Method for preparing nanofiber from whey protein isolate and application in improving stability of beta-carotene powder

Abstract

The application discloses a method for preparing nanofibers from whey protein isolate and application of the nanofibers in improving the stability of beta-carotene powder, and comprises the following steps: (1) hydrolyzing whey protein isolate by pepsin to obtain a pepsin hydrolysate; (2) preparing a nanofiber solution by heating the pepsin hydrolysate under an acidic condition; and (3) then adjusting the pH value, mixing the nanofiber solution with a beta-carotene ethanol solution, and rotating to evaporate ethanol to obtain an embedding material.

Description

Technical Field

[0001] This invention relates to a method for preparing nanofibers, and more particularly to a method for preparing nanofibers from whey protein isolate and its application in improving the stability of β-carotene powder. Background Technology

[0002] To improve protein functionality, enzymatic hydrolysis has been widely used to modify protein structures, such as solubility, emulsification, gelation, and foaming properties, and releases amphiphilic structures during hydrolysis. In recent years, amphiphilic structures have self-assembled into various nanostructures, such as nanotubes, nanofibers, and microparticles. These nanostructures possess intrinsic bioactivity and sustained safety. By incorporating hydrophobic components into nanostructures, they can serve as natural encapsulation materials for food matrices and can also be used to deliver bioactive substances. Therefore, nanofiber materials broaden the application of food proteins in functional foods, nutritional supplements, and pharmaceuticals.

[0003] Proteins have a wide range of functions and can form different structures under varying conditions, with shapes ranging from linear to spherical. Research has found that many globular proteins, such as soy protein, can self-assemble into amyloid fibrous polymers under high-temperature heating at low pH and ionic strength. Amyloid fibrils are rich in small-diameter and long (nm~μm) β-sheet filamentous protein structures and have wide applications in food, medicine, cosmetics, and other fields.

[0004] β-carotene is a secondary metabolite synthesized by plants and is commonly used as a food coloring additive in the food industry. β-CE is a fat-soluble nutrient that promotes health benefits. Furthermore, the CE molecule contains unsaturated conjugated double bonds, making it highly susceptible to environmental influences.

[0005] Therefore, how to utilize WPI to prepare self-assembled nanofibers and encapsulate β-CE is a technical problem that needs to be solved. Furthermore, how to present a good nanofiber solution system with uniform distribution and high aspect ratio, how to achieve better adsorption of β-CE, and how to extend the shelf life of the WPIF / β-CE lyophilized powder are even more important technical problems to be solved. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for preparing nanofibers from whey protein isolate, comprising the following steps:

[0007] (1) Whey protein isolate was hydrolyzed by pepsin to obtain a protease hydrolysate. (2) The protease hydrolysate was heated under acidic conditions to prepare a nanofiber solution.

[0008] Furthermore, the whey protein isolate is whey protein isolate 80 or higher.

[0009] Furthermore, the enzymatic hydrolysis time is 1.5-3.5 hours, preferably around 2 hours.

[0010] Furthermore, the enzymatic hydrolysis temperature is 35-45℃, pH 2-3, preferably 40℃, pH 2.5.

[0011] Furthermore, the enzyme is inactivated after hydrolysis.

[0012] Furthermore, in step (2), the acidic conditions are pH 1.0-3.0, preferably pH 2.0.

[0013] Furthermore, the heating temperature in step (2) is 60-95℃, preferably 90℃.

[0014] Furthermore, the heating treatment time in step (2) is 5h-20h, preferably 10h.

[0015] Furthermore, the nanofibers are starch-like nanofibers.

[0016] Furthermore, the diameter of the nanofibers is approximately 20-200 nm.

[0017] Furthermore, the β-sheet content in the nanofibers is higher than 30%.

[0018] The present invention also provides an amyloid whey protein nanofiber, which is prepared by the above method. The nanofiber has a diameter of about 20-200 nm and a β-sheet content of more than 30%.

[0019] This invention also provides an application of nanofibers to improve the stability of β-carotene powder.

[0020] Furthermore, the pH of the nanofiber solution was adjusted, and then mixed with a β-carotene ethanol solution. The ethanol was then rotary evaporated to obtain the encapsulated material.

[0021] Further, adjust the pH to 2.5-4, further, 3-3.5, further, 3.2±0.02.

[0022] Furthermore, the content of β-CE is between 0.1% and 1 wt%.

[0023] Furthermore, the mixing temperature is 30-70℃, preferably 50℃; the mixing time is 0.5-3h, preferably 1h.

[0024] Furthermore, the temperature for preparing the β-carotene ethanol solution is 30-70℃, preferably 50℃; the stirring time is 0.5-3h, preferably 1h.

[0025] The present invention also provides a method for preparing whey protein isolate nanofibers and β-carotene encapsulated material, comprising (1) hydrolyzing whey protein isolate with pepsin to obtain a protease hydrolysate, (2) heating the protease hydrolysate under acidic conditions to prepare a nanofiber solution, (3) adjusting the pH, mixing with a β-carotene ethanol solution, and rotary evaporating the ethanol to obtain the encapsulated material.

[0026] The present invention also provides whey protein isolate nanofibers and β-carotene encapsulated material, which are prepared by the above method.

[0027] Beneficial effects: The whey protein nanofibers prepared by this invention have a diameter of over 200 nm, are relatively uniformly distributed and have a high aspect ratio, and have a β-sheet content of over 30%; by encapsulating β-carotene, they exhibit a longer shelf life.

[0028] Terminology Explanation:

[0029] Whey protein isolate, also known as whey protein isolate, is a protein extracted from milk. It is characterized by high nutritional value, easy digestibility and absorption, and the presence of various bioactive components, making it a recognized high-quality protein supplement for the human body. Whey protein is extracted from milk, and the bioactive compounds it contains, such as α-lactalbumin, β-lactoglobulin, lactoferrin, and immunoglobulins, can regulate the body's immunity. In this article, WPI is an abbreviation for whey protein isolate.

[0030] Whey protein isolate nanofibers are nanofibers prepared from whey protein isolate. In this article, WPIF is an abbreviation for whey protein isolate nanofibers.

[0031] β-carotene is a secondary metabolite synthesized by plants. In the food industry, it is commonly used as a food additive for coloring. In this article, β-CE is the abbreviation for β-carotene, a fat-soluble nutrient food that promotes health functions. Attached Figure Description

[0032] Figure 1 The effects of different enzymes (trypsin, neutral protease, pepsin, and papain) on the degree of hydrolysis of 3.0% WPI solution at different hydrolysis times (1, 2, 3, 4, and 5 h);

[0033] Figure 2 The effect of proteases (trypsin, pepsin, papain, and neutral protease) on the Th t value of WPI solution self-assembled solution under different hydrolysis times (1, 2, 3, 4, and 5 h) pretreatment was investigated.

[0034] Figure 3 The structure of WPI without enzymatic hydrolysis after heat treatment under acidic conditions for 10 hours;

[0035] Figure 4 The structures of WPI after pretreatment with trypsin at different hydrolysis times (1, 2, 3, 4 and 5 h) followed by heat treatment under acidic conditions for 10 h.

[0036] Figure 5 The structures of WPI after pretreatment with pepsin for different hydrolysis times (1, 2, 3, 4 and 5 h) followed by heat treatment under acidic conditions for 10 h.

[0037] Figure 6 The structures of WPI after pretreatment with different hydrolysis times (1, 2, 3, 4 and 5 h) by papain enzymatic hydrolysis followed by heat treatment under acidic conditions for 10 h.

[0038] Figure 7 The structures of WPI after pretreatment with neutral protease for different hydrolysis times (1, 2, 3, 4 and 5 h) followed by heat treatment under acidic conditions for 10 h.

[0039] Figure 8 The effects of four different proteases (trypsin, pepsin, papain, and neutral protease) on the zeta potential of WPIF solution under different hydrolysis times (1, 2, 3, 4, and 5 h) were investigated.

[0040] Figure 9 The effects of pretreatment with four proteases (neutral protease, pepsin, papain and trypsin) at different hydrolysis times (1, 2, 3, 4 and 5 h) on the free sulfhydryl content of WPIF solution were investigated.

[0041] Figure 10 Storage stability of WPIF / β-CE after 2 h of pretreatment with trypsin, pepsin, papain and neutral protease. Detailed Implementation

[0042] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0043] 3g of whey protein isolate was dissolved in distilled water to prepare a 3.0% (w / v) solution. The solution was stirred at room temperature for 3 hours to achieve complete hydration. Four enzymes were added to the protein solution at an enzyme / substrate ratio of 1 / 30, and the solution was adjusted to the optimal hydrolysis conditions for the four enzymes: pepsin (40℃, pH 2.5), trypsin (55℃, pH 7.5), neutral protease (40℃, pH 7.5), and papain (55℃, pH 6.0), with all four enzymes having the same activity. The enzymes were added to the whey protein isolate solution and enzymatically hydrolyzed in an incubator. To investigate the effect of the degree of enzyme hydrolysis on the self-assembly of nanofibers, samples were taken at intervals of 1, 2, 3, 4, and 5 hours. Subsequently, the enzymes were inactivated at 90℃ for 10 minutes and immediately cooled in ice water to terminate the reaction. To prepare nanofibers, the pH of the protein hydrolysate was adjusted to 2.0 using 6 mol / L HCl and 1 mol / L HCl. The hydrolysate was then transferred to a 50 mL threaded-sealed tube and heated in a constant-temperature water bath at 90 °C for 10 h. After the reaction, the tube was immediately cooled in ice water for 20 min to terminate the reaction, and the sample was stored at 4 °C for future analysis. Untreated 3.0% (w / v) whey protein isolate was used as a control group.

[0044] The effect of adding four proteases and different hydrolysis times on the degree of hydrolysis (DH) of whey protein isolate was analyzed by determining the content of free amino groups using the OPA method. The OPA solution was prepared as follows: 3.81 g sodium tetraborate, 100 mg sodium dodecyl sulfate (SDS), 80 mg OPA (dissolved in 2 mL anhydrous ethanol), and 88 mg dithiothreitol (DTT). 400 μL of a 100-fold diluted WPI hydrolysate was added to 3 mL of the OPA solution, and the reaction was carried out in the dark for 2 min. The absorbance was measured at 340 nm using a spectrophotometer.

[0045]

[0046] In the formula: DH represents the degree of hydrolysis, h1 represents the concentration of hydrolyzed amino acids, h0 represents the concentration of unhydrolyzed amino acids, and htot represents the total amino acid concentration of WPI (meqv / g protein), which is 7.8 in this experiment.

[0047] Compared to natural whey powder, the degree of enzyme hydrolysis is typically controlled between 10% and 90%. The effects of different enzymes (trypsin, neutral protease, pepsin, and papain) on the degree of hydrolysis of 3.0% WPI solution at different hydrolysis times (1, 2, 3, 4, and 5 hours) are shown below. Figure 1 As shown, the degree of protein hydrolysis increases sharply in the initial stage (0-3h) and tends to stabilize as the hydrolysis time increases (4-5h), which may be related to the saturation of the number of enzyme-substrate binding regions. Figure 1The study also demonstrated that the maximum degree of hydrolysis of WPI reached 80.0% with trypsin after 5 hours of hydrolysis, while the maximum degrees of hydrolysis for neutral protease, pepsin, and papain were 61.6%, 10.0%, and 6.3%, respectively. Compared with the other three enzyme treatments, trypsin exhibited stronger hydrolytic ability for WPI than neutral protease, pepsin, and papain.

[0048] The β-sheet content was determined using the ThT fluorescence assay. The ThT stock solution was prepared as follows: 2.8 g NaCl, 0.07 g KCl, 0.504 g disodium phosphate, and 0.084 g potassium monophosphate were dissolved in 350 mL distilled water to obtain a 10 mmol / L (pH = 7) phosphate buffer solution (PBS). Then, 280 mg ThT was dissolved in 350 mL PBS and filtered through a 0.22 μm filter. The ThT stock solution was stored at 4 °C protected from light for future use. The ThT stock solution was diluted with distilled water at a ratio of 1:100 to obtain the ThT working solution. 24 μL of the nanofiber self-assembled sample was mixed with 4 mL of the ThT working solution and vortexed for 1 min. The ThT fluorescence of the sample was detected using a fluorescence spectrophotometer under the following conditions: excitation wavelength 365 nm, emission wavelength scanning range 400-600 nm (slit width = 10 nm).

[0049] β-sheets are a major secondary structure in the self-assembly of proteins to form nanofibers; therefore, β-sheet content is considered a fundamental parameter for determining nanofiber formation. Thioflavin T fluorescent dye can bind to the β-sheets of protein secondary structures without affecting the aggregation kinetics of nanofibers. Figure 2 As shown, the fluorescence intensity increased significantly when the enzyme hydrolysis time was 1 hour, which may be due to the aggregation of proteins into a certain number of nanofibers.

[0050] The applicant unexpectedly discovered that the enzyme with the best β-sheet effect was not trypsin, which has the highest degree of hydrolysis, but pepsin. The Tht value reached its maximum after 2 hours of hydrolysis, and the fluorescence intensity of the four enzymes was in the order of pepsin > papain > neutral protease > trypsin. However, after 2 hours, the Tht value of the WPIF solution decreased slightly with increasing hydrolysis time, suggesting that excessive hydrolysis may damage the β-sheet structure of the nucleus. The Tht values ​​of the pepsin, papain, and neutral protease groups decreased significantly with increasing hydrolysis time; the Tht value of the trypsin pretreatment group showed a slight change, with its maximum value lower than that of the other three enzyme treatments. In summary, the following conclusions can be drawn: both enzyme type and hydrolysis time affect β-sheet formation; excessive enzymatic hydrolysis reduces the β-sheet content, which is detrimental to nanofiber formation; the pretreatment group with pepsin hydrolysis for 2 hours showed the highest fluorescence intensity, and it can be inferred from the experimental results that the solution contained the highest nanofiber content under these conditions.

[0051] Electron microscopy: The sample solution was diluted to 0.3% (w / v) with HCl at pH 2. One drop of the diluted sample was placed on a copper grid, dried, and then negatively stained with 2.0% phosphotungstic acid solution. Excess solution was blotted off with filter paper, and the sample was dried at room temperature for 15 min. The structure of the WPIF sample was observed under a transmission electron microscope at 100 kV.

[0052] When a whey protein (WPI) hydrolysis solution at pH 2.0 is heated to 90°C, the hydrolyzed peptides and monomers self-assemble into nanofibers via hydrophobic forces. The microstructure of the protein nanosystems was observed using transmission scanning electron microscopy (TEM), with TEM images of the samples magnified up to 8000x. The effects of different proteases (trypsin, neutral protease, papain, and pepsin) on the formation of whey protein nanofibers at different treatment times (1, 2, 3, 4, and 5 h) are shown below. Figure 3 As shown. From Figure 3 As can be seen, the aggregation morphology and size distribution of WPIF samples pretreated with the four enzymes are significantly different.

[0053] like Figure 3 As shown, unmodified WPI, after heat treatment under acidic conditions for 10 hours, did not form linear or tortuous fibrous structures. Different protein structures were obtained by heat treating different enzyme hydrolysates under the same conditions. Figure 4 It can be seen that after 2 hours of hydrolysis, a small number of cross-linked nanofiber structures are generated in the solution, and the diameter of the fibers varies considerably; however, as the hydrolysis time of WPI by trypsin increases, the degree of protein aggregation after heat treatment increases, the fibers become interconnected, the fibrous structure gradually decreases, and a network structure appears. Figure 6 and Figure 7As can be seen, neutral proteases and papain also undergo structural changes with prolonged enzymatic hydrolysis time, forming twisted nanofibers. At 5 hours of hydrolysis, only a large number of spherical particles are formed. Figure 5 As shown, after 2 hours of pepsin hydrolysis, whey protein isolate (WPI) forms nanofibers with diameters of approximately 20-200 nm. After 2-4 hours of heat treatment, even longer nanofibers (length > 1 μm) appear, forming a stable system. In contrast, more spherical aggregates (diameter > 2 μm) are observed after 5 hours of hydrolysis, because the increased hydrolysis time further accelerates the exposure of hydrophobic protein residues. The nanofibers formed after 2 hours of pepsin hydrolysis exhibit the best structure and better semi-flexibility, indicating that 2 hours of pretreatment with pepsin hydrolysis of whey protein isolate can form long and ordered structures.

[0054] Different enzymatic hydrolysis methods affect the morphology of nanofibers because different enzymes hydrolyze whey protein isolate to produce different molecular protein monomers and peptides, which is a key factor in nanofiber formation. Therefore, based on TEM images and results, it is boldly hypothesized (and can be inferred) that whey protein isolate hydrolyzed by protease forms a continuous polymer through electrostatic interactions between nanofibers. This has never been reported before, and this is the first time the applicant has discovered and drawn this conclusion. Therefore, the nanofiber structure formed after 2 hours of pepsin hydrolysis is so suitable as an embedding material that it would not have been expected by those skilled in the art.

[0055] Zeta potential (ξ-potential) analysis: The ξ-potential was measured using a zeta potential meter at 25℃. The sample was diluted with distilled water with pH 2.5 to a concentration of 1 mg / mL. 1 mL of the sample was taken into the sample cell. The temperature of the sample cell was controlled to be below 50℃, the voltage was 150V, and the frequency was 250Hz.

[0056] Zeta potential is an important parameter for characterizing the stability of nanofibers in solution systems. Fiberization leads to an increase in the positive charge of nanofibers, possibly due to the exposure of charged groups caused by the hydrolysis and partial unfolding of whey protein isolates during fiber aggregation. Figure 8As shown, compared with unhydrolyzed WPI (~21.0 mV), pre-treatment significantly increased the zeta potential of the WPIF sample solution. At a protein hydrolysis time of 2 h, the zeta potential values ​​reached approximately 48.0 mV (pepsin), 44.9 mV (papain), 40.1 mV (neutral protease), and 27.0 mV (trypsin), respectively (p<0.05). After pepsin hydrolysis, whey protein is further decomposed into small molecules with a positive net charge, leading to a significant increase in the induced potential. Nanofibers exhibit a higher charge capacity. Amino acid systems with more surface charge are more stable. The increased potential may also be due to the increased number of nanoangles during heat treatment under acidic conditions, leading to increased hydrophobicity and surface charge on the nanofiber solution surface, thus enhancing the zeta potential. This can be explained by the stable dispersion characteristics of the nanofibers. However, prolonged enzymatic hydrolysis gradually decreased the zeta potential. The zeta potential values ​​of the nanofiber solutions after 5 hours of enzymatic hydrolysis with pepsin, papain, neutral protease, and trypsin were 32.5, 34.8, 31, and 22 mV, respectively. This indicates that hydrolyzed whey protein, upon heating, forms larger spherical or mesh-like structures and reduces the number of protofibrils, leading to system instability. These structural changes make the protein more prone to precipitation; therefore, the zeta potential value of WPIF decreases with prolonged enzymatic hydrolysis time. In conclusion, the WPIF sample pretreated with pepsin for 2 hours exhibited better nanofiber solution stability than the other three enzymes after 2 hours.

[0057] FTIR analysis of nanofiber solution: The enzymatically hydrolyzed protein powder is highly hygroscopic. To prevent quality changes during storage, a vacuum freeze dryer was used to remove excess moisture. Fourier transform infrared spectroscopy was used to analyze the secondary structure of WPI and WPIF. The sample was mixed with KBr powder at a 1:100 ratio, ground into powder, then graded using a 300 μm sieve and compressed into thin layers for testing. The secondary structure of the WPIF sample was found to be between 4000 and 400 cm⁻¹. -1 Within the wavenumber range of 4cm -1 Scan at a resolution of [resolution value].

[0058] The raw data was imported into EZ OMNIC version 8.0 software, and its FTIR spectral baseline was adjusted. The spectral region (amide band I) was selected at 1600-1700 cm⁻¹. -1 The secondary structure of WPI and WPIF was analyzed. The original spectra were smoothed, and then Fourier deconvolution and second derivatives were used to calculate the number, location, and area of ​​component bands.

[0059] The secondary structures of WPI and WPIF proteins were studied using FTIR. The FTIR spectrum of WPI showed an absorption band in the 400-4000 cm⁻¹ range, with peaks in this region corresponding to the stretching vibrations of olefins (C=C) and the stretching vibrations of hydrogen-containing olefins (=CH). FTIR spectroscopy analysis was performed on WPIF nanofibers pretreated with enzymatic hydrolysis. Since the amide I band of proteins consists of β-sheets, irregular structures, α-helices, and β-turns, the amide I band of WPIF changed compared to native WPI. Therefore, the amide I region (1600-1700 cm⁻¹) in the FTIR spectrum was observed to be different. -1 The changes in the secondary structure of the WPIF protein were analyzed: β-sheet, 1620-1640 cm⁻¹ -1 Irregular structure, 1640-1644cm -1 α-helix, 1650-1656 cm -1 β-turn, 1660-1700cm -1 .

[0060] The secondary structures of WPIF under different enzymatic hydrolysis times are shown in Table 1. After solution pretreatment via protease hydrolysis, the β-sheet content in the amyloid nanofibers gradually increased. The contents treated with papain, pepsin, neutral protease, and trypsin reached their maximum values ​​of 27.65%, 30.44%, 24.21%, and 25.31% at 2, 2, 3, and 4 h, respectively. However, excessive hydrolysis led to changes in the (amino acid and peptide) component ratios, which significantly affected WPIF formation; for example, β-sheet formation was accompanied by the exposure of hydrophobic regions. Furthermore, Table 2.4 shows that the α-helix content decreased with increasing hydrolysis time, likely due to the disruption of hydrogen bonds in the α-helix during WPIF formation. Secondary FTIR analysis indicated that heat treatment increased the β-sheet content and decreased the α-helix content, suggesting that WPIF formation is related to the conversion between α-helices and β-sheets. Additionally, the random coils and β-turns in WPIF exhibited different trends during hydrolysis. After treatment with pepsin for 2 hours, nanofibers with a diameter of less than 200 nm and a β-sheet content of more than 30% were obtained. Both of these properties make them very suitable as nanofiber materials for encapsulating oil-soluble active substances, which is something that those skilled in the art could not have predicted.

[0061] Table 1. Curve fitting results of nanofiber amide I band.

[0062]

[0063] Protease pretreatment of whey protein isolate (WPI) hydrolysates results in different secondary structures for two main reasons: First, different proteases act on different sites on WPI, leading to variations in the amino acids and polypeptides obtained, thus resulting in different hydrolysates. Second, hydrolysis disrupts some covalent bonds, hydrogen bonds, and disulfide bonds, causing the unfolding of the protein's three-dimensional structure, followed by depolymerization and rearrangement of the protein molecules. In summary, all FTIR results indicate that when proteases are used for treatment, hydrolysis alters the secondary structure of WPI, resulting in differences in the nanofiber structures formed by protein self-assembly.

[0064] Thiol content determination: To determine the SH content, 5 mL of Tris-Gly buffer (0.086 M Tris, 0.09 M glycine, and 0.004 M EDTA, pH 8.0) containing 8 M urea was added to 0.3 mL of 3% (w / v) WPI sample. Then, 20 μL of 2,2′-dinitro-5,5′-dithiobenzoate (DTNB) was added; after 15 min, the absorbance at 412 nm was measured spectrophotometrically. A blank was prepared using the supernatant from the DTNB-free buffer. Calculations are as follows:

[0065]

[0066] Where A412 represents the absorbance at a wavelength of 412 nm, C represents the sample concentration in mg / mL, D represents the dilution factor, and 73.53 is derived from 106 / (1.36×104), where 1.36×104 represents the molar absorbance, and 106 is the conversion from molar basis to μM / mL basis and from mg to g for solids.

[0067] The effects of different enzymatic hydrolysis methods and different hydrolysis times on the free sulfhydryl group content of WPIF, such as Figure 9 As shown, almost all free thiol groups in WPI are exposed, and the content of exposed free thiol groups did not change significantly during the reaction (p>0.05). The total free thiol content of natural WPI is approximately 40.0 μmol / g, consistent with previous results (40.6 μmol / g). Figure 9The effects of four enzyme pretreatments on thiol content were compared. The thiol content decreased over time, with the order of thiol content being: trypsin > neutral protease > papain > pepsin. Furthermore, within the first 3 hours, the total free thiol content of WPIF pretreated with trypsin showed no significant change, while the pepsin group showed the most significant decrease (p<0.05). The significant decrease in thiol content after 2 hours may be related to protein oxidation during prolonged heating. Combining the results in 2.4.5 and 2.4.6, it can be concluded that protease hydrolysis of WPI not only leads to changes in secondary structure and surface hydrophobicity but also to changes in thiol content. A decrease in thiol content in the solution leads to an increase in hydrophobicity; therefore, a decrease in thiol content is beneficial for nanofiber formation.

[0068] The pH of the 3% (w / v) enzymatically digested nanofiber solutions treated for 1, 2, 3, 4, and 5 hours was adjusted to 3.2 ± 0.02. 82 mg of β-CE was dissolved in 41 mL of ethanol and stirred in a 50 °C water bath for 1 hour. Then, 1 mL of β-CE was mixed with the WPIF solution and mixed at 50 °C for 1 hour using an air bath with constant temperature shaking. Next, the samples were rotated at 45 °C for 10 minutes using a rotary vacuum evaporator to remove excess ethanol. All samples were stored at 4 °C for analysis.

[0069] β-carotene was first dispersed in the organic phase ethanol, and then mixed with starch-like nanofibers. The content of β-CE in the system was between 0.1% and 1 wt%. Studies have shown that nanofibers can stably adsorb β-CE, and finally form WPIF / β-CE.

[0070] Storage stability: The lyophilized samples were sealed and stored in the dark at three different temperatures: room temperature, 45℃, and 60℃. Samples were taken at 1, 2, 3, 4, and 5 weeks of storage to measure the remaining amount of β-carotene in the samples. 2 mg WPIF / β-carotene was added to 1 mL of a mixed organic solvent (ethanol:hexane = 2:3), the mixture was shaken for 60 s, and centrifuged at 10000 r / min for 10 min. The supernatant was collected to measure the absorbance at a wavelength of 450 nm.

[0071] The degradation of β-CE is mainly due to photo-oxidation, which produces oxidation products including lactones and ketones. Figure 10The data showed the trend of β-CE residual content changes with storage time (i.e., 0, 1, 2, 3, 4, and 5 weeks). During the 5-week storage period, the residual β-CE content gradually decreased with increasing storage time, indicating that β-CE in the sample began to degrade. Compared with other groups, the residual β-CE content in the pepsin hydrolysis 2h pretreatment group changed the least in the first 4 weeks, indicating that the nanofibers in the pepsin hydrolysis 2h pretreatment group had the best adsorption strength and protective effect on β-CE.

[0072] It should be understood that the above detailed description of the technical solutions of the present invention with reference to preferred embodiments is illustrative and not restrictive. Those skilled in the art can modify the technical solutions described in the embodiments or make equivalent substitutions for some of the technical features based on reading this specification; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing whey protein isolate nanofibers and β-carotene encapsulated material, comprising the following steps: (1) whey protein isolate is hydrolyzed by pepsin to obtain a protease hydrolysate; (2) the protease hydrolysate is heated under acidic conditions to prepare a nanofiber solution; (3) the pH is then adjusted, and the solution is mixed with a β-carotene ethanol solution, and the ethanol is rotary evaporated to obtain the encapsulated material. In step (1), the pepsin hydrolysis time is 1.5-3h, the temperature is 35-45℃, and the pH is 2-3. In step (2), the heating temperature is 60-95℃, the heating time is 5h-20h, and the acidic condition is pH 1.0-3.

0. The diameter of the nanofibers is 20-200 nm; The nanofibers contain more than 30% β-sheets.

2. The method according to claim 1, characterized in that, The whey protein isolate has a content of 80% or higher. And / or, inactivation is performed after enzymatic hydrolysis; And / or, the heating temperature in step (2) is 90 ℃; And / or, the heat treatment time in step (2) is 10 hours; And / or, the nanofibers are starch-like nanofibers.

3. An application of starch-like whey protein nanofibers in improving the stability of β-carotene powder; the application includes the following steps: (1) whey protein isolate is hydrolyzed by pepsin to obtain a protease hydrolysate, (2) the protease hydrolysate is heated under acidic conditions to prepare a nanofiber solution, (3) then the pH is adjusted, mixed with a β-carotene ethanol solution, and the ethanol is rotary evaporated to obtain an encapsulated material; In step (1), the pepsin hydrolysis time is 1.5-3h, the temperature is 35-45℃, and the pH is 2-3. In step (2), the heating temperature is 60-95℃, the heating time is 5h-20h, and the acidic condition is pH 1.0-3.

0. The diameter of the nanofibers is 20-200 nm; The nanofibers contain more than 30% β-sheets.

4. The application according to claim 3, characterized in that, In step (3), the pH is adjusted to 2.5-4; And / or, the mixing temperature is 30-70°C, and the mixing time is 0.5-3h; And / or, the temperature for preparing the β-carotene ethanol solution is 30-70°C, and the stirring time is 0.5-3h.

5. A whey protein isolate nanofiber and β-carotene encapsulation compound, prepared by the method described in claim 1.

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