represents the normalized kinetic curve of pea protein isolate fiberization after t0.1 ultrasonic treatment, and B represents the hysteresis duration of samples at different ultrasonic intensities at t0.1. 1 / 2 and t growth The test results are shown in Figure C, which represents the maximum ThT fluorescence intensity of pea amyloid fibers treated with different ultrasonic intensities at t0.1. The results show that the t0.120% and t0.130% groups exhibit unique kinetic characteristics. Specifically, their growth curves initially show a longer lag phase, followed by a significant increase in growth rate upon entering the logarithmic growth phase, particularly in the t0.130% group. This phenomenon can be attributed to the acid hydrolysis and nucleation processes during the lag phase, during which protein molecules initially assemble into oligomers. These oligomers serve as templates for fiber growth and can rapidly elongate during the logarithmic growth phase. For samples treated with 20% and 30% high ultrasonic intensities, the ultrasound applied at the end of the lag phase may have triggered oligomer deconstruction. The oligomers require further elongation to enter the growth phase, thus increasing the time required for nucleation and consequently prolonging the lag phase. However, the broken oligomers can provide more active ends during subsequent fiber growth, significantly accelerating the growth rate during the logarithmic growth phase. Figure 3 As shown in C, there was no significant difference in the maximum ThT fluorescence intensity between the control group and each ultrasound group, indicating that t0.1 ultrasound had no significant effect on fiber conversion rate.

[0084] Figure 4The figures show the kinetics of pea amyloid protein fiber formation after ultrasonic treatment at t0.9, where A is the normalized kinetic curve of pea amyloid protein fiber formation after ultrasonic treatment at t0.9, and B is the maximum ThT fluorescence intensity of pea amyloid protein fibers treated with different ultrasonic intensities at t0.9. The results show that, given that the fiber formation reaction has reached saturation, the differences in fiber growth kinetics among the samples after ultrasonic treatment are small. Specifically, the t0.9 1% sample showed a slightly accelerated fiber growth rate and a slightly increased maximum ThT fluorescence intensity compared to the control group (P>0.05), indicating a slight increase in fiber conversion rate. This phenomenon can be attributed to the partial fragmentation of the fiber structure caused by ultrasonic treatment; these fragmented fiber segments act as nucleation templates, accelerating the fiber self-assembly process to some extent. For other samples in the t0.9 group, although further increases in ultrasonic intensity resulted in more fiber fragments and an increase in the number of fiber ends during the nucleation-growth process, the concentration of free protein participating in fiber growth was low, leading to a slower fiber growth rate in these samples. Therefore, optimizing the intensity of ultrasound requires balancing the generation of fiber fragments with the concentration of free proteins to maximize the growth efficiency of pea amyloid fibers.

[0085] 2. Statistical analysis of pea amyloid protein fiber morphology

[0086] To quantitatively analyze the regulatory effect of ultrasonic treatment on the morphology of pea amyloid fibers, this test case used FiberApp to perform statistical analysis on TEM images of pea amyloid fibers. Specifically, pea amyloid fiber samples were diluted with ultrapure water at pH 2.0 to a protein concentration of 0.1 mg / mL. The diluted sample was then dropped onto a 300-mesh copper grid with a carbon support film, stained with 5 μL of 2% (w / v) phosphotungstic acid for 30 s, and dried in air. The samples were then observed at 80 kV and TEM images were captured at 3000x magnification. At least three representative TEM images were selected from each sample and analyzed using FiberApp. Each sample had a dataset of at least 100 fibers, from which the profile length (Lc) and duration (Lp) of each fiber were obtained, thus yielding the profile length distribution. If the order of magnitude of Lc (lower limit) of a fiber is less than the order of magnitude of Lp (lower limit), it is classified as a rigid fiber; if the order of magnitude of Lc is equal to the order of magnitude of Lp, it is classified as a semi-flexible fiber; if the order of magnitude of Lc is greater than the order of magnitude of Lp, it is classified as a flexible fiber. Refer to the following formula for specific determination:

[0087] M = int(log 10 (L c ))-int(log 10 (L p ));

[0088] Where M<0, it is a rigid fiber; M=0, it is a semi-flexible fiber; M>0, it is a flexible fiber.

[0089] Based on this, the ratio of rigid fibers, semi-flexible fibers, and flexible fibers can be obtained.

[0090] Figure 5 TEM images of pea amyloid fiber samples are shown, where t0 represents the start of the fibrillation process (0h), t0.1 represents the end of the lag phase in the fibrillation process (5h), and t0.9 represents the end of the logarithmic growth phase in the fibrillation process (26.5h). t0(1–30%)F, t0.1(1–30%)F, and t0.9(1–30%)F represent pea amyloid fiber samples formed after sonication at t0, t0.1, and t0.9, respectively. The results show that the pea amyloid fibers in all samples exhibit diverse morphological characteristics, encompassing three main categories: rigid, semi-flexible, and flexible fibers.

[0091] Figure 6 The images show the outline length distribution of pea amyloid protein fibers obtained by ultrasonic treatment. In the images, A shows the outline length distribution of fiber samples under different heating times, B shows the outline length distribution of fibers obtained by ultrasonic treatment and incubation at the beginning of the lag period (0h), C shows the outline length distribution of fibers obtained by ultrasonic treatment and incubation at the end of the lag period (5h), and D shows the outline length distribution of fibers obtained by ultrasonic treatment and incubation at the end of the growth period (26.5h).

[0092] Figure 7 The following are statistical charts showing the proportions of flexible, semi-flexible, and rigid fibers in pea amyloid fibers obtained through ultrasonic treatment: A shows the proportions of rigid, semi-flexible, and flexible fibers in fibers obtained after ultrasonic treatment and subsequent incubation at the beginning of the lag phase (0h); B shows the proportions of rigid, semi-flexible, and flexible fibers in fibers obtained after ultrasonic treatment and subsequent incubation at the end of the lag phase (5h); and C shows the proportions of rigid, semi-flexible, and flexible fibers in fibers obtained after ultrasonic treatment and subsequent incubation at the end of the exponential growth phase (26.5h). Specific results are listed in Table 2.

[0093] Table 2. Proportion of rigid fibers, semi-flexible fibers, and flexible fibers in pea amyloid fibers.

[0094]

[0095]

[0096] The results show that the profile length of pea amyloid fibers increases with increasing reaction time during the fibrosis process. However, ultrasonic treatment at time points t0, t0.1, and t0.9 significantly shortens the fiber profile length. This is attributed to the fiber fragmentation caused by ultrasonic treatment, which provides more active ends for fiber elongation but also limits the overall profile length increase. Figure 7 The ultrasonic treatment also significantly altered the proportions of fibers with different morphologies. For samples ultrasonically treated at time t0, the proportion of rigid fibers decreased significantly at high ultrasonic power, while the proportions of flexible and semi-flexible fibers increased significantly, especially at t0 / 10%F. Compared to t0 / t0.1 F, the proportion of flexible fibers increased from 5.83% to 16.83%, and the proportion of semi-flexible fibers increased from 24.17% to 39.60%. At lower ultrasonic power, the proportion of rigid fibers increased slightly. All ultrasonic intensities in the t0.1 group significantly reduced the proportion of rigid fibers, but the proportion of flexible fibers remained at a low level, except at t0.1 / 10%F, where compared to t0 / t0.1 F, the proportion of flexible fibers increased from 5.83% to 13.00%, and the proportion of semi-flexible fibers increased from 24.17% to 35.00%. In the t0.9 group, the proportion of flexible fibers increased significantly with the increase of ultrasonic power. The proportion of flexible fibers in t0.930%F increased from 8.67% to 47.00% compared to t0.9 F, while the proportion of rigid fibers decreased significantly. The proportion of rigid fibers in t0.930%F decreased from 66.00% to 35.00% compared to t0.9 F.

[0097] Example 2

[0098] The preparation of a pea starch-like protein fiber-chitosan composite film includes the following steps:

[0099] Chitosan (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was mixed with acetic acid solution (volume concentration of 2%) at a ratio of 3g:100mL and stirred until the chitosan was completely dissolved. The pH of the system was adjusted to 2.0 using 3M HCl to obtain a chitosan solution (3%, w / v).

[0100] The pea starch-like protein fiber prepared in Example 1 was mixed with water at a ratio of 1g:100mL to obtain a pea starch-like protein fiber dispersion (1%, w / v);

[0101] Take 15 mL of the pea starch-like protein fiber dispersion and mix it with 15 mL of the chitosan solution. Add 0.5 mL of glycerol and place the resulting mixture in a 60°C water bath and stir for 30 min. Then place it in an ultrasonic cleaner and sonicate for 30 min to fully degas it to obtain a membrane solution. Pour 30 mL of the membrane solution into a 9 mm diameter petri dish and dry it in a 50°C forced-air drying oven for 18 h. Then place it in a desiccator to equilibrate for 24 h to obtain the pea starch-like protein fiber-chitosan composite film.

[0102] A control group and a blank group were set up. The control group used 15 mL of pea protein isolate solution (1%, w / v) instead of the pea amyloid protein fiber dispersion. The blank group used 15 mL of ultrapure water with a pH of 2.0 instead of the pea amyloid protein fiber dispersion. Other operations were the same as in Example 2.

[0103] The naming convention for the pea amyloid protein fiber-chitosan composite films prepared using pea amyloid protein fibers is the same as that for pea amyloid protein fibers, and they are respectively denoted as t0 / t0.1 F, t010%F, t0.110%F, t0.9 F, and t0.930%F; the composite film prepared using unfibrinated pea protein isolate is denoted as control t0 (i.e., control group); and the film prepared using the blank group is denoted as CS.

[0104] The thicknesses of each composite film are shown in Table 3.

[0105] Table 3 Thickness of Composite Films

[0106] Composite thin film sample types Thickness (mm) CS 0.153±0.001 controlt0 0.157±0.026 t0 / t0.1F 0.151±0.021 t010%F 0.149±0.007 t0.110%F 0.150±0.014 t0.9F 0.146±0.010 t0.930%F 0.146±0.012

[0107] Test Example 2: Evaluation of Thin Film Mechanical Properties

[0108] The film sample was cut into strips of 60 mm × 10 mm, and its mechanical properties were measured using a texture analyzer. The initial spacing was set to 30 mm, and the tensile speed was set to 50 mm / min until the film sample broke. The tensile strength (TS) and elongation at break (E) of the film were calculated using the following formulas:

[0109]

[0110] Where F is the maximum tensile force at break (N), a is the width of the film (mm), b is the thickness of the film (mm), L is the length of the film at break (mm), and L0 is the initial length of the film (mm).

[0111] Figure 8The figures show the tensile strength and elongation at break of the films. The left side shows the tensile strength test results, and the right side shows the elongation at break test results. The results show that the CS (chitosan film) group has poor tensile strength and elongation at break, at 3.32 MPa and 64.93%, respectively. This indicates that when chitosan alone is used as the film-forming matrix, its mechanical properties are poor. After adding pea protein isolate to the chitosan film system (control t0 group), this composite film system slightly improved the tensile strength and elongation at break compared to CS, but the difference was not significant. This may be because the natural conformation of pea protein isolate can generate weak intermolecular interactions with chitosan molecules and cannot stack together to form a network structure. After adding pea amyloid fibers to the chitosan film system, the tensile strength and elongation at break of this composite film system were significantly improved. The t010%F, t0.110%F, control48h, and t0.930%F groups showed significant improvements compared to CS, while the t0 / t0.1F group did not show a significant improvement in mechanical properties compared to CS. Further analysis of these results revealed that the pea amyloid fiber samples added in the t0 / t0.1F and control48h groups were untreated fiber samples. The heating time for t0 / t0.1F was shorter than that for control48h, resulting in shorter fiber profiles (e.g., ...). Figure 6 As shown in the figure, the stiffness and flexibility of the two groups of fiber samples were statistically similar, indicating that under the premise of similar fiber stiffness and flexibility, the profile length of the fiber sample is positively correlated with the mechanical properties of the composite film. This may be because fiber samples with longer profile lengths can form network structures with more cross-linking points than fiber samples with shorter profile lengths. The mechanical properties of the network structure are positively correlated with the stiffness and flexibility of the macromolecules and the number of cross-linking points. Therefore, fibers with longer profile lengths will have an advantage in mechanical properties. On the other hand, comparing the three groups t0 / t0.1 F, t010%F, and t0.110%F, it can be found that the mechanical properties of t010%F and t0.110%F treated by ultrasound are better than those of t0 / t0.1 F without ultrasound treatment. After ultrasonic treatment, the profile length of the fiber samples formed by pea protein isolate decreased, but the proportion of flexible fibers and semi-flexible fibers contained increased significantly (e.g., Figure 7 As shown in the figure, this indicates that the rigidity or flexibility of the fiber sample has a significant impact on the mechanical properties of the composite film system. Specifically, when the fiber sample contains a high proportion of flexible or semi-flexible fibers, the fibers are more prone to intermolecular entanglement, which also increases the number of crosslinking points in the network structure, thus improving the mechanical properties of the composite film system.

[0112] Figure 9The figure shows the correlation analysis results between the mechanical properties of the film and the stiffness and flexibility of the fibers. Specifically, the stiffness and flexibility of the fiber-added samples were correlated with the tensile strength and elongation at break of the resulting composite film. The results show that the proportion of rigid fibers in the fiber samples is negatively correlated with both mechanical properties of the composite film, while the proportion of semi-flexible fibers to flexible fibers is positively correlated with both mechanical properties. Specifically, compared with t010%F and t0.110%F, the ultrasonically treated t010%F and t0.110%F have a higher content of flexible fibers and a lower content of rigid fibers. The composite films prepared using these two sets of fiber samples have higher mechanical strength than those prepared with t0 / t0.1F.

[0113] Example 3

[0114] The preparation of the composite coating preservative includes the following steps:

[0115] The membrane solution was prepared according to the method in Example 2. 375 μL of epigallocatechin gallate (EGCG) solution (concentration of 20 mg / mL, pH value of 2.0) was added to the membrane solution to make the mass concentration ratio of pea starch-like protein fiber to EGCG in the system 20:1, thus obtaining the composite coating preservative.

[0116] Test Example 3

[0117] 1. Evaluation of in vitro antioxidant activity

[0118] This test used DPPH to determine the antioxidant activity of pea protein isolate and pea amyloid cellulose samples. First, DPPH was dissolved in anhydrous ethanol to obtain a 0.1 mM DPPH working solution. 200 μL of pea protein isolate dispersion (20 mg / mL, pH 2.0, control group) or pea amyloid cellulose dispersion (20 mg / mL, pH 2.0, experimental group) was mixed with 800 μL of DPPH working solution. For the blank group, 200 μL of ultrapure water (pH 2.0) was mixed with 800 μL of DPPH working solution. The resulting solutions were incubated at 25°C in the dark for 30 min, and the absorbance of the samples at 517 nm was measured using a microplate reader. The DPPH free radical scavenging activity of the samples was calculated using the following formula:

[0119]

[0120] Where A0 is the absorbance value of the blank group and A1 is the absorbance value of the sample.

[0121] Figure 10The figure shows the DPPH free radical scavenging rate test results for pea protein isolate and pea amyloid fibers. The results show that the DPPH free radical scavenging rate was significantly improved after pea protein isolate was prepared into pea amyloid fibers. This may be because after the native conformation of pea protein isolate is transformed into pea amyloid fibers, more cysteine ​​residues located inside the pea protein isolate are exposed, and cysteine ​​residues have certain antioxidant activity. Among the different pea amyloid fiber samples, the DPPH free radical scavenging rates of the t0.9 F group and the t0.930% F group were higher, at 30.14±0.88% and 32.23±1.86%, respectively. This indicates that the longer the incubation time of pea amyloid fibers, the more cysteine ​​residues are exposed. The higher antioxidant activity of pea amyloid fibers makes them suitable for the preservation of fresh-cut fruits that are prone to browning.

[0122] 2. Edible coating preservation application for fresh-cut apples

[0123] The composite coating preservative is used as a coating for preserving fresh-cut apples, as follows:

[0124] After washing the fresh apples, peel them and cut them into uniform strips. Immediately immerse the cut apples in the composite coating preservative, wait 30 seconds, and then remove them. Place them in an environment with a temperature of 25°C and a relative humidity of 75%, take pictures at 12-hour intervals, and measure the color of all fresh-cut apple samples after 48 hours.

[0125] Meanwhile, a blank group and a control group were set up; the blank group was a fresh-cut apple sample without coating treatment; the control group (CS) was a chitosan control group, specifically, when preparing the composite coating preservative, 15 mL of ultrapure water with a pH of 2.0 was used instead of pea starch-like protein fiber dispersion, and then the fresh-cut apples were coated and preserved using the composite coating preservative according to the above method.

[0126] Figure 11 The image shows the storage results of fresh-cut apples. The results indicate that after coating preservation, the shape of the fresh-cut apples remained relatively consistent before and after storage, and the browning rate was inhibited to varying degrees. After 12 hours of storage, the control group showed significant browning, while the browning of all samples with added pea amyloid fiber was inhibited, with little difference compared to the 0-hour samples. This trend became more pronounced with prolonged storage time: the CS group was superior to the control group, the control group was slightly superior to the CS group, and the groups with added pea amyloid fiber were superior to the other groups, consistent with the trend in the samples' antioxidant capacity. Furthermore, pea amyloid fiber has a stronger ability to bind EGCG compared to pea protein isolate, thus exhibiting stronger anti-browning ability.

[0127] Table 4 shows the colorimetric values ​​of fresh-cut apples after 48 hours of storage. L* represents the brightness of the sample color; a* represents the red-green hue of the sample color; b* represents the yellow-blue hue of the sample color (a higher b* value indicates a more yellowish tint); △E represents the difference between the sample color and the standard color. The results showed that the b* distribution of the groups with added pea protein isolate and pea amyloid fiber was as follows: control t0>t0 10%F>t0 / t0.1 F>t0.9 F>t0.9 30%F>t0.1 10%F. This is similar to the relationship between the antioxidant activities of the corresponding pea protein isolate and pea amyloid fiber samples (e.g., ...). Figure 10 As shown in the figure, this indicates that pea amyloid fiber samples with high antioxidant activity can effectively inhibit browning in fresh-cut apples. Among them, the longer the incubation time of the pea amyloid fiber samples, the stronger the anti-browning ability (t0 / t0.1 F).<t0.9 F,P> (0.05), longer incubation time results in longer fiber profiles, thus enabling them to carry more EGCG and providing a certain degree of sustained release during storage. Differences in fiber morphology also affect their resistance to browning. After ultrasonic treatment, the two groups with higher flexible fiber content, t0.110%F and t0.930%F, were both higher than their respective fiber control groups (t0.110%F was higher than t0 / t0.1 F, and t0.930%F was higher than t0.9 F). In particular, t0.110%F showed a significant difference compared to the control group, indicating that samples with a higher proportion of flexible and semi-flexible fibers can carry more EGCG. Furthermore, due to the higher proportion of flexible and semi-flexible fibers, the network structure formed between the fibers is more compact, which also improves the sustained release effect of EGCG.

[0128] Table 4. Color values ​​of fresh-cut apples after 48 hours of storage.

[0129] Sample types L* a* b* △E blank 81.01 ± 2.47 ab ]] 13.95 ± 1.37 a ]] 41.61 ± 0.58 a ]] 92.15 ± 2.20 a ]] CS 70.83 ± 7.22 b ]] 11.78 ± 0.77 ab ]] 38.07 ± 0.64 b ]] 81.34 ± 5.99 b ]] controlt0 75.29 ± 4.35 b ]] 10.13 ± 1.09 b ]] 35.69 ± 1.76 b ]] 84.01 ± 3.17 ab ]] t0 / t0.1F 78.86 ± 4.36 ab ]] 9.05 ± 0.71 bc ]] 34.19 ± 1.06 b ]] 86.44 ± 4.12 ab ]] t010%F 81.44 ± 0.81 ab ]] 9.99 ± 0.50 bc ]] 34.96 ± 0.30 b ]] 89.18 ± 0.82 ab ]] t0.110%F <![CDATA[88.15±0.91 a ]]> <![CDATA[5.76±0.20 c ]]> <![CDATA[27.90±0.95 c ]]> <![CDATA[92.64±1.16 a ]]> t0.9F <![CDATA[86.23±1.17 a ]]> <![CDATA[7.39±1.54 c ]]> <![CDATA[31.18±1.95 bc ]]> <![CDATA[92.02±0.54 a ]]> t0.930%F <![CDATA[86.98±2.31 a ]]> <![CDATA[6.11±0.10 c ]]> <![CDATA[28.05±1.53 c ]]> <![CDATA[91.59±2.65 a ]]>

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing plant amyloid protein fibers, comprising the following steps: Plant protein isolate is mixed with water to obtain an aqueous solution of plant protein isolate; the plant protein isolate is pea protein isolate. The pH of the plant protein isolate aqueous solution was adjusted to 1.5-2.5, followed by solid-liquid separation to obtain a liquid material containing plant protein. The plant protein-containing liquid material is subjected to fiber growth treatment to obtain the plant amyloid protein fiber; The fiber growth process includes sequentially performing a first incubation treatment, an ultrasonic treatment, and a second incubation treatment, wherein the first incubation treatment and the second incubation treatment are performed under non-ultrasonic conditions; The temperatures for the first and second incubation treatments are independently 75–85°C, and the total time for the first and second incubation treatments is 45–50 h; the first and second incubation treatments are carried out under stirring conditions. The conditions for the ultrasonic treatment include: a temperature of 0–4°C, a total time of 1–3 min, and a pause of 1–3 s after every 1–3 s of ultrasonic treatment; The fiber growth treatment is any one of the following three cases: Case 1: The incubation time for the first incubation treatment is 0 hours, and the ultrasonic power density for the ultrasonic treatment is 2.6–4 W / mL; Scenario 2: The incubation time for the first incubation treatment is 4-5 hours, and the ultrasonic power density for the ultrasonic treatment is 3-4 W / mL; Case 3: The incubation time for the first incubation treatment is 25 to 26.5 hours, and the ultrasonic power density for the ultrasonic treatment is 2 to 2.2 W / mL.

2. The preparation method according to claim 1, characterized in that, The ratio of plant protein isolate to water is 1-3 g: 100 mL.

3. The plant amyloid protein fiber prepared by the preparation method according to claim 1 or 2 includes flexible fibers, semi-flexible fibers and rigid fibers, wherein the percentage of flexible fibers in the plant amyloid protein fiber is 4-50%, the percentage of semi-flexible fibers is 10-45%, and the percentage of rigid fibers is 35-65.8%.

4. A composite film comprising a chitosan matrix and plant amyloid fibers distributed in the chitosan matrix, wherein the plant amyloid fibers are the plant amyloid fibers of claim 3.

5. A composite coating preservative, comprising chitosan, plant amyloid protein fiber, epigallocatechin gallate and solvent, wherein the plant amyloid protein fiber is the plant amyloid protein fiber of claim 3.

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