Plant amyloid protein fiber, preparation method of plant amyloid protein fiber, composite film and composite coating preservative

Abstract

The invention provides a plant amyloid protein fiber, a preparation method thereof, a composite film and a composite coating preservative, and belongs to the technical field of protein fibers. The method comprises the following steps: mixing plant protein isolate with water, adjusting the pH value to 1.5-2.5, then carrying out solid-liquid separation, and carrying out fiber growth treatment on the obtained liquid material containing the plant protein to obtain the plant amyloid protein fiber; according to a growth kinetic curve, the fiber growth treatment comprises first incubation treatment, ultrasonic treatment and second incubation treatment in sequence, the time of the first incubation treatment is 0 h to the end of the logarithmic phase, and the first incubation treatment and the second incubation treatment are carried out under a non-ultrasonic condition; 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. According to the method, the growth process and morphology of the fibers can be conveniently and simultaneously regulated, and the proportion of rigid fibers can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of protein fibers, and in particular to a plant amyloid protein fiber and a preparation method thereof, a composite film, and a composite coating preservative. Background Art

[0002] In recent years, the supply of animal protein has been limited, and plant protein has attracted widespread attention as an alternative protein due to its wide range of sources. Pea protein isolate, a byproduct of pea starch extraction, is the second largest source of protein in plants after soybeans. It has high nutritional value and low allergenicity. Pea protein isolate is mostly globulin. When the globular structure undergoes self-assembly transformation into a fibrous structure with a high aspect ratio to form pea amyloid protein fibers, the application scenarios of pea protein isolate can be expanded.

[0003] At present, the relevant technology usually directly transforms the spherical plant protein into a fibrous structure by self-assembly under heating conditions. However, the rigid fibers account for a large proportion of the protein fibers prepared by this method. Summary of the invention

[0004] The object of the present invention is to provide a plant amyloid protein fiber and a preparation method thereof, a composite film and a composite coating preservative. The method of the present invention is used to prepare the plant amyloid protein fiber, which is convenient for simultaneously regulating the growth process and morphology of the plant amyloid protein fiber, and can reduce 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] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0007] Mixing the plant protein isolate with water to obtain a plant protein isolate aqueous solution;

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

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

[0010] According to the growth kinetics curve of the plant amyloid protein fiber, the fiber growth process includes a hysteresis phase, a logarithmic growth phase and a plateau phase in sequence;

[0011] The fiber growth treatment includes a first incubation treatment, an ultrasonic treatment and a second incubation treatment in sequence, wherein the first incubation treatment is performed from 0 h to the end of the logarithmic growth phase, and the first incubation treatment and the second incubation treatment are performed under non-ultrasonic conditions; when the first incubation treatment is performed for 0 h, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL.

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

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

[0014] Preferably, the temperatures 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 h; and the first incubation treatment and the second incubation treatment are carried out under stirring conditions.

[0015] Preferably, the first incubation treatment lasts from 0 h to the end of the hysteresis period.

[0016] Preferably, based on the start of the fiber growth treatment, the hysteresis period ranges from 0 to 5 hours, and the logarithmic growth period ranges from 5 to 26.5 hours.

[0017] Preferably, the fiber growth process includes any one of the following three situations:

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

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

[0020] Case 3: The time of the first incubation treatment is 25 to 26.5 h, and the ultrasonic power density of 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 in the above technical scheme, including flexible fibers, semi-flexible fibers and rigid fibers. 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] The invention provides a composite coating preservative, comprising chitosan, plant amyloid protein fibers, epigallocatechin gallate and a solvent, wherein the plant amyloid protein fibers are the plant amyloid protein fibers described in the above technical solution.

[0024] The present 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 value of the plant protein isolate aqueous solution to 1.5-2.5, then performing solid-liquid separation to obtain a plant protein-containing liquid material; subjecting the plant protein-containing liquid material to a 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 hysteresis period, a logarithmic growth period and a plateau period; the fiber growth treatment includes sequentially performing a first incubation treatment, an ultrasonic treatment and a second incubation treatment, the first incubation treatment time being 0h to the end of the logarithmic growth period, the first incubation treatment and the second incubation treatment being performed under non-ultrasonic conditions; when the first incubation treatment time is 0h, the ultrasonic power density of the ultrasonic treatment is ≥2.5W / mL. The method provided by the present invention is convenient for simultaneously regulating the growth process and morphology of plant amyloid protein fibers. In the present invention, specifically, the ultrasonic treatment can accelerate the growth of plant amyloid protein fibers, which is of great significance for the high energy consumption production process of preparing plant amyloid protein fibers, and is conducive to improving production efficiency and reducing energy consumption; and the ultrasonic treatment can regulate the morphology of plant amyloid protein fibers and reduce the proportion of rigid fibers. The test results show that ultrasonic treatment increases the proportion of flexible fibers and semi-flexible fibers in pea amyloid protein fibers. The composite film prepared using the plant amyloid protein fibers of the present invention has good mechanical properties; the composite coating preservative prepared using the plant amyloid protein fibers of the present invention has good preservation performance. The test results show that the composite coating preservative has a better inhibitory effect on the browning of fresh-cut apples, and the browning inhibition effect is better when the content of flexible fibers and semi-flexible fibers is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a comparison diagram of the preparation process of pea amyloid protein fibers;

[0026] Figure 2 This is the result of the test on the fibrillation kinetics of pea protein isolate treated with ultrasound at t0;

[0027] Figure 3 This is the result of the test on the fibrillation kinetics of pea protein isolate treated with ultrasound at t0.1;

[0028] Figure 4 This is the result of the kinetic test of pea protein isolate fibrillation treated with ultrasound at t0.9;

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

[0030] Figure 6 is the contour length distribution diagram of pea amyloid protein fibers obtained by ultrasonic treatment;

[0031] Figure 7 The figure is a statistical diagram of the number ratio of flexible fibers, semi-flexible fibers and rigid fibers in pea amyloid fibers obtained by ultrasonic treatment;

[0032] Figure 8 The graph is the test result of the tensile strength and elongation at break of the film;

[0033] Fig. 9 This is the result of the correlation analysis between the mechanical properties of the film and the rigidity and flexibility of the fiber;

[0034] Fig.10 The DPPH free radical scavenging rate test results of pea protein isolate and pea amyloid protein fiber are shown in the figure

[0035] Fig.11 This is a picture showing the storage results of fresh-cut apples. DETAILED DESCRIPTION

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

[0037] Mixing the plant protein isolate with water to obtain a plant protein isolate aqueous solution;

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

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

[0040] According to the growth kinetics curve of the plant amyloid protein fiber, the fiber growth process includes a hysteresis phase, a logarithmic growth phase and a plateau phase in sequence;

[0041] The fiber growth treatment includes a first incubation treatment, an ultrasonic treatment and a second incubation treatment in sequence, wherein the first incubation treatment is performed from 0 h to the end of the logarithmic growth phase, and the first incubation treatment and the second incubation treatment are performed under non-ultrasonic conditions; when the first incubation treatment is performed for 0 h, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL.

[0042] The present invention facilitates simultaneous regulation of the growth process and morphology of plant amyloid protein fibers through ultrasonic treatment. Specifically, ultrasound is an efficient, low-energy and environmentally friendly physical processing technology, which can change the protein structure at all levels without introducing exogenous substances through the input of external energy, and improve the functional characteristics of the protein. The present invention uses ultrasound to regulate the different stages of plant amyloid protein fiber growth (such as the initial t0, the end of the hysteresis period t0.1, and the end of the growth period t0.9), and the regulation time point is flexible; and the rigidity and flexibility index of the plant amyloid protein fiber morphology is quantified, and then the morphology and function of the plant amyloid protein fiber are associated, and on this basis, the performance differences of plant amyloid protein fibers with different morphologies in the protein-chitosan composite film system and the composite coating preservative are further explored, wherein the plant amyloid protein fiber samples with higher content of flexible fibers and semi-flexible fibers have better mechanical properties, antioxidant properties and preservation properties. The preparation method of the plant amyloid protein fiber described in the present invention is described in detail below.

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

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

[0045] As an embodiment of the present invention, the material-liquid ratio of the pea protein powder and water when mixed can be 0.5-1.5g:10mL, specifically 1g:10mL; the time of the first stirring treatment can be 1.5-2.5h, specifically 2h; the reagent used to adjust the pH value to 7.5-8.5 (specifically 8.0) can be a NaOH solution, and the concentration of the NaOH solution can be 3M; the time of the second stirring treatment can be 1.5-2.5h, specifically 2h. As an embodiment of the present invention, the first solid-liquid separation and the second solid-liquid separation can be centrifuged; the centrifugal temperature can be 3-6°C, specifically 4°C; the rotation speed can be 6000-10000rpm, specifically 8000rpm; the time can be 15-25min, specifically 20min. As an embodiment of the present invention, the pH value of the liquid material is adjusted to 4-5 (specifically 4.5), the reagent may be hydrochloric acid, and the concentration of the hydrochloric acid may be 3M; the time of the first stirring treatment may be 0.5-1.5h, specifically 1h; the solid material obtained after the second solid-liquid separation is mixed with water, and the material-liquid ratio may be 0.5-1.5g:5-15mL, specifically 1g:10mL; the reagent used for adjusting the pH value to 6.5-7.5 (specifically 7.0) may be NaOH solution, and the concentration of the NaOH solution may be 3M. As an embodiment of the present invention, the conditions of the dialysis include: the molecular weight cutoff of the dialysis bag may be 8-14kDa; the dialysate may be water, specifically deionized water; the temperature is 3-6°C, specifically 4°C; the time may be 42-54h, specifically 48h; the dialysate is preferably replaced every 6h during the dialysis process. The present invention does not specifically limit the conditions of the freeze-drying, and the conditions familiar to those skilled in the art may 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 a plant protein isolate aqueous solution. As an embodiment of the present invention, the amount ratio of the plant protein isolate to water can be 1-3 g:100 mL, specifically 2 g:100 mL; the present invention has no special limitation on the way of mixing the plant protein isolate with water, as long as the plant protein isolate can be fully dissolved in water.

[0047] After obtaining the plant protein isolate aqueous solution, the present invention adjusts the pH value of the plant protein isolate aqueous solution to 1.5-2.5, and then separates the solid and liquid to obtain a plant protein-containing liquid material. As one embodiment of the present invention, the pH value of the plant protein isolate aqueous solution is adjusted to 1.5-2.5 (specifically 2.0). The reagent used can be hydrochloric acid, and the concentration of the hydrochloric acid can be 3M; the present invention adjusts the pH value of the plant protein isolate aqueous solution to the above range, which can make the plant protein isolate acid hydrolyzed during the subsequent incubation process, which is beneficial to the subsequent preparation of plant amyloid protein fibers. As one embodiment of the present invention, the solid-liquid separation method can be centrifugation; the centrifugation temperature can be 3-6°C, specifically 4°C; the rotation speed can be 6000-10000rpm, specifically 8000rpm; the time can be 15-25min, specifically 20min.

[0048] After obtaining the plant protein-containing liquid material, the present invention performs a fiber growth treatment on the plant protein-containing liquid material to obtain the plant amyloid protein fiber. As an embodiment of the present invention, the plant amyloid protein fiber may include pea amyloid protein fiber; in the embodiment of the present invention, the plant protein isolate is pea protein isolate as an example, and pea amyloid protein fiber is finally prepared. In the present invention, according to the growth kinetics curve of the plant amyloid protein fiber, the fiber growth treatment includes a hysteresis period, a logarithmic growth period and a plateau period in sequence. The hysteresis period of the present invention specifically refers to the initial stage of amyloid protein fiber growth, at which time the protein in the natural conformation is hydrolyzed into protein monomers or short peptides under a high temperature and high acid environment. These protein monomers can be polymerized through non-covalent interactions to form oligomers rich in cross-β folding structures, which is the nucleation process in the fiber growth process; in the process of protein monomers self-assembling into oligomers, they are characterized by a thioflavin T fluorescent dye that can specifically bind to the cross-β folding structure of amyloid fibers. It is generally believed that 0 to 10% of the maximum fluorescence intensity corresponds to the hysteresis period of amyloid protein fiber growth. The logarithmic growth phase described in the present invention specifically refers to the stage of rapid growth of amyloid fibers. At this time, the oligomers grow to a certain size, and with it as a template, the protein monomers self-assemble at the ends of the oligomers through π-π interactions and hydrophobic interactions to form fibrils with a high aspect ratio. It is generally believed that 10 to 90% of the maximum fluorescence intensity corresponds to the logarithmic growth phase. The plateau phase described in the present invention specifically refers to the mature stage of amyloid fiber growth. At this time, the protein monomer concentration in the system can no longer support the rapid self-assembly of amyloid fibers, and the growth rate slows down. At this stage, the fibrils begin to influence each other and self-assemble into mature amyloid fibers by entanglement, orderly arrangement, etc. It is generally believed that more than 90% of the maximum fluorescence intensity corresponds to the plateau phase. The method for obtaining the growth kinetics curve of the pea amyloid fiber is described in detail in the following text of the present invention, which will not be repeated here. In the embodiment of the present invention, taking pea amyloid protein fiber as an example, according to its growth kinetics curve, the time range of the hysteresis phase is 0 to 5 hours, the time range of the logarithmic growth phase is 5 to 26.5 hours, and the period after 26.5 hours is the plateau phase.

[0049] In the present invention, the fiber growth treatment includes sequentially performing a first incubation treatment, an ultrasonic treatment and a second incubation treatment, wherein the first incubation treatment is performed from 0 h to the end of the logarithmic growth phase, and the first incubation treatment and the second incubation treatment are performed under non-ultrasonic conditions.

[0050] The time of the first incubation treatment of the present invention is 0h to the end of the logarithmic growth phase. As an embodiment of the present invention, the time of the first incubation treatment can further be 0h to the end of the hysteresis phase, specifically 0h (that is, the first incubation treatment is not performed at this time, and the subsequent ultrasonic treatment is directly performed). As an embodiment of the present 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 350-450rpm, specifically 400rpm.

[0051] As an embodiment of the present invention, the conditions of the ultrasonic treatment include: the temperature can be 0-4°C, further can be 0-2°C; the total time can be 1-3min, specifically can be 2min; every ultrasonic 1-3s pause 1-3s, specifically can be every ultrasonic 2s pause 2s; when the time of the first incubation treatment is 0h, the ultrasonic power density of the ultrasonic treatment is ≥2.5W / mL, further can be 2.5-4W / mL, specifically can be 2.5W / mL, 2.6W / mL, 2.7W / mL, 3W / mL, 3.5W / mL or 4W / mL; when the time of the first incubation treatment is not 0h, the ultrasonic power density of the ultrasonic treatment can be 2-4W / mL, specifically can be 2W / mL, 2.2W / mL, 2.4W / mL, 2.5W / mL, 2.6W / mL, 2.7W / mL, 3W / mL, 3.5W / mL or 4W / mL. In an embodiment of the present invention, the diameter of the horn used for the ultrasonic treatment is specifically 10mm. In the embodiment of the present invention, the effects of different ultrasonic intensities on the growth of plant amyloid protein fibers are specifically studied, and the ultrasonic intensity can be 1-30%, specifically 1%, 5%, 10%, 20% and 30%, and the corresponding actual ultrasonic power densities are 2.1766W / mL, 2.3909W / mL, 2.6588W / mL, 3.1946W / mL and 3.7304W / mL, respectively. In the embodiment of the present invention, the rated power of the ultrasonic equipment is 900W when the ultrasonic intensity is 100%.

[0052] As an embodiment of the present invention, the total time of 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 450rpm, specifically 400rpm.

[0053] As an embodiment of the present invention, the fiber growth process includes any one of the following three situations:

[0054] Case 1: The time of the first incubation treatment is 0h, and the ultrasonic power density of the ultrasonic treatment is 2.6-4W / mL. In the embodiment of the present invention, the plant protein-containing liquid material is subjected to ultrasonic treatment and second incubation treatment in sequence; the ultrasonic intensity during the ultrasonic treatment can be 10%, 20% or 30% (the corresponding actual ultrasonic power densities are 2.6588W / mL, 3.1946W / mL and 3.7304W / mL, respectively), the temperature of the ultrasonic treatment is provided by ice water, the total duration of the ultrasonic treatment is 2min, and the treatment is paused for 2s every 2s; the temperature of the second incubation treatment is 80°C, and the time is 48h; the second incubation treatment is carried out under stirring conditions, and the stirring speed is 400rpm.

[0055] Case 2: The time of the first incubation treatment is 4 to 5 hours, and the ultrasonic power density of the ultrasonic treatment is 3 to 4 W / mL. In the embodiment of the present invention, the plant protein-containing liquid material is subjected to the first incubation treatment, the ultrasonic treatment and the second incubation treatment in sequence; 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% (the corresponding actual ultrasonic power densities are 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 the treatment is paused for 2 seconds every 2 seconds; 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] Case three: The time of the first incubation treatment is 25 to 26.5 hours, and the ultrasonic power density of the ultrasonic treatment is 2 to 2.2 W / mL. In the embodiment of the present invention, the plant protein-containing liquid material is subjected to the first incubation treatment, the ultrasonic treatment and the second incubation treatment in sequence; 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% (the corresponding actual ultrasonic power density is 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 the treatment is paused for 2 seconds every 2 seconds; 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 the present invention, ultrasonic treatment (the rated power of the ultrasonic equipment is 900W, and the ultrasonic intensity is 1%, 5%, 10%, 20% and 30%) is performed at different stages (starting t0, end of the hysteresis period t0.1, and end of the growth period t0.9) during the growth process of plant amyloid protein fibers, so that the growth process and morphology thereof can be regulated. Specifically, with respect to the fiber growth process, high-intensity ultrasonic treatment (10%, 20% and 30%) at t0 significantly accelerates the fiberization process without significantly reducing the fiber yield; high-intensity ultrasonic treatment (20% and 30%) at t0.1 significantly increases the growth rate of plant amyloid protein fibers in the logarithmic growth phase; low ultrasonic intensity (1%) at t0.9 can increase the growth rate of plant amyloid protein fibers. In terms of fiber morphology, high-intensity ultrasound (10%, 20% and 30%) at t0 can significantly reduce the proportion of rigid fibers, and increase the proportion of flexible fibers 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 dialyzes and freeze-dries the resulting product system in sequence to obtain the plant amyloid protein fibers. As one embodiment of the present invention, the conditions of the dialysis include: the molecular weight cutoff of the dialysis bag can be 8 to 14 kDa; the dialysate can be water (the pH value can be specifically 2.0), specifically deionized water; the temperature is 3 to 6°C, specifically 4°C; the time can be 42 to 54h, specifically 48h; the dialysate is preferably replaced every 6h during the dialysis process. The present invention does not specifically limit the conditions for the freeze-drying, and the conditions familiar to those skilled in the art can be used.

[0059] The present invention provides plant amyloid protein fibers prepared by the preparation method described in the above technical scheme, including flexible fibers, semi-flexible fibers and rigid fibers, wherein the number percentage of flexible fibers in the plant amyloid protein fibers is 4-50%, further can be 10-45%, further can be 20-40%, and in the embodiments, specifically can be 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 number percentage of semi-flexible fibers is 10-45%, further can be 20-40%, further can be 30 ~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 embodiments; the number percentage of rigid fibers is 35-65.8%, further can be 40-56%, further can be 45-50%, 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 embodiments.

[0060] 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. As an embodiment of the present invention, the mass ratio of the chitosan matrix to the plant amyloid protein fibers in the composite film can be 2 to 4:1, specifically 3:1; the thickness of the composite film can be 0.140 to 0.160 mm. The composite film provided by the present invention can be used for preserving fresh-cut fruits and vegetables.

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

[0062] As an embodiment of the present invention, the preparation method of the chitosan solution may include the following steps: mixing chitosan and acetic acid solution (volume concentration may be 2%) in a ratio of 2 to 4 g: 100 mL (specifically 3 g: 100 mL), stirring until the chitosan is completely dissolved, and adjusting the pH value of the system to 1.8 to 2.0 with hydrochloric acid (concentration may be 3M) to obtain the chitosan solution. As an embodiment of the present invention, the plant amyloid fiber dispersion may be obtained by mixing plant amyloid fiber and water in a ratio of 0.8 to 1.2 g: 100 mL (specifically 1 g: 100 mL). As an embodiment of the present invention, the volume ratio of the chitosan solution, the plant amyloid fiber dispersion and glycerol may be 15:13 to 17:0.3 to 0.7, specifically 15:15:0.5. In an embodiment of the present invention, the mold may be a culture dish with a diameter of 9 mm; the drying temperature may be 45 to 55°C, specifically 50°C; the drying time may be 15 to 20 hours, specifically 18 hours. In the embodiment of the present invention, after drying, the obtained film material is placed in a dryer for equilibration for 24 hours to obtain the composite film.

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

[0064] As an embodiment of the present invention, the mass ratio of chitosan, plant amyloid fiber and EGCG can be 2-4:0.5-1.5:1-3, specifically 3:1:2. As an embodiment of the present invention, the concentration of plant amyloid fiber in the composite coating preservative can be 8-12 mg / mL, specifically 10 mg / mL. As an embodiment of the present invention, 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 preserving fresh-cut fruits and vegetables, for example, it can be used for preserving fresh-cut apples. The test results of the present invention show that the fiber group with a higher content of flexible fibers and semi-flexible fibers (t0.110%; t0.930%) has the best browning inhibition effect, and the b* value is significantly lower than that of the original isolated protein and the protein fiber group obtained without ultrasound.

[0065] The present invention has no special limitation on the preparation method of the composite coating preservative, and the components can be mixed evenly. In the embodiment of the present invention, the membrane liquid can be prepared by referring to the above technical scheme, and then the membrane liquid is mixed with the EGCG solution to obtain the composite coating preservative; the pH value of the EGCG solution can be 1.8 to 2.0, and the concentration and volume of the EGCG solution can ensure that the concentrations of the components in the obtained composite coating preservative meet the above requirements.

[0066] In the embodiments of the present invention, pea protein isolate is taken as an example to study the influence of ultrasonic power density and ultrasonic treatment time nodes in the fiberization process on the fiberization kinetics and final product morphology of pea protein isolate, analyze the physicochemical properties and structural differences of the original protein isolate and amyloid protein fibers before and after ultrasonic treatment, reveal the mechanism of ultrasonic regulation of pea protein isolate fiberization, and further explore the performance differences of pea amyloid protein fibers with different morphologies in protein-chitosan composite film systems and coating preservatives.

[0067] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0069] Pea protein powder (purchased from Xi'an Virgin Biotechnology Co., Ltd., with a purity of 80wt%) was mixed with deionized water at a ratio of 1g:10mL, stirred for 2h, and then the pH value of the system was adjusted to 8.0 with a 3M NaOH solution, stirred for another 2h to completely dissolve the pea protein powder, centrifuged at 8000rpm for 20min at 4°C, and the supernatant was collected; the pH value of the supernatant was adjusted to 4.5 with 3M HCl, stirred for 1h, and then centrifuged at 8000rpm for 10mi at 4°C. n, collecting the precipitate; mixing the precipitate with deionized water at a ratio of 1 g: 10 mL, adjusting the pH value of the system to 7.0 with a 3 M NaOH solution under continuous stirring, and after the precipitate is completely dissolved, dialyzing the resulting solution at 4° C. for 48 h, the molecular weight cutoff of the dialysis bag used for the dialysis is 8 to 14 kDa, the dialysis fluid used for the dialysis is deionized water, and the dialysis fluid is replaced every 6 h during the dialysis process, and finally freeze-drying the purified product obtained by dialysis to obtain the pea protein isolate.

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

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

[0072] 8.0 mg of thioflavin 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 filter membrane to obtain a ThT stock solution (0.08%, w / v), which was stored at 4°C in the dark to prevent degradation; the ThT stock solution was diluted 50 times with PBS to obtain a ThT working solution;

[0073] During the incubation process, 15 μL of sample (2%, w / v) obtained at regular intervals was mixed with 135 μL of ultrapure water with a pH value of 2.0, and 2850 μL of ThT working solution was added. The mixture was incubated at 25°C in the dark for 10 min, and then the Th content of the sample was measured using an ELISA reader at an excitation wavelength of 460 nm and an emission wavelength of 490 nm. T fluorescence intensity; the data of ThT fluorescence intensity (incubation time and fluorescence intensity) were analyzed using the AmyloFit website (http: / / www.amylofit.ch.cam.ac.uk) to obtain a pea amyloid protein fiber growth kinetic curve; according to the pea amyloid protein fiber growth kinetic curve, the time points corresponding to the three stages of pea amyloid protein fiber growth were obtained: the hysteresis period was from 0 to the time point corresponding to 10% of the maximum fluorescence intensity, the logarithmic growth period was from the time point corresponding to 10% of the maximum fluorescence intensity to the time point corresponding to 90% of the maximum fluorescence intensity, and the plateau period was from the time point corresponding to 90% of the maximum fluorescence intensity to the end of the incubation treatment; wherein, the time point corresponding to 10% of the maximum fluorescence intensity was recorded as t0.1, and the time point corresponding to 90% of the maximum fluorescence intensity was recorded as t0.9; after determining these time points, the pea amyloid protein fiber intermediate was subjected to ultrasonic treatment specifically according to the relevant time points in the subsequent Example 1.

[0074] Example 1

[0075] The method for preparing pea amyloid protein fibers comprises the following steps:

[0076] The pea protein isolate was mixed with deionized water at a ratio of 2g:100mL, and magnetic stirring was performed at 25°C for 2h to fully dissolve the pea protein isolate to obtain a pea protein isolate solution (2%, w / v); the pH value of the pea protein isolate solution was adjusted to 2.0 with 3M HCl, and the solution was centrifuged at 8000rpm for 10min at 4°C to collect the supernatant; the supernatant was placed in an 80°C water bath, and the first incubation treatment was performed at 0h (t0), 5h (end of the hysteresis period, t0.1) and 26.5h (end of the logarithmic growth period, t0.9) at a stirring speed of 400rpm, and then each group of samples (10mL) were taken and immersed in ice water, and each group of samples was subjected to ultrasonic treatment, wherein a 10mm diameter amplitude rod was used for the ultrasonic treatment, and the ultrasonic intensity of each group of samples was 2.5:1 and 2.5:1 during the ultrasonic treatment. The ultrasonic treatment was carried out at 1%, 5%, 10%, 20% and 30% (the rated power of the ultrasonic equipment was 900W with the ultrasonic intensity as 100%; the actual ultrasonic power density corresponding to each ultrasonic intensity is specifically shown in Table 1), the total ultrasonic treatment time was 2min, and the treatment was paused for 2s after each treatment, and each group of samples was recorded as t01-30% group, t0.11-30% group and t0.91-30% group respectively; after the ultrasonic treatment was completed, each group of samples was returned to an 80°C water bath, and a second incubation treatment was carried out at a stirring speed of 400rpm to obtain the pea amyloid protein fiber; wherein the total time of the first incubation treatment and the second incubation treatment was 48h, and during the second incubation treatment, samples were taken at regular intervals to determine the ThT fluorescence intensity according to the aforementioned method, and the pea amyloid protein fiber growth kinetics 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 comparison chart of the preparation process of pea amyloid protein fibers. Figure 1The lower middle part is a flowchart of preparing pea amyloid protein fibers by ultrasonic treatment, and the upper part is a flowchart of preparing pea amyloid protein fibers without ultrasonic treatment; PPI represents pea protein isolate; control t0, control t0.1, control t0.9 and control48h (t0.9F) represent the samples obtained by incubating the PPI samples at 80℃ for 0h, 5h, 26.5h and 48h, respectively. The time points are determined according to the growth kinetic curve of pea amyloid protein fibers; t0(1-30%)U, t0.1(1-30%)U and t0.9(1-30%)U represent the samples obtained by ultrasonic treatment after incubating the PPI samples at 80℃ for 0h, 5h and 26.5h, respectively. The ultrasonic intensities are 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 finally formed by t0(1-30%)U, t0.1(1-30%)U and t0.9(1-30%)U, respectively. In order to be closer to the actual production, Figure 1 As shown in the upper middle part, when ultrasonic treatment is omitted, the pea amyloid protein fiber products finally selected from the t0 group and the t0.1 group are samples corresponding to 80% of the maximum ThT fluorescence intensity, while the pea amyloid protein fiber products finally selected from the t0.9 group are samples incubated for 48 hours.

[0080] Test Example 1

[0081] 1. Growth Kinetics of Pea Amyloid Fibers Treated with Ultrasonication

[0082] Figure 2 Figure 2 is the result of the kinetic test of pea protein isolate fibrillation by ultrasonic treatment at t0, where A is the normalized kinetic curve of pea protein isolate fibrillation by ultrasonic treatment at t0, and B is the hysteresis period of the sample at different ultrasonic intensities at t0, t 1 / 2 and t growth Test result diagram, C is the maximum ThT fluorescence intensity test result diagram of pea protein isolate fiber treated with different ultrasonic intensities at t0; t 1 / 2 represents the time required to reach half of the maximum ThT fluorescence intensity, t growth The results showed that the fibrosis process of pea protein isolate treated with ultrasound at t0 showed significant differences. Specifically, when the ultrasound intensity was ≥10% (t010%, 20% and 30%), the fibrosis process was significantly accelerated compared with the control group, which was consistent with the Figure 2 The shortened lag phase and half-maximum fluorescence time (t 1 / 2). On the contrary, the fibrosis process at t01% and t05% ultrasound intensities slowed down, indicating that high-power density ultrasound treatment at t0 can effectively promote the fibrosis process. This phenomenon may be attributed to the fact that high-power ultrasound promotes the conformational unfolding of pea protein molecules before heating, reduces the energy threshold required for subsequent protein acid hydrolysis, and then promotes the "nucleation" process of self-assembly between protein molecules, ultimately accelerating the fibrosis process. Figure 2 As shown in Figure C, when the ultrasonic power increases, the maximum ThT fluorescence intensity of the samples in the t0 group decreases slightly, but there is no significant difference with the control group, which shows that ultrasound has little effect on the fibrillation degree of pea amyloid protein. The maximum fluorescence intensity of t01% is slightly higher than that of the control group (P>0.05), indicating that more fibers are generated. In summary, the use of different powers of ultrasound at t0 can regulate the fibrillation process of pea protein, and high-intensity ultrasound accelerates the fibrillation process without significantly reducing the yield of fibers.

[0083] Figure 3 The results of the kinetic test of pea protein isolate fibrillation by ultrasonic treatment at t0.1 are shown in Figure 1, where A is the normalized kinetic curve of pea protein isolate fibrillation by ultrasonic treatment at t0.1, and B is the hysteresis period of the sample at different ultrasonic intensities at t0.1, t 1 / 2 and t growth The test result diagram, C is the maximum Th T fluorescence intensity test result diagram of pea amyloid protein fibers treated with different ultrasonic intensities at t0.1. The results show that the t0.120% group and the t0.130% group present unique kinetic characteristics. Specifically, the initial stage of their growth curves is accompanied by a longer hysteresis period, and then when entering the logarithmic growth phase, the growth rate increases significantly, especially the t0.130% group. This phenomenon can be attributed to the acid hydrolysis and nucleation process during the hysteresis period, during which protein molecules are initially assembled into oligomers. These protein oligomers can serve as templates for fiber growth and can elongate rapidly during the logarithmic growth phase. For samples treated with 20% and 30% high ultrasonic intensities, the ultrasound applied at the end of the hysteresis period may have triggered the deconstruction of the oligomers, and the oligomers need to further elongate before entering the growth phase, thereby increasing the time required for nucleation and prolonging the hysteresis period. However, the broken oligomers can provide more active ends in the subsequent fiber growth process, significantly accelerating the growth rate in the logarithmic growth phase. As shown in 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 obvious effect on the fiber conversion rate.

[0084] Figure 4The figure is the test result of the fibrillation kinetics of pea protein isolate treated with ultrasound at t0.9, where A is the normalized fibrillation kinetics curve of pea protein isolate treated with ultrasound at t0.9, and B is the test result of the maximum ThT fluorescence intensity of pea amyloid protein fibers treated with different ultrasound intensities at t0.9. The results show that since the fibrillation reaction has entered the saturation stage, the difference in the fiber growth kinetics of this group of samples after ultrasound treatment is small. Among them, the fiber growth rate of the t0.91% sample is slightly accelerated compared with the control group, and the maximum ThT fluorescence intensity is slightly increased (P>0.05), indicating that the fiber conversion rate is slightly improved. This phenomenon can be attributed to the partial fragmentation of the fiber structure caused by ultrasound treatment. These broken fiber fragments serve as nucleation templates, which accelerate the fiber self-assembly process to a certain extent. For other samples in the t0.9 group, as the ultrasound intensity is further increased, although more fiber fragments are produced to increase the number of fiber ends in the nucleation-growth process, the concentration of free protein that can participate in fiber growth in the system is low at this time, so that the fiber growth rate of these samples finally shows a slowing trend. Therefore, the optimization of ultrasound intensity needs to balance the generation of fiber fragments and the free protein concentration to maximize the growth efficiency of pea amyloid protein fibers.

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

[0086] In order to quantitatively analyze the regulatory effect of ultrasonic treatment on the morphology of pea amyloid protein fibers, this test example uses FiberApp to perform statistical analysis on the TEM images of pea amyloid protein fibers. Specifically, the pea amyloid protein fiber sample was diluted with ultrapure water with a pH value of 2.0 to a protein concentration of 0.1 mg / mL, and then the diluted sample was dropped onto a 300-mesh copper grid with a carbon support film, stained with 5 μL of 2% (w / v) phosphotungstic acid for 30 seconds, and after drying in the air, the sample was observed at a voltage of 80 kV and TEM images were captured at a magnification of 3000 times; at least 3 representative TEM images were selected for each sample and analyzed using FiberApp. Each sample had a data set of at least 100 fibers, based on which the contour length (Lc) and persistence length (Lp) of each fiber were obtained, and then the contour length distribution was obtained. If the order of magnitude of Lc of a fiber is less than the order of magnitude of Lp, it is judged to be a rigid fiber; if the order of magnitude of Lc of a fiber is equal to the order of magnitude of Lp, it is judged to be a semi-flexible fiber; if the order of magnitude of Lc of a fiber is greater than the order of magnitude of Lp, it is judged to be a flexible fiber. Refer to the following formula for specific judgment:

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

[0088] Among them, M<0 means rigid fiber; M=0 means semi-flexible fiber; M>0 means flexible fiber.

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

[0090] Figure 5 The TEM images of pea amyloid fiber samples, where t0 represents the start of the fibrillation process (0h), t0.1 represents the end of the hysteresis 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 the pea amyloid fiber samples formed after ultrasonic treatment at t0, t0.1, and t0.9, respectively. The results show that the pea amyloid fibers in all samples exhibit diverse morphological characteristics, covering three categories: rigid, semi-flexible, and flexible fibers.

[0091] Figure 6 The figure is the contour length distribution diagram of pea amyloid protein fibers obtained by ultrasonic treatment, wherein A is the contour length distribution diagram of the fiber samples at different heating times, B is the contour length distribution diagram of the fiber obtained by ultrasonic treatment and incubation at the beginning of the hysteresis period (0h), C is the contour length distribution diagram of the fiber obtained by ultrasonic treatment and incubation at the end of the hysteresis period (5h), and D is the contour length distribution diagram of the fiber obtained by ultrasonic treatment and incubation at the end of the growth period (26.5h).

[0092] Figure 7 The figure is a statistical diagram of the number ratio of flexible fibers, semi-flexible fibers and rigid fibers in the pea amyloid protein fibers obtained by ultrasonic treatment, wherein A is a statistical diagram of the number ratio of rigid fibers, semi-flexible fibers and flexible fibers in the fibers obtained by ultrasonic treatment at the beginning of the hysteresis period (0h) and subsequent incubation, B is a statistical diagram of the number ratio of rigid fibers, semi-flexible fibers and flexible fibers in the fibers obtained by ultrasonic treatment at the end of the hysteresis period (5h) and subsequent incubation, and C is a statistical diagram of the number ratio of rigid fibers, semi-flexible fibers and flexible fibers in the fibers obtained by ultrasonic treatment at the end of the exponential growth period (26.5h) and subsequent incubation. The specific results are listed in Table 2.

[0093] Table 2 The ratio of rigid fibers, semi-flexible fibers and flexible fibers in pea amyloid protein fibers

[0094]

[0095]

[0096] The above results show that during the fibrillation process, the contour length of pea amyloid protein fibers increases with the increase of reaction time. However, ultrasonic treatment at t0, t0.1 and t0.9 can significantly shorten the contour length of the fibers. This is attributed to the fiber fragmentation caused by ultrasonic treatment, which provides more active ends for fiber elongation, but also limits the growth of the overall contour length. Figure 7 The ultrasonic treatment also significantly changed the proportion of fibers with different morphologies. For the samples treated with ultrasound at time t0, the proportion of rigid fibers decreased significantly under high ultrasonic power, while the proportion of flexible fibers and semi-flexible fibers increased significantly, especially at t010%F. Compared with 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%; while at lower ultrasonic power, the proportion of rigid fibers increased slightly. All intensities of ultrasound in the t0.1 group significantly reduced the proportion of rigid fibers, but the proportion of flexible fibers remained at a low level, except for t0.110%F. Compared with 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, with the increase of ultrasonic power, the proportion of flexible fibers increased significantly, and the proportion of flexible fibers in t0.930%F increased from 8.67% to 47.00% compared with t0.9 F, while the proportion of rigid fibers decreased significantly, and the proportion of rigid fibers in t0.930%F decreased from 66.00% to 35.00% compared with t0.9 F.

[0097] Example 2

[0098] The method for preparing a pea amyloid protein fiber-chitosan composite film comprises the following steps:

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

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

[0101] Take 15 mL of the pea amyloid protein fiber dispersion and mix it with 15 mL of the chitosan solution, add 0.5 mL of glycerol, place the obtained mixed liquid in a 60°C water bath and stir for 30 minutes, then place it in an ultrasonic cleaner for 30 minutes to fully degas, and obtain a membrane liquid; pour 30 mL of the membrane liquid into a culture dish with a diameter of 9 mm, dry it in a 50°C forced air drying oven for 18 hours, and then place it in a dryer for equilibration for 24 hours to obtain the pea amyloid protein fiber-chitosan composite film.

[0102] A control group and a blank group were set up at the same time; 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 value of 2.0 instead of the pea amyloid protein fiber dispersion; other operations were the same as the steps in Example 2.

[0103] Among them, the group naming method of the pea amyloid protein fiber-chitosan composite film prepared using pea amyloid protein fiber is the same as the naming method of the pea amyloid protein fiber, which is respectively recorded as t0 / t0.1 F, t010%F, t0.110%F, t0.9 F and t0.930%F; the composite film prepared using unfibrillated pea protein isolate is recorded as control t0 (i.e., control group); the film prepared in the blank group is recorded as CS.

[0104] The thickness of each composite film is shown in Table 3.

[0105] Table 3 Thickness of composite films

[0106] Composite 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 film mechanical properties

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

[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 results of the tensile strength and elongation at break of the film are shown in the figure on the left, and the elongation at break of the film is shown on the right. The results show that the tensile strength and elongation at break of the CS (chitosan film) group are both poor, 3.32MPa and 64.93% respectively, which indicates that the mechanical properties of the film system are poor when chitosan alone is used as the film-forming matrix. After adding pea protein isolate to the chitosan film system (control t0 group), the tensile strength and elongation at break of this composite film system are slightly improved compared with CS, but there is no significant difference. This may be because the natural conformation pea protein isolate can produce weak intermolecular interactions with chitosan molecules and cannot stack with each other to form a network structure. After adding pea amyloid fiber to the chitosan film system, the tensile strength and elongation at break of the composite film system were significantly improved, among which the t010%F, t0.110%F, control48h and t0.930%F groups were significantly improved compared with CS, but the mechanical properties of the t0 / t0.1 F group were not significantly improved compared with CS. Further analysis of this result showed that the pea amyloid fiber samples added to the t0 / t0.1 F and control 48h groups were fiber samples that had not been ultrasonically treated. The heating time of t0 / t0.1 F was shorter than that of control 48h, and the contour length of the formed fiber sample was also shorter (such as Figure 6 As shown), the rigidity and flexibility of the two groups of fiber samples are statistically similar, which means that under the premise of similar rigidity and flexibility of the fibers, the contour length of the fiber samples is positively correlated with the mechanical properties of the composite film. This may be because fiber samples with longer contour lengths can form a network structure with more cross-linking points than fiber samples with shorter contour lengths, and the mechanical properties of the network structure are positively correlated with the rigidity and flexibility of the macromolecules and the number of cross-linking points. Therefore, fibers with longer contour lengths will have more advantages in mechanical properties. On the other hand, by comparing the three groups of 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 with ultrasound are better than those of t0 / t0.1 F not treated with ultrasound. After the pea protein isolate is treated with ultrasound, the contour length of the fiber sample formed is reduced, but the proportion of flexible fibers and semi-flexible fibers contained is significantly increased (as shown in Figure 2). Figure 7 This indicates that the rigidity and flexibility of the fiber sample have a significant effect on the mechanical properties of the composite film system. Specifically, when the proportion of flexible fibers or semi-flexible fibers in the fiber sample is high, the fibers are more likely to generate intermolecular entanglements, which also increases the number of cross-linking points in the network structure, thereby improving the mechanical properties of the composite film system.

[0112] Fig. 9The figure is the result of the correlation analysis between the mechanical properties of the film and the rigidity and flexibility of the fiber. Specifically, the rigidity and flexibility of the added fiber sample are correlated with the tensile strength and elongation at break of the formed composite film. The results show that the ratio of rigid fibers in the fiber sample is negatively correlated with the two indicators of the mechanical properties of the composite film, and the ratio of semi-flexible fibers to flexible fibers is positively correlated with the two indicators of the mechanical properties of the composite film. Among them, compared with t010%F and t0.110%F, the flexible fiber content in t010%F and t0.110%F treated with ultrasound is higher, while the rigid fiber content is lower. The mechanical strength of the composite film prepared with these two groups of fiber samples is higher than that of t0 / t0.1F.

[0113] Example 3

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

[0115] A film liquid was prepared by referring to the method of Example 2, and 375 μL of epigallocatechin gallate (EGCG) solution (concentration of 20 mg / mL, pH value of 2.0) was added to the film liquid so that the mass concentration ratio of pea amyloid protein fiber to EGCG in the system was 20:1, and the composite coating preservative was obtained.

[0116] Test Example 3

[0117] 1. Evaluation of Antioxidant Activity in Vitro

[0118] This test example uses DPPH to determine the antioxidant activity of pea protein isolate and pea amyloid protein fiber samples. First, DPPH was dissolved in anhydrous ethanol to obtain a DPPH working solution with a concentration of 0.1mM. Take 200μL of pea protein isolate dispersion (20mg / mL, pH value is 2.0, control group) or pea amyloid protein fiber dispersion (20mg / mL, pH value is 2.0, experimental group) and mix it with 800μL DPPH working solution. The blank group is 200μL ultrapure water with a pH value of 2.0 mixed with 800μL DPPH working solution; the mixed liquid was kept at 25°C in the dark for 30 minutes, and the absorbance of the sample at 517nm was determined using an enzyme marker. The calculation formula for the DPPH free radical scavenging activity of the sample is as follows:

[0119]

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

[0121] Fig.10The figure shows the test results of DPPH radical scavenging rate of pea protein isolate and pea amyloid fiber. The results show that after pea protein isolate is prepared into pea amyloid fiber, its DPPH radical scavenging rate is significantly improved. This may be because after the natural conformation of pea protein isolate is converted into pea amyloid fiber, more cysteine ​​residues located inside pea protein isolate are exposed, and cysteine ​​residues have certain antioxidant activity. Among different pea amyloid fiber samples, the DPPH radical scavenging rates of t0.9 F group and t0.930% F group are higher, which are 30.14±0.88% and 32.23±1.86% respectively, which indicates that the longer the incubation time of pea amyloid fiber, the more cysteine ​​residues will be exposed. The higher antioxidant activity of pea amyloid fiber enables it to be used for the preservation of fresh-cut fruits that are prone to browning.

[0122] 2. Application of edible coating for fresh-cut apples

[0123] The composite coating preservative is used as a coating to preserve fresh-cut apples, specifically as follows:

[0124] Fresh apples were cleaned, peeled and cut into strips of uniform shape, and then the cut apples were immediately immersed in the composite coating preservative, taken out after waiting for 30 seconds, and placed in an environment with a temperature of 25°C and a relative humidity of 75%, and photographed at intervals of 12 hours. After 48 hours, the colorimetry of all fresh-cut apple samples was measured.

[0125] A blank group and a control group were set up at the same time; the blank group was a fresh-cut apple sample that was not treated with a coating; the control group (CS) was a chitosan control group, specifically, when preparing the composite coating preservative, 15 mL of ultrapure water with a pH value of 2.0 was used instead of the pea amyloid protein fiber dispersion, and then the composite coating preservative was used to coat and preserve the fresh-cut apples according to the above method.

[0126] Fig.11 The results of storage of fresh-cut apples show that after the fresh-cut apples were coated for preservation, their shapes were relatively consistent before and after storage, and the browning rate was inhibited to varying degrees. When the fresh-cut apples were placed for 12 hours, the blank group had already undergone obvious browning, while the browning of each group of samples with pea amyloid protein fibers was inhibited, with little difference from the samples at 0 hours. As the storage time of fresh-cut apples increased, the trend became more obvious: the CS group was better than the blank group, the controlt0 group was slightly better than the CS group, and the group with pea amyloid protein fibers added was better than the other groups, which was the same as the trend of the antioxidant capacity of the samples. In addition, compared with pea protein isolate, pea amyloid protein fibers have a stronger ability to bind EGCG, so they have a stronger anti-browning ability.

[0127] Table 4 shows the chromaticity values ​​of fresh-cut apples after 48 hours of storage, where L* represents the brightness of the sample color; a* represents the red-green color of the sample color; b* represents the yellow-blue color of the sample color. The higher the b* value, the more yellow the sample color; △E represents the difference between the sample color and the standard color. The results show that the b* size distribution of the groups with added pea protein isolate and pea amyloid protein fiber samples is as follows: control t0>t010%F>t0 / t0.1 F>t0.9 F>t0.930%F>t0.110%F, which is similar to the size relationship of the antioxidant activity of the corresponding pea protein isolate and pea amyloid protein fiber samples (such as Fig.10 This indicates that the pea amyloid protein fiber samples with high antioxidant activity can effectively inhibit the browning of fresh-cut apples. Among them, the longer the incubation time, the stronger the anti-browning ability of the pea amyloid protein fiber samples (t0 / t0.1 F<t0.9 F,P> 0.05), the longer the incubation time, the longer the fiber outline length, so it can load more EGCG, and play a certain role in sustained release during storage. The difference in fiber morphology also affects its anti-browning ability. The two groups of t0.110%F and t0.930%F with higher flexible fiber content obtained after ultrasonic treatment are higher than their respective fiber control groups (t0.110%F is higher than t0 / t0.1 F, t0.930%F is higher than t0.9 F), especially t0.110%F has a significant difference compared with the control group, indicating that the samples with a high proportion of flexible fibers and semi-flexible fibers can load more EGCG, and due to the high proportion of flexible fibers and semi-flexible fibers, the network structure formed between the fibers is more compact, and the sustained release effect of EGCG is also improved.

[0128] Table 4 Chromaticity values ​​of fresh-cut apples after 48 h storage

[0129] Sample Type L* a* b* △E blank <![CDATA[81.01±2.47 ab ]]> <![CDATA[13.95±1.37 a ]]> <![CDATA[41.61±0.58 a ]]> <![CDATA[92.15±2.20 a ]]> CS <![CDATA[70.83±7.22 b ]]> <![CDATA[11.78±0.77 ab ]]> <![CDATA[38.07±0.64 b ]]> <![CDATA[81.34±5.99 b ]]> controlt0 <![CDATA[75.29±4.35 b ]]> <![CDATA[10.13±1.09 b ]]> <![CDATA[35.69±1.76 b ]]> <![CDATA[84.01±3.17 ab ]]> t0 / t0.1F <![CDATA[78.86±4.36 ab ]]> <![CDATA[9.05±0.71 bc ]]> <![CDATA[34.19±1.06 b ]]> <![CDATA[86.44±4.12 ab ]]> t010%F <![CDATA[81.44±0.81 ab ]]> <![CDATA[9.99±0.50 bc ]]> <![CDATA[34.96±0.30 b ]]> <![CDATA[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 is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing plant amyloid fiber, comprising the following steps: Mixing the plant protein isolate with water to obtain a plant protein isolate aqueous solution; The pH value of the plant protein isolate aqueous solution is adjusted to 1.5 to 2.5, followed by solid-liquid separation to obtain a plant protein-containing liquid material; The plant protein-containing liquid material is subjected to a fiber growth treatment to obtain the plant amyloid protein fiber; According to the growth kinetics curve of the plant amyloid protein fiber, the fiber growth process includes a hysteresis phase, a logarithmic growth phase and a plateau phase in sequence; The fiber growth treatment includes a first incubation treatment, an ultrasonic treatment and a second incubation treatment in sequence, wherein the first incubation treatment is performed from 0 h to the end of the logarithmic growth phase, and the first incubation treatment and the second incubation treatment are performed under non-ultrasonic conditions; when the first incubation treatment is performed for 0 h, the ultrasonic power density of the ultrasonic treatment is ≥2.5 W / mL.

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

3. The preparation method according to claim 2, characterized in that: The conditions of the ultrasonic treatment include: a temperature of 0 to 4°C, a total time of 1 to 3 minutes, and a pause of 1 to 3 seconds after each ultrasonic treatment of 1 to 3 seconds; when the first incubation treatment time is 0 hours, the ultrasonic power density of the ultrasonic treatment is 2.5 to 4 W / mL; when the first incubation treatment time is not 0 hours, the ultrasonic power density of the ultrasonic treatment is 2 to 4 W / mL.

4. The preparation method according to claim 3, characterized in that: The temperatures 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.

5. The preparation method according to claim 4, characterized in that: The first incubation treatment time is from 0 h to the end of the hysteresis period.

6. The preparation method according to any one of claims 1 to 5, characterized in that: Counted from the beginning of the fiber growth treatment, the time range of the lag phase is 0 to 5 hours, and the time range of the logarithmic growth phase is 5 to 26.5 hours.

7. The preparation method according to claim 6, characterized in that: The fiber growth process includes any one of the following three situations: Case 1: The time of the first incubation treatment is 0 h, and the ultrasonic power density of the ultrasonic treatment is 2.6-4 W / mL; Case 2: The time of the first incubation treatment is 4 to 5 hours, and the ultrasonic power density of the ultrasonic treatment is 3 to 4 W / mL; Case 3: The time of the first incubation treatment is 25 to 26.5 h, and the ultrasonic power density of the ultrasonic treatment is 2 to 2.2 W / mL.

8. The plant amyloid protein fibers prepared by the preparation method according to any one of claims 1 to 7 include flexible fibers, semi-flexible fibers and rigid fibers, wherein the number percentage of flexible fibers in the plant amyloid protein fibers is 4 to 50%, the number percentage of semi-flexible fibers is 10 to 45%, and the number percentage of rigid fibers is 35 to 65.8%. 9 . 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 according to claim 8 .

10. A composite coating preservative, comprising chitosan, plant amyloid protein fibers, epigallocatechin gallate and a solvent, wherein the plant amyloid protein fibers are the plant amyloid protein fibers according to claim 8.

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