A method for preparing plant protein nanoparticles

Abstract

The present invention discloses a method for preparing plant protein nanoparticles, which belongs to the field of biotechnology. The preparation method of the present invention is as follows: the plant protein is dispersed in water so that the mass fraction of the plant protein is 2 to 15%, and protease is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis liquid is centrifuged, and the supernatant is freeze-dried to obtain the enzymatically hydrolyzed plant protein. The enzymatically hydrolyzed plant protein is dissolved in water so that the mass fraction of the enzymatically hydrolyzed plant protein is 0.5 to 3%, and then a macroporous adsorption resin is added and adsorbed by oscillation. The macroporous adsorption resin adsorbed with the enzymatically hydrolyzed plant protein is filtered and separated, washed and desalted with water, and the desalted macroporous adsorption resin is oscillated and desorbed using an ethanol aqueous solution. The desorbed liquid is freeze-dried to obtain plant protein nanoparticles. The preparation method of the present invention is simple and effective, and has low cost. The prepared plant protein nanoparticles have good water dispersibility and emulsification properties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for preparing plant protein nanoparticles. Background Art

[0002] Nanoparticles can be used as delivery vehicles for nutrients. Currently, most nanoparticles used in the food and pharmaceutical industries are inorganic particles or synthetic polymers, which have poor biocompatibility and pose a potential threat to human health. In contrast, protein-based nanoparticles have excellent biocompatibility. Plant protein is a high-quality protein resource due to its wide availability, low cost, high bioavailability, and cholesterol-free nature. However, most plant proteins have poor water solubility, making them difficult to disperse in water, limiting their application in the food industry. The antisolvent method for preparing nanoparticles is an effective means of enhancing the functional properties of plant proteins, particularly their emulsifying properties. However, due to their low water solubility, most plant proteins, such as rice protein and pea protein, produce nanoparticles with low yields, are also relatively rigid, and have poor water dispersibility. Patent CN 110129395 A prepares nanoparticles using a restricted enzyme modification technique, but the free enzyme cannot be recovered after the enzymatic hydrolysis reaction, increasing production costs. Furthermore, the protein hydrolysis process introduces a large amount of salt ions, which can affect the formation and stability of nanoparticles, thereby hindering their application in delivery systems. Therefore, how to desalt and prepare more stable plant protein nanoparticles has become an urgent problem to be solved. Summary of the Invention

[0003] The present invention provides a method for preparing plant protein nanoparticles, comprising the following steps:

[0004] The plant protein is dispersed in water to a mass fraction of 2-15%, and protease is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis solution is centrifuged, and the supernatant is freeze-dried to obtain the enzymatically hydrolyzed plant protein. The enzymatically hydrolyzed plant protein is dissolved in water to a mass fraction of 0.5-3%, and then a macroporous adsorption resin is added and adsorbed by oscillation. The macroporous adsorption resin adsorbed with the enzymatically hydrolyzed plant protein is separated by suction, rinsed with water for desalination, and the desalted macroporous adsorption resin is desorbed by oscillation using an ethanol aqueous solution. The desorbed solution is freeze-dried to obtain plant protein nanoparticles.

[0005] In the present invention, plant proteins include but are not limited to rice protein, pea protein, chickpea protein and peanut protein.

[0006] In the present invention, the protease is selected from one or more of trypsin, alkaline protease, papain, and composite protease. In order to facilitate the recovery and reuse of the protease, the enzyme can be prepared as an immobilized enzyme. Preferably, magnetic chitosan microspheres can be used to immobilize the protease. Specifically, the present invention provides a method for preparing chitosan magnetic microsphere immobilized enzyme, the steps of which are as follows:

[0007] (1) Preparation of chitosan magnetic microspheres

[0008] 1g of Fe3O4 magnetic nanoparticles, 10mL of chitosan solution (250g / L, dissolved in 2% acetic acid), 8mL of Tween-80, and 50mL of oil phase were dispersed at 1500-3000 rpm for 0.5-2 hours to form an oil-in-water emulsion. Subsequently, 20mL of citral solution (50g / L) in acetone was added for cross-linking, and the reaction was allowed to proceed for 4-6 hours. The microspheres were collected, rinsed sequentially with petroleum ether, isopropyl alcohol, and distilled water, and vacuum dried to obtain the immobilized support, i.e., chitosan magnetic microspheres.

[0009] (2) Preparation of immobilized enzyme

[0010] 1 g of chitosan magnetic microspheres was placed in 20 mL of phosphate buffer (pH 7-11) to fully swell. Then, 2 g of protease solution and chitosan magnetic microspheres were mixed, and citral solution was added. The mixture was shaken in a constant temperature water bath at 40-60°C for 1-2 h, allowed to stand, and the supernatant liquid was discarded. The mixture was rinsed with ultrapure water until the supernatant had no UV absorption to obtain chitosan magnetic microsphere-immobilized protease, which was then stored at 4°C.

[0011] In the present invention, the mass ratio of enzyme to substrate is selected from 1:100 (w / w).

[0012] In the present invention, the enzymatic hydrolysis conditions are: pH = 7.0-8.0, enzymatic hydrolysis temperature 50° C., and enzymatic hydrolysis time within 60 min.

[0013] Preferably, when the protease is trypsin, the enzymatic hydrolysis conditions are: pH = 8.0, enzymatic hydrolysis temperature 50°C, and enzymatic hydrolysis time 1 to 26 min; when the protease is alkaline protease, the enzymatic hydrolysis conditions are: pH = 8.0, enzymatic hydrolysis temperature 50°C, and enzymatic hydrolysis time 1 to 22 min; when the protease is papain, the enzymatic hydrolysis conditions are: pH = 7.0, enzymatic hydrolysis temperature 50°C, and enzymatic hydrolysis time 2 to 58 min; when the protease is a composite protease, the enzymatic hydrolysis conditions are: pH = 7.0, enzymatic hydrolysis temperature 50°C, and enzymatic hydrolysis time 1 to 28 min.

[0014] In the present invention, the degree of hydrolysis of the enzymatically hydrolyzed plant protein is 2-6%.

[0015] In the present invention, the centrifugal conditions are: centrifugal force 3000-5000g, centrifugal time 10-30min.

[0016] In the present invention, the mass ratio of the macroporous adsorption resin to the enzymatically hydrolyzed plant protein solution is 1:3 to 1:5.

[0017] In the present invention, the oscillation adsorption time is 10 to 12 hours.

[0018] In the present invention, the volume fraction of ethanol in the ethanol aqueous solution is 50 to 80%.

[0019] In the present invention, the oscillation desorption time is 10 to 16 hours.

[0020] The plant protein nanoparticles prepared by the above method are spherical and can be used as carriers of functional factors (such as curcumin, resveratrol, lycopene or lutein, etc.), improving the stability of the functional factors. They also have excellent water dispersibility and emulsification properties and can also be used as stabilizers for Pickering emulsions.

[0021] Based on the above content, the present invention also provides an application of the plant protein nanoparticles prepared by the above method in improving the stability of functional factors or Pickering emulsions.

[0022] The beneficial effects of the present invention are:

[0023] The present invention utilizes the adsorption properties of a macroporous adsorption resin to desalt enzymatically hydrolyzed proteins. The enzymatically hydrolyzed proteins are then desorbed and self-assembled into plant protein nanoparticles using an ethanol aqueous solution. The preparation method is simple, effective, and low-cost. The plant protein nanoparticles prepared by the method have excellent water dispersibility and emulsification properties. They can be used to carry functional factors in the form of particles or Pickering emulsion stabilizers, improving their stability and thus increasing the added value of plant protein and broadening its application channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The scanning electron micrographs of the protein nanoparticles described in Examples 1 to 4 and Comparative Examples 1 to 2 are shown;

[0025] Figure 2 is the particle size of the protein nanoparticles described in Examples 1 to 4 and Comparative Examples 1 to 2;

[0026] Figure 3 is the potential of the protein nanoparticles described in Examples 1 to 4 and Comparative Examples 1 to 2;

[0027] Figure 4 The surface hydrophobicity of the protein nanoparticles described in Examples 1 to 4 and Comparative Examples 1 to 2;

[0028] Figure 5 The interfacial tensions of the protein nanoparticles of Examples 1 to 4 and Comparative Examples 1 to 2 are shown in FIG. 1 , wherein the curves in the figure are Comparative Example 1, Comparative Example 2, Example 2, Example 3, Example 1, and Example 4 from top to bottom, respectively;

[0029] Figure 6 The particle sizes of the Pickering emulsions prepared from the protein nanoparticles described in Examples 1 to 4 and Comparative Examples 1 to 2 at 0 and 7 days of storage;

[0030] Figure 7 These are laser confocal images of the Pickering emulsions prepared from the protein nanoparticles described in Examples 1 to 4 and Comparative Examples 1 to 2 after storage for 0 and 7 days;

[0031] Figure 8 The loading rates of the protein nanoparticles on curcumin in Examples 1 to 4 and Comparative Examples 1 to 2 are shown. DETAILED DESCRIPTION

[0032] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0033] Example 1

[0034] Prepare rice protein nanoparticles as follows:

[0035] 30g rice protein was dispersed in 500mL distilled water for 1h, immobilized trypsin was added, and enzymolysis was carried out under pH=8.0 and 50 ℃ of conditions for 1.5min to obtain a hydrolyzate with a degree of hydrolysis of about 2%. Then the pH of the enzymolysis solution was rapidly adjusted to 7, and 3000rpm was centrifuged for 15min to reclaim the immobilized enzyme, and the supernatant was taken and freeze-dried to obtain enzymolysis rice protein. 3.75g enzymolysis rice protein was dispersed in 500mL distilled water, stirred for 1h, hydrated for 8h at 4 ℃, and then 100g macroporous adsorption resin was added and placed in a 1L Erlenmeyer flask. It was adjusted to 25 ℃ for a constant temperature oscillator with a rotating speed of 150rpm for 12h of oscillation adsorption. After the adsorption is completed, the liquid is discarded, and the macroporous adsorption resin is rinsed three times with distilled water for desalination. Then, the macroporous adsorption resin is mixed with 500 mL of 50% ethanol aqueous solution by volume fraction in a 1 L conical flask, and the constant temperature oscillator is adjusted to 25° C. and the rotation speed is 150 rpm. The oscillation desorption is carried out for 12 hours. The desorbed liquid is separated from the macroporous adsorption resin by suction filtration, and the desorbed liquid is rotary evaporated at 45° C. for 30 minutes and freeze-dried to obtain rice protein nanoparticles.

[0036] Example 2

[0037] The steps for preparing pea protein nanoparticles are as follows:

[0038] 30 g of pea protein was dispersed in 500 mL of distilled water for 1 hour, and immobilized alkaline protease was added. Enzymatic hydrolysis was carried out at pH = 8.0 and 50°C for 5 minutes to obtain a hydrolyzate with a degree of hydrolysis of approximately 3%. The pH of the hydrolyzate was then quickly adjusted to 7, the immobilized enzyme was recovered, and the mixture was centrifuged at 4500 rpm for 10 minutes. The supernatant was collected and freeze-dried to obtain enzymatically hydrolyzed rice protein. 3.75g enzymolysis rice protein is dispersed in 500mL distilled water, stirred for 1h, after 4℃ of hydration for 12h, 150g macroporous adsorption resin is added, placed in a 1L Erlenmeyer flask, regulating constant temperature oscillator is 25℃, and the rotating speed is 150rpm, and adsorption is shaken for 10h. After adsorption terminates, discard liquid, the macroporous adsorption resin is rinsed 3 times with distilled water with desalination, then the macroporous adsorption resin and 500mL ethanol volume fraction are mixed with 75% ethanol aqueous solution in a 1L Erlenmeyer flask, regulating constant temperature oscillator is 25℃, and the rotating speed is 150rpm, and desorption is shaken for 10h. The desorbent is separated from the macroporous adsorption resin by suction filtration, and the desorbent is rotary evaporated at 45℃ for 30min, and freeze-dried to obtain pea protein nanoparticles.

[0039] Example 3

[0040] The steps for preparing rice protein nanoparticles are as follows:

[0041] 30 g of rice protein was dispersed in 500 mL of distilled water for 1 hour, and immobilized papain was added. The mixture was enzymatically hydrolyzed at pH 7.0 and 50° C. for 58 minutes to obtain a hydrolyzate with a degree of hydrolysis of approximately 6%. The pH of the enzymatic hydrolyzate was then rapidly adjusted to 7, the immobilized enzyme was recovered, and the mixture was centrifuged at 4000 rpm for 30 minutes. The supernatant was collected and freeze-dried to obtain enzymatically hydrolyzed rice protein. 3.75g enzymolysis rice protein is dispersed in 500mL distilled water, stirred for 1h, 4 ℃ of hydration 8h, add 125g macroporous adsorbent resin, be placed in 1L conical flask, regulating constant temperature oscillator is 25 ℃, and rotating speed is 150rpm, vibration adsorption 12h, after adsorption finishes, discard liquid, macroporous adsorbent is rinsed 3 times with desalination with distilled water, then macroporous adsorbent and 500mL ethanol volume fraction are mixed with 80% ethanol water in 1L conical flask, regulating constant temperature oscillator is 25 ℃, rotating speed is 150rpm, vibration desorption 12h, desorbent is separated with macroporous adsorbent resin by suction filtration, desorbent is revolved 30min at 45 ℃, freeze drying obtains rice protein nanoparticles.

[0042] Example 4

[0043] Preparation of peanut protein nanoparticles, the steps are as follows:

[0044] 30 g of peanut protein was dispersed in 500 mL of distilled water for 1 hour, and immobilized composite protease was added. Enzymatic hydrolysis was carried out at pH = 7.0 and 50°C for 1 minute to obtain a hydrolyzate with a hydrolysis degree of approximately 2%. The pH of the enzymatic hydrolyzate was then quickly adjusted to 7, the immobilized enzyme was recovered, and the mixture was centrifuged at 5000 rpm for 20 minutes. The supernatant was collected and freeze-dried to obtain enzymatically hydrolyzed peanut protein. 3.75g enzymolysis rice protein is dispersed in 500mL distilled water, stirred for 1h, hydrated at 4℃ for 8h, 166g macroporous adsorbent resin is added, placed in a 1L Erlenmeyer flask, regulating constant temperature oscillator is 25℃, and the rotating speed is 150rpm, and adsorption is shaken for 11h. After adsorption finishes, discard liquid, the macroporous adsorbent resin is rinsed 3 times with distilled water with desalination, then the macroporous adsorbent resin and 500mL ethanol volume fraction are mixed with 70% ethanol water in a 1L Erlenmeyer flask, regulating constant temperature oscillator is 25℃, and the rotating speed is 150rpm, and desorption is shaken for 11h. The desorbent is separated from the macroporous adsorbent resin by suction filtration, and the desorbent is rotary evaporated at 45℃ for 30min, and freeze-dried to obtain peanut protein nanoparticles.

[0045] Comparative Example 1

[0046] The steps for preparing rice protein nanoparticles are as follows:

[0047] 30g rice protein was dispersed in 500mL distilled water for 1h, immobilized trypsin was added, and enzymolysis was carried out under pH=8.0 and 50 ℃ of conditions for 1.5min to obtain a hydrolyzate with a degree of hydrolysis of about 2%. Then the pH of the enzymolysis solution was rapidly adjusted to 7, and 3000rpm was centrifuged for 15min to reclaim the immobilized enzyme, and the supernatant was taken and freeze-dried to obtain enzymolysis rice protein. 3.75g enzymolysis rice protein was dispersed in 500mL distilled water, stirred for 1h, hydrated for 8h at 4 ℃, and then 100g macroporous adsorption resin was added and placed in a 1L Erlenmeyer flask. It was adjusted to 25 ℃ for a constant temperature oscillator with a rotating speed of 150rpm for 12h of oscillation adsorption. After the adsorption is completed, the liquid is discarded, and the macroporous adsorption resin is rinsed three times with distilled water for desalination. Then, the macroporous adsorption resin is mixed with 500 mL of ethanol with a volume fraction of 20% ethanol aqueous solution in a 1 L conical flask, and the constant temperature oscillator is adjusted to 25° C. and the rotation speed is 150 rpm. The oscillation desorption is carried out for 12 hours. The desorbed liquid is separated from the macroporous adsorption resin by suction filtration, and the desorbed liquid is rotary evaporated at 45° C. for 30 minutes and freeze-dried to obtain rice protein nanoparticles.

[0048] Comparative Example 2

[0049] The steps for preparing rice protein nanoparticles are as follows:

[0050] 30g rice protein was dispersed in 500mL distilled water for 1h, immobilized trypsin was added, and enzymolysis was carried out under pH=8.0 and 50 ℃ of conditions for 1.5min to obtain a hydrolyzate with a degree of hydrolysis of about 2%. Then the pH of the enzymolysis solution was rapidly adjusted to 7, and 3000rpm was centrifuged for 15min to reclaim the immobilized enzyme, and the supernatant was taken and freeze-dried to obtain enzymolysis rice protein. 3.75g enzymolysis rice protein was dispersed in 500mL distilled water, stirred for 1h, hydrated for 8h at 4 ℃, and then 100g macroporous adsorption resin was added and placed in a 1L Erlenmeyer flask. It was adjusted to 25 ℃ for a constant temperature oscillator with a rotating speed of 150rpm for 12h of oscillation adsorption. After the adsorption is completed, the liquid is discarded, and the macroporous adsorption resin is rinsed three times with distilled water for desalination. Then, the macroporous adsorption resin is mixed with 500 mL of 100% ethanol aqueous solution with an ethanol volume fraction in a 1 L conical flask, and the constant temperature oscillator is adjusted to 25° C. and the rotation speed is 150 rpm. The oscillation desorption is carried out for 12 hours. The desorbed liquid is separated from the macroporous adsorption resin by suction filtration, and the desorbed liquid is rotary evaporated at 45° C. for 30 minutes and freeze-dried to obtain rice protein nanoparticles.

[0051] The protein nanoparticles prepared in Examples 1 to 4 and Comparative Examples 1 to 2 were tested as follows:

[0052] 1. Scanning electron microscopy analysis

[0053] The protein nanoparticle samples were evenly smeared and fixed on the conductive double-sided tape, and then the morphology was observed using a field emission scanning electron microscope (SU9000) after gold spraying. The results are as follows: Figure 1 shown.

[0054] Depend on Figure 1 It can be seen that when the volume fraction of ethanol in the ethanol aqueous solution is 50-80%, the prepared nanoparticles have regular shapes and are spherical nanoparticles (Examples 1-4); when the volume fraction of ethanol in the ethanol aqueous solution is 20%, no nanoparticles are formed (Comparative Example 1); when the volume fraction of ethanol in the ethanol aqueous solution is 100%, the surface of the formed nanoparticles becomes rough (Comparative Example 2).

[0055] 2. Nanoparticle size and potential analysis

[0056] The average particle size and potential of protein nanoparticles were measured using a nanoparticle size potential analyzer (Zetasizer Lab). Figure 2 and Figure 3 shown.

[0057] Depend on Figure 2 and Figure 3It can be seen that the average particle size of the nanoparticles prepared in Examples 1 to 4 is between 500 and 990 nm, and the potential is between -25 and -30 mV. The average particle size of the nanoparticles prepared in Comparative Example 1 is 486 nm, and the potential is -18.20 mV. The average particle size of the nanoparticles prepared in Comparative Example 2 is 557 nm, and the potential is -36.45 mV, which may be due to the high concentration of ethanol promoting the exposure of the charged groups of the nanoparticles. In addition, the particle size of the nanoparticles in Example 1 is larger, which may be due to the fact that a certain concentration of ethanol can promote the aggregation of proteins. The particle size of the nanoparticles in Comparative Example 1 is the smallest, which may be due to the fact that large aggregates are broken into small particles.

[0058] 3. Surface hydrophobicity analysis

[0059] The nanoparticles were diluted to a concentration between 0.01 and 0.1 mg / mL using a phosphate buffer solution at pH 7.0. 4 mL of the diluted protein sample was mixed with 20 μL of 8.0 mM ANS solution and the fluorescence intensity was measured using a fluorescence spectrophotometer (F2700). The fluorescence intensity was plotted against the protein concentration, and the slope was the surface hydrophobicity index of the protein. The results were as follows: Figure 4 shown.

[0060] Depend on Figure 4 The surface hydrophobicity of the particles in Comparative Example 2 was the highest, while that in Comparative Example 1 was the lowest. This is likely due to the ethanol-induced gradual unfolding of the protein structure, exposing more hydrophobic groups. While a certain degree of hydrophobic group exposure is beneficial for protein adsorption at the oil-water interface, excessive exposure is detrimental to protein adsorption at the oil-water interface.

[0061] 4. Interfacial tension analysis

[0062] The interfacial tension of protein nanoparticles was measured using an optical contact angle meter (KRUSS DSA100). Figure 5 shown.

[0063] Depend on Figure 5 It can be seen that the interfacial tension of protein nanoparticles decreases with time, and the particles are gradually adsorbed on the oil-water interface. The interfacial tensions of Comparative Examples 1 and 2 are the largest, indicating that their ability to reduce the oil-water interface is poor.

[0064] 5. Water dispersibility

[0065] The water dispersibility of the protein nanoparticles was determined using the Folin-phenol method. The protein nanoparticles prepared in Examples 1-4 and Comparative Examples 1-2 were dispersed in a phosphate buffer solution at pH 7.0. The sample dispersions were centrifuged (3000 rpm for 15 minutes). The dispersibility of the protein nanoparticles was expressed as the percentage of the protein concentration in the supernatant to the total protein concentration. The results are shown in Table 1.

[0066] Table 1

[0067]

[0068]

[0069] As shown in Table 1, the dispersibility of the protein nanoparticles obtained in Examples 1 to 4 is all above 90%, indicating good dispersibility.

[0070] 6. Pickering emulsion stability

[0071] The protein nanoparticles prepared in Examples 1 to 4 and Comparative Examples 1 to 2 were dispersed in phosphate buffer (pH = 7) to a concentration of 4%, and 10% by volume of corn oil was added. The mixture was dispersed at 10,000 rpm for 5 minutes, and then ultrasonicated at 450 W for 5 minutes to obtain a Pickering emulsion.

[0072] The particle size of the emulsions stored for 0 and 7 days was measured using a nanoparticle size potential analyzer (Zetasizer Lab).

[0073] 1 mL of the emulsion was stained by adding 20 μL of Nile red solution (1 mg / mL in ethanol) and 20 μL of Nile blue solution (1 mg / mL in ultrapure water). The microstructure of the emulsion was observed using a laser confocal microscope (TCS SP5).

[0074] The test results are as follows Figure 6 and Figure 7 shown.

[0075] The average particle size of the emulsion prepared by the protein nanoparticles described in Examples 1 to 4 was smaller before and after storage, and the microstructure ( Figure 7 ) indicates that the droplet distribution is relatively uniform, indicating that the resulting Pickering emulsion has high storage stability. In contrast, the emulsions prepared using the protein nanoparticles described in Comparative Examples 1 and 2 exhibited larger average droplet sizes before and after storage, indicating that the resulting Pickering emulsions exhibited poor stability. This suggests that the protein nanoparticles prepared in Examples 1-4 have high surface activity, which contributes to the stability of the emulsions.

[0076] After the emulsions were stored at room temperature for 7 days, the stability index (SI) of the emulsions of different samples was measured: SI = 100% × H t / H0. H tis the height of the emulsion layer after storage / cm, H0 refers to the total height of the emulsion / cm, see Table 1. As can be seen from Table 1, the emulsion stability index of Comparative Example 1 with an ethanol volume fraction of 20% is the lowest, while the emulsion stability indexes of Examples 1 to 4 are all above 90%, indicating high stability.

[0077] 7. Bioavailability of curcumin in Pickering emulsion

[0078] The protein nanoparticles prepared in Examples 1 to 4 and Comparative Examples 1 to 2 were dispersed in water to a concentration of 4%. 10% by volume of corn oil (containing 0.2% curcumin) was added, and the mixture was dispersed at a high speed of 10,000 rpm for 5 minutes. The mixture was then ultrasonicated at an ultrasonic power of 450 W for 5 minutes to obtain a Pickering emulsion.

[0079] An in vitro simulated gastrointestinal digestion experiment of emulsion was carried out using an in vitro simulated digestion model. After the digestion was completed, the in vitro simulated small intestinal digestive fluid was taken, and after centrifugation at 15000rpm for 30min, the solution was divided into 3 layers, the upper layer was the undigested oil phase, the middle layer was the micelles, and the bottom layer was the sediment layer. The middle micelle layer solution was taken. The micelles and the initial emulsion were mixed with anhydrous ethanol at a mass ratio of 1:4, and after thorough mixing, they were centrifuged at 5000rpm for 15min, and the organic phase was collected, and repeated three times. The collected curcumin solution was measured for its absorbance at 419nm using a UV-visible spectrophotometer, and the curcumin content was calculated based on the standard curve of curcumin in ethanol. The bioavailability of curcumin was calculated using the following formula: Bioavailability (%) = 100×C 胶束 / C 乳液 .

[0080] The test results are shown in Table 1. The bioavailability of Comparative Examples 1 to 2 was less than 34%, while the bioavailability of Examples 1 to 4 was greater than 45%. The results show that the Pickering emulsion prepared from the protein nanoparticles of the present invention can serve as a good carrier for the oil-soluble functional factor curcumin and improve its bioavailability.

[0081] 8. Loading rate of curcumin on nanoparticles

[0082] The protein nanoparticles prepared in Examples 1 to 4 and Comparative Examples 1 to 2 were dispersed in water to a concentration of 2%; curcumin was dissolved in anhydrous ethanol (2 mg / mL); the protein nanoparticle dispersion was then mixed with the curcumin solution at a volume ratio of 50:1, stirred for 2 hours in a dark environment, and the mixture was centrifuged at 10,000 rpm for 20 minutes. The supernatant was lyophilized to obtain composite nanoparticles. The composite nanoparticles were dissolved in distilled water, mixed with ethanol, vortexed to extract the curcumin loaded on the particles, centrifuged (10,000 rpm, 20 minutes), and the absorbance of the supernatant at 419 nm was measured using an ultraviolet spectrophotometer. The content of nanoparticle-loaded curcumin was calculated using a standard curve of curcumin in ethanol.

[0083] Load factor (%) = 100 × C 上清 / C 总 .

[0084] The test results are as follows Figure 8 As shown. The results show that the protein nanoparticle loading rates of Examples 1 to 4 are all above 88%, which shows that the protein nanoparticles prepared by the technical solution of the present invention can better load oil-soluble active ingredients. The loading rates of Comparative Examples 1 and 2 are lower, which may be due to the difference in surface hydrophobicity of different protein nanoparticles. Hydrophobic interaction is the main force between curcumin and protein particle carriers. A moderate increase in surface hydrophobicity is conducive to the combination of nanoparticles and curcumin, but too high surface hydrophobicity will lead to poor water dispersibility of protein nanoparticles, and thus cannot better combine with curcumin.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A method for preparing plant protein nanoparticles, characterized in that: Here are the steps: The plant protein is dispersed in water to a mass fraction of 2-15%, and an immobilized enzyme is added for enzymolysis. After the enzymolysis is completed, the enzymolysis solution is centrifuged, and the supernatant is freeze-dried to obtain the enzymolyzed plant protein. The enzymolyzed plant protein is dissolved in water to a mass fraction of 0.5-3%, and then a macroporous adsorption resin is added and adsorbed by oscillation. The macroporous adsorption resin adsorbed with the enzymolyzed plant protein is separated by filtration, washed with water for desalination, and the desalted macroporous adsorption resin is desorbed by oscillation using an ethanol aqueous solution. The desorbed solution is freeze-dried to obtain plant protein nanoparticles. The plant protein is selected from one or more of rice protein, pea protein and peanut protein; the enzyme in the immobilized enzyme is selected from one or more of trypsin, alkaline protease, papain and composite protease; the enzymatic hydrolysis conditions are: pH=7.0-8.0, enzymatic hydrolysis temperature 50°C, and enzymatic hydrolysis time within 60 minutes; the hydrolysis degree of the enzymatically hydrolyzed plant protein is 2-6%; the mass ratio of the macroporous adsorption resin to the enzymatically hydrolyzed plant protein solution is 1:3-1:5; and the volume fraction of ethanol in the ethanol aqueous solution is 50-80%.

2. Plant protein nanoparticles prepared by the method according to claim 1.

3. Use of the plant protein nanoparticles according to claim 2 in the preparation of Pickering emulsion.

4. Use of the plant protein nanoparticles according to claim 2 in preparing a curcumin Pickering emulsion.

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