CN115286691A

CN115286691A - Barley peptide nano-carrier and preparation method and application thereof

Info

Publication number: CN115286691A
Application number: CN202210740580.2A
Authority: CN
Inventors: 宋洪东; 管骁; 靳祖珑; 汪新月
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2022-06-27
Filing date: 2022-06-27
Publication date: 2022-11-04

Abstract

The invention relates to a barley peptide nano-carrier and a preparation method and application thereof. The barley peptide P1 is QQPTIQL, and the amino acid sequence is Gln-Gln-Pro-Thr-Ile-Gln-Leu; the barley peptide P2 is GVGSVSV, and the amino acid sequence is Gly-Val-Gly-Pro-Ser-Val-Gly-Val. The invention also provides a preparation method of the barley peptide nano-carrier, which comprises the steps of preparing barley protein by using barley as a raw material, carrying out enzymolysis on the barley protein by using trypsin or alpha-chymotrypsin to obtain barley peptide, and preparing the barley peptide nano-carrier by using an ultrasonic method in an auxiliary manner. The barley peptide nano-carrier provided by the invention can be used for embedding food functional factors and can obviously enhance the water solubility and stability of the food functional factors. Compared with the prior art, the barley peptide nano-carrier provided by the invention is simple to prepare, has no chemical solvent residue, high safety and low cost, and has important application prospects in the fields of food, medicine and the like.

Description

Barley peptide nano-carrier and preparation method and application thereof

Technical Field

The invention relates to the field of nano material technology and biotechnology, in particular to a barley peptide nano carrier and a preparation method and application thereof.

Background

The nano-carrier is a novel carrier with the particle size of 10-1000nm, plays an important role in embedding and delivering functional components or medicaments so as to maintain the stability of the functional components or the medicaments and promote the absorption of the functional components or the medicaments, and is a research hotspot in the fields of food, biological medicine and the like. The self-assembled peptide is an important material for constructing the nano-carrier, and has the advantages of good biocompatibility, adjustable size and shape, nutritive value, biological activity and the like. The nano-carrier formed by the self-assembly peptide has wide application prospect in the fields of biomedicine, materials, chemistry, food and the like. At present, people mainly prepare self-assembled peptides by an artificial chemical synthesis method so as to prepare nano-carriers with different structures and functions, and the nano-carriers are used for embedding and loading functional components/medicines. However, artificial chemical synthesis of self-assembled peptides has many problems, such as that chemical synthesis of self-assembled peptides uses a large amount of organic harmful reagents, which limits the application of the self-assembled peptides in the fields of food and the like; the cost is high, and generally, the cost for synthesizing gram-grade self-assembled peptide is often thousands of yuan, and the cost is increased along with the increase of the chain length of the peptide; the preparation method cannot be carried out on a large scale, and the practical application of the peptide-based self-assembly nano-carrier is limited. Therefore, there is an urgent need to find other methods to prepare safe, low-cost self-assembling peptide nanocarriers on a large scale.

Barley is the fourth crop of grain worldwide, second only to wheat, rice and corn, with annual global barley yields of over 1 million tons. The barley protein has rich source and low price, mainly comes from byproducts of barley starch processing and beer production, but cannot be fully utilized. Barley protein is rich in water-insoluble prolamines and glutelins and is a potential source of self-assembling peptides due to its abundance of hydrophobic amino acids. However, no research and report on the preparation of peptide nanocarriers using barley protein has been found.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a barley peptide nano-carrier and a preparation method and application thereof.

The invention aims to utilize abundant and cheap food-borne barley protein to obtain barley peptide through biological enzyme hydrolysis, and then prepare a peptide nano carrier through a self-assembly method, and further apply the peptide nano carrier to the solubilization and stabilization of functional components.

The purpose of the invention can be realized by the following technical scheme:

in a first aspect of the present invention, there is provided a barley peptide nanocarrier, which is self-assembled from any one or more of the following barley self-assembly peptides:

the barley peptide P1 is QQPTIQL, and has an amino acid sequence of Gln-Gln-Pro-Thr-Ile-Gln-Leu;

the barley peptide P2 is GVGSVSV, and the amino acid sequence is Gly-Val-Gly-Pro-Ser-Val-Gly-Val.

Preferably, the barley peptide nano-carrier has the particle size of less than 100nm and moderate hydrophilicity and hydrophobicity.

In a second aspect of the invention, there is provided a polynucleotide encoding a barley peptide selected from one or more of:

the barley peptide P1 is QQPTIQL, and has the amino acid sequence of Gln-Gln-Pro-Thr-Ile-Gln-Leu;

the barley peptide P2 is GVGSVSV, and the amino acid sequence is Gly-Val-Gly-Pro-Ser-Val-Gly-Val. In a third aspect of the present invention, there is provided a method for preparing a barley peptide nanocarrier, the method comprising the steps of:

1) Preparation of barley protein:

2) Preparation of the barley protein peptide:

performing enzymolysis on barley protein by adopting one or a combination of trypsin and alpha-chymotrypsin to obtain barley peptide;

3) Preparing a barley peptide nano-carrier:

and dispersing the barley peptide in deionized water, and performing ultrasonic treatment to prepare the barley peptide nano carrier.

In one embodiment of the present invention, in step 1), the preparation method of the barley protein comprises: pulverizing barley, sieving, defatting, mixing defatted barley flour with 1-3% NaCl solution (w/w), stirring, and centrifuging to obtain solid residue; and then dispersing the solid residue in 70% volume fraction ethanol solution, performing water bath extraction, centrifuging, collecting precipitate, finally re-dispersing the precipitate in deionized water, adjusting the pH value to 10-12, performing extraction, centrifuging to obtain supernatant, adjusting the pH value of the supernatant to 4.4-4.6, standing at low temperature for precipitation, washing with water, and performing freeze drying to obtain the barley protein.

In one embodiment of the present invention, in step 2), the conditions for enzymatic hydrolysis of the barley protein are as follows: the pH value of the enzymolysis environment is 7-9, the enzymolysis temperature is 30-60 ℃, and the ratio of enzyme to substrate is 1:25 to 100, and the enzymolysis time is 0.5 to 4 hours.

In one embodiment of the invention, in the step 2), after the barley protein is subjected to enzymolysis, the enzymolysis product is heated to 60-100 ℃, and then is sequentially cooled, the pH value is adjusted to be neutral, and the barley peptide is obtained through rotary evaporation concentration and freeze drying.

In one embodiment of the present invention, in step 3), the barley protein peptide is dispersed in deionized water at a concentration of 0.1 to 5mg/mL, and after dissolution, the precipitate is first removed by centrifugation at 500 to 4000rpm, and the supernatant is collected and then subjected to sonication.

In one embodiment of the invention, in the step 3), the supernatant is placed in an ultrasonic cleaning instrument, and the barley peptide can be orderly self-assembled to form the nano-carrier under the action of ultrasonic waves, wherein the ultrasonic wave power is 50-600W, and the ultrasonic wave time is 5-45min.

In one embodiment of the present invention, after the barley peptide nanocarrier is obtained in step 3), step 4) of separating and purifying the barley protein peptide nanocarrier is further performed, and the specific method of step 4) is as follows:

separating the barley protein peptide nanoparticles by using a chromatographic column, detecting the particle size of each elution peak in the elution process by using deionized water as a mobile phase, and collecting a first peak component, namely the purified barley protein peptide nano-carrier.

In one embodiment of the invention, in step 4), the chromatography column is a sephadex G-75 glass chromatography column.

In one embodiment of the invention, in step 4), the absorbance value is measured at 214nm and the first peak component is collected.

The fourth aspect of the invention provides an application of a barley peptide nano-carrier in embedding, solubilizing and stabilizing food functional factors.

Compared with the prior art, the barley peptide nano-carrier provided by the invention is derived from barley peptide, and the barley peptide is prepared from abundant and cheap barley protein, so that the barley peptide nano-carrier has the advantages of low cost, simple process, easiness in large-scale production and the like; the whole process flow for preparing the barley peptide nano carrier provided by the invention has no toxic and harmful reagent residue, the whole process flow meets the requirement of food grade, and the barley peptide nano carrier can be used in the fields of food, health-care food and the like besides the field of biological medicine, and has the advantages that chemically synthesized peptides do not have; the barley peptide nano-carrier provided by the invention can be used for loading and embedding food functional factors, remarkably improves the water solubility and stability of embedded components, and has wide application prospect and important application value in the fields of food, biological medicine and the like.

Drawings

FIG. 1 hydrolysis curves of barley protein under alpha-chymotrypsin and trypsin;

FIG. 2 Transmission Electron Microscopy (TEM) of barley peptide self-assembled nanocarriers prepared with different enzymes, a, α -chymotrypsin; b, trypsin;

FIG. 3 surface tension of an aqueous solution of barley peptide nanocarriers;

in FIG. 3, B represents barley protein; B-Chy represents a barley peptide nanocarrier prepared using alpha-chymotrypsin; B-Tps represents barley peptide nanocarriers prepared with trypsin;

FIG. 4 is a diagram of the separation and purification of barley peptide nanocarriers;

in FIG. 4, a, α -chymotrypsin; b, trypsin;

FIG. 5 is a diagram showing the particle size distribution of purified barley peptide self-assembled nano-carriers prepared by different enzymes;

in FIG. 5, a, α -chymotrypsin; b, trypsin;

FIG. 6 MS/MS profile of barley self-assembling peptide QQPTIQL;

FIG. 7 MS/MS profile of barley self-assembly peptide GVGGPSVSV;

FIG. 8 is an appearance diagram of solutions obtained after quercetin loading of barley self-assembly peptides and barley proteins respectively;

in fig. 8, reference numeral 1 is a barley self-assembly peptide; reference numeral 2 is barley protein;

FIG. 9 changes in CAPE retention rates in free CAPE and CAPE-loaded barley peptide nanocarriers during 28 days of low temperature (4 ℃) storage.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

EXAMPLE 1 preparation and enzymatic hydrolysis of barley protein

(1) Preparation of barley protein

Defatted barley flour was dispersed in a 2% by mass NaCl solution at a feed/liquid ratio (w/v) of 1. The mixture was centrifuged at 4000rpm for 15 minutes in a low-speed centrifuge to separate solid and liquid, and the supernatant was discarded. The residue obtained by centrifugation was redispersed in 1000mL of a 70% volume fraction ethanol solution and stirred at 500rpm for 1 hour in a thermostatic water bath at 50 ℃. After completion, the mixture was centrifuged at 4000rpm for 15 minutes, and the precipitate was collected. The precipitate was redispersed in deionized water and adjusted to pH 10.5-11 with 1M NaOH solution and stirred at 500rpm for 1 hour at room temperature. Centrifuging at 4000rpm for 15 min with a low speed centrifuge for solid-liquid separation, adjusting pH to 5.2 with 1M hydrochloric acid, and standing at 4 deg.C for 30 min to precipitate protein. After the completion of the reaction, the precipitate was collected by centrifugation at 4000rpm for 15 minutes. Further dispersing the precipitate with deionized water at a ratio of 1.

(2) Enzymatic hydrolysis of barley protein

Carrying out enzymolysis on alpha-chymotrypsin: the barley protein was dispersed in deionized water, adjusted to pH 7.8 with 0.5M NaOH solution, heated to 50 ℃ in a water bath on a magnetic stirrer and kept stable. 10mg of chymotrypsin was weighed precisely, dispersed in 1mL of 20mM sodium carbonate buffer solution having pH 7.8, and enzymatic buffer was added to the protein solution to start the enzymatic hydrolysis, the ratio of enzyme to substrate was 2:100. during the enzymatic hydrolysis, the pH of the enzymatic system was kept constant at 7.8 by titration with a 0.5M NaOH solution and the volume of NaOH solution consumed was recorded. And when the pH value of the enzymolysis system is not changed within 5 minutes or is changed by less than 0.01 within 15 minutes, the enzymolysis is determined to be complete, the enzymolysis liquid is boiled for 5 minutes to inactivate the enzyme so as to stop the enzymolysis, the pH value is adjusted to be neutral, a low-speed centrifuge is used for centrifuging for 10 minutes at the rotating speed of 1000rpm to remove partial insoluble matters or large particles, and the supernatant is frozen and dried to obtain the barley protein peptide hydrolyzed by the alpha-chymotrypsin.

And (3) carrying out enzymolysis by trypsin: 500mg of barley protein powder was dispersed in 50mL of deionized water, adjusted to pH 8.0 with 0.5M NaOH solution, heated to 37 ℃ in a water bath on a magnetic stirrer and kept stable. 20mg of trypsin was weighed precisely, dispersed in 1mL of 20mM sodium carbonate buffer solution at pH 8.0, and the enzyme buffer solution was added to the protein solution to start the enzyme hydrolysis at a ratio of 4:100. during the enzymatic hydrolysis, the pH of the enzymatic system was kept constant at 8.0 by titration with a 0.5M concentration NaOH solution and the volume of NaOH solution consumed was recorded. And when the pH value of the enzymolysis system is not changed within 5 minutes or is changed by less than 0.01 within 15 minutes, the enzymolysis is determined to be complete, the enzymolysis solution is boiled for 5 minutes to inactivate the enzyme so as to stop the enzymolysis, the pH value is adjusted to be neutral, a low-speed centrifuge is used for centrifuging for 10 minutes at the rotating speed of 1000rpm to remove partial insoluble matters or large particles, and the supernatant is frozen and dried to obtain the barley protein peptide hydrolyzed by the trypsin.

The degree of proteolysis was calculated from the volume consumed by the 0.5M NaOH solution recorded in the proteolysis experiment. The volumes of the NaOH solutions consumed cumulatively at the time of the enzymatic hydrolysis reaching 10, 30, 60, 90, 120, 150, 180, 210, 240 minutes were recorded, the degree of hydrolysis was calculated by the pH-stat method, and a hydrolysis curve was plotted with the enzymatic hydrolysis time as the abscissa and the degree of hydrolysis as the ordinate.

FIG. 1 is a hydrolysis curve of barley protein under 2 different enzymes, alpha-chymotrypsin and trypsin specifically hydrolyze hydrophobic amino acid and hydrophilic amino acid respectively, and have strong specificity, so that the final hydrolysis degrees are both less than 5%, and the final hydrolysis degrees of the two enzymes are relatively similar.

Example 2 preparation and characterization of a barley protein peptide nanocarrier

The barley peptide was dispersed in deionized water at a concentration of 1mg/mL, and after dissolution, the precipitate was first removed by centrifugation at 3000 rpm, and the supernatant was collected. And then placing the supernatant in an ultrasonic cleaning instrument, and setting the ultrasonic condition as that the ultrasonic power is 300W and the ultrasonic time is 20min. Under the action of ultrasound, the barley peptide can be orderly self-assembled to form a nano carrier. After the end, measuring the nanometer size and the poly dispersion coefficient (PDI) by using a dynamic light scattering instrument; and observing the morphology of the peptide nano carrier by adopting a transmission electron microscope. FIG. 2 is a transmission electron microscope image of barley peptide self-assembled nano-carriers prepared by different enzymes, and Table 1 shows the size and PDI value of barley protein peptide nano-carriers obtained by hydrolysis of 2 different enzymes. From these results, it can be seen that 2 different barley protein peptide nanocarriers were all less than 100nm in size, and were uniformly dispersed, with the nanocarriers being approximately spherical. Importantly, the barley peptide nano-carrier prepared by the method has good stability in aqueous solution, is not easy to precipitate, is clear and transparent, and overcomes the defects that a pure barley protein nano-carrier is easy to be unstable and easy to aggregate. In addition, researches report that the size of the nano-carrier significantly influences the uptake of cells to the nano-carrier, and the nano-carrier with the size less than about 100nm is more easily taken up. Therefore, the barley protein peptide nano-carrier prepared by the method can be easily absorbed by organisms.

TABLE 1

Note that Chy and Tps represent alpha-chymotrypsin and trypsin, respectively

In addition, the air-water surface tension of the barley peptide nanocarrier was measured. Preparing a peptide nano-carrier mother solution with the concentration of 5mg/mL, diluting the peptide nano-carrier mother solution into gradient concentrations of 0.01, 0.1, 1, 5, 10, 50, 100, 500, 1000, 3000 and 5000 mu g/mL, dripping enough large liquid drops at room temperature, and photographing to determine the surface tension after the nano-carrier in the solution is adsorbed and balanced on the air-water surface. Protein solutions of the same concentration were used as controls. The results of the experiment are shown in FIG. 3. In general, peptide nanocarriers prepared by enzymatic hydrolysis have both hydrophilic and lipophilic properties, and can be classified into 3 types according to the difference in surface tension at a concentration of 5 mg/mL: relatively hydrophobic (tensile force is more than 30mN/m and less than 40 mN/m), moderately hydrophilic (tensile force is more than 40mN/m and less than 50 mN/m), and relatively hydrophilic (tensile force is more than 50 mN/m). Therefore, it can be determined that the currently prepared barley peptide nanocarrier has moderate hydrophilicity and hydrophobicity.

Example 3 purification and characterization of barley nanocarriers

Prepare the sephadex G-75 glass chromatographic column by itself. 20mg of the barley peptide nanocarrier prepared in the above step was dissolved in 2mL of deionized water, and the solution was filtered through a 0.45 μm filter and then loaded. And (3) eluting the nano-carrier at the flow rate of 1mL/min by using deionized water as a mobile phase, detecting by using an ultraviolet detector at the wavelength of 220nm, and recording a size exclusion chromatogram by using a chromatographic analyzer. Collecting one tube of eluent every 5 minutes, diluting the eluent by a certain multiple, and determining the particle size of each elution peak component.

FIG. 4 is a chromatogram of the separation of 2 barley protein peptide nanocarriers to purify a sample to obtain self-assembled peptides by removing a portion of the peptides not involved in self-assembly and a portion of impurities. Combining size exclusion chromatogram and particle size analysis, it can be found that most of the self-assembled nano-carriers can not enter the pores (less than 10 nm) of the gel particles and can be rapidly eluted from the gaps of the gel particles, and the first elution peak is the nano-carriers formed by self-assembly of the peptides with self-assembly ability. The substance eluted for a long time is hydrophilic peptide which does not undergo self-assembly, or combined pigment and partial salt released in proteolysis, and the impurities can be removed in the separation process. FIG. 5 is a distribution diagram of the particle size of the first peak, which is found to have a particle size of less than 100nm, which is consistent with the particle size distribution of the unpurified peptide nanocarriers, and also indicates that the first peak is indeed a barley peptide nanocarrier.

Example 4 sequence analysis of peptides in barley peptide nanocarriers

And analyzing and identifying the sequence of the peptide in the barley peptide nano-carrier by using an LC-MS/MS technology. The purified barley nanocarriers were redissolved in 20. Mu.L of 0.1% formic acid aqueous solution, separated using a reverse phase chromatography column (150. Mu.m i.d.. Times.150mm, packed with Acclaim PepMap RPLC 18, 1.9. Mu.m,

) Mobile phase A is 0.1% formic acid water solution, mobile phase B is 0.1% formic acid/80% acetonitrile solution, gradient elution is carried out at the flow rate of 600nL/min, and separation gradient is as follows: 4% by weight B (0 min) -8% by weight B (2 min); 8% by weight B (2 min) -40% by weight B (45 min); 40% by weight B (45 min) -60% by weight B (55 min); 60% by weight B (55 min) -95% by weight B (56 min); 95% B (56 min) -95% B (66 min). The ion source type is electrospray ionization source (ESI), positive ion scanning mode, spray voltage 2200V, capillary temperature 270 ℃. Setting primary mass spectrum parameters: scanning range 100-2000m/z, maximum resolution 70000, and automatic gain parameter 3000000. Setting secondary mass spectrum parameters: scanning range 50-2000m/z, maximum resolution 17500 and automatic gain parameter 100000.

The barley peptide nano-carrier obtained by enzymolysis of alpha-chymotrypsin is detected by mass spectrometry to mainly have m/z =827.46 and a charged ion peak, and secondary mass spectrometry is carried out on the ion peak. The secondary mass spectrum of this ion is shown in FIG. 6. The amino acid sequence of the self-assembly peptide is Gln-Gln-Pro-Thr-Ile-Gln-Leu by database matching and manual analysis calculation, namely the P1 peptide QQPTIQL.

The barley peptide nano-carrier obtained by trypsinization is detected by mass spectrum, and mainly has m/z =671.37 and an ion peak with a charge, secondary mass spectrum analysis is carried out on the ion peak, and the secondary mass spectrum of the ion is shown in figure 7. Through database matching and manual analysis and calculation, the amino acid sequence of the self-assembly peptide is Gly-Val-Gly-Pro-Ser-Val-Gly-Val, namely the P2 peptide GVGGPSVCV.

Example 5 embedding of barley peptide nanocarriers with Quercetin

The barley peptide-quercetin composite nano system is prepared by adopting a liquid-liquid dispersion method and an ultrasonic auxiliary method. Firstly, preparing a quercetin mother solution: accurately weigh 20mg quercetin into a 1.5mL centrifuge tube, add 1mL dimethyl sulfoxide, vortex and shake well. Preparing a barley self-assembly peptide aqueous solution: a certain amount of barley self-assembly peptide is weighed, 10mL of pure water is added, and the mixture is gently shaken up. When the barley peptide-quercetin composite nano system is prepared, setting the core material ratio (w/w) to be 1. Taking 50 mu L of quercetin mother liquor, slowly dropwise adding the quercetin mother liquor into a barley self-assembly peptide aqueous solution, applying ultrasonic treatment by using an ultrasonic cell disruption instrument, using a 30w (watt) horn, controlling the power to be 80%, controlling the working time to be 3s each time, stopping the working time to be 2s in the middle, treating each sample for 5min, placing the sample in an ice bath in the ultrasonic process, absorbing the energy overflowing from the ultrasonic, and avoiding excessive temperature rise and denaturation of the sample. Centrifuging (1000g, 2min) for five times, removing foams generated by ultrasonic treatment, unencapsulated quercetin and impurity precipitate, and taking supernatant to obtain composite nanoparticle aqueous solution. And measuring the content of the quercetin in the solution by using an ultraviolet spectrophotometer under the maximum absorption wavelength 374nm of the quercetin. The encapsulation efficiency, the drug loading capacity and the solubilization times are calculated according to the following formulas:

table 2 shows the embedding of quercetin by graded concentrations of barley self-assembly peptide. It can be seen that when the core material ratio is greater than 1; when the core material ratio is less than 1. Therefore, the core material ratio of the barley self-assembly peptide-quercetin composite nano-particles prepared by the liquid-liquid dispersion self-assembly and ultrasonic-assisted methods is about 1. Under the condition of a core material ratio of 1.

TABLE 2

A comparison of the construction of the nano-loading system using barley protein and barley peptide is shown in FIG. 8. Under the current preparation conditions or methods, the barley protein cannot be directly dissolved in water, and a large amount of organic solvent is needed to participate in the construction of a load system, and the barley protein spontaneously aggregates and precipitates in the water environment and cannot be embedded, so that the barley protein is not suitable for being directly constructed in the water environment in a self-assembly manner. Compared with the barley protein, the barley peptide has stronger amphipathy due to hydrophobic and hydrophilic groups exposed by enzymolysis, can be directly dissolved in water and self-assembled to form a nano-loading system, is clear and transparent in water and is not easy to precipitate.

Example 6 barley peptide nanocarriers entrap coffee phenyl acetate (CAPE)

20mg of Caffeic Acid Phenethyl Ester (CAPE) was weighed out and dissolved in 1mL of ethanol to prepare a mother liquor. 40mg of the barley protein was weighed, self-assembled and dispersed in deionized water, and after 100. Mu.L of CAPE ethanol solution was added, sonication was performed for 5 minutes at a power of 240W in a sonicator. And centrifuging at 1000 Xg for 2 minutes and repeating for 5 times to separate out unencapsulated CAPE and remove bubbles on the solution, wherein the supernatant is the CAPE-peptide nano carrier solution. Taking 100 mu L of nano carrier solution, and using ethanol to fix the volume to 1mL. A standard curve was prepared by preparing 1, 5, 10, 15, 20. Mu.g/mL CAPE ethanol solutions from the CAPE ethanol mother liquor. CAPE content was determined by HPLC using a 250mm × 4.6 μm C18 reverse phase chromatography column with an 80% methanol solution (methanol: water: formic acid =80: 0.1, v). The sample solution was filtered through a 0.45 μm organic phase filter and 15 μ L of the sample was applied each time. As a control, a saturated aqueous solution of CAPE was prepared by adding 100. Mu.L of the CAPE ethanol mother liquor to 10mL of deionized water and sonicating and centrifuging at 1000 Xg to remove insoluble components. And (3) drawing a standard curve by taking the CAPE concentration as an abscissa and taking the liquid chromatogram peak area as an ordinate. The envelope rate and the drug loading rate are calculated according to the following formula:

CAPE is a small molecular active substance and has various biological activity functions, such as antioxidant, anticancer, antiviral and anti-inflammatory effects. However, CAPE has poor water solubility and is easily oxidized by light, oxygen and the like, and the application of CAPE is greatly limited. As shown in Table 3, the solubility of CAPE in water is 1.80. Mu.g/mL, and then the barley self-assembly peptide nano-carrier can well embed CAPE, greatly enhance the solubility of CAPE, increase to 168.4. Mu.g/mL, and increase by 93.5 times. These results indicate that the barley self-assembly peptide nanocarrier is an excellent carrier for loading functional factors for solubilizing food.

TABLE 3

Fresh CAPE-peptide nanocarriers were prepared as described above, unencapsulated CAPE was removed by centrifugation and sterilized with 0.45 μm filter, sealed and stored at 4 ℃. Meanwhile, the storage stability of the CAPE-peptide nano-carrier is evaluated by storing the free non-embedded CAPE aqueous solution under the refrigeration condition. A tube of the sample was taken on

days

0, 3, 7, 14, 21, and 28 to determine CAPE levels. 100. Mu.L of the solution was diluted to 1mL with ethanol, and the absorbance at 323nm was measured with a microplate reader. A standard curve was generated by measuring 1, 5, 10, 15, 20. Mu.g/mL CAPE in ethanol. The CAPE retention rate is calculated by the following formula:

the change in CAPE retention in aqueous solutions of free CAPE and CAPE-loaded peptide nanocarrier at 4 ℃ and below is shown in FIG. 9. Free CAPE is readily oxidized in water, is difficult to stabilize, and has a retention of about 20% after 28 days of storage at 4 ℃. The CAPE retention rate of the CAPE-loaded peptide nano-carrier aqueous solution is about 80% at 4 ℃, which shows that the barley peptide nano-carrier can effectively protect the CAPE and has the function of stabilizing the CAPE.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. A barley peptide nano-carrier is characterized by being prepared by self-assembling any one or more of the following barley self-assembly peptides:

2. The barley peptide nanocarrier of claim 1, wherein the barley peptide nanocarrier has a particle size of less than 100nm and a moderate hydrophilicity and hydrophobicity.

3. A polynucleotide encoding a barley peptide selected from one or more of the following:

4. The method for preparing the barley peptide nanocarrier according to claim 1, comprising the steps of:

1) Preparation of barley protein:

2) Preparation of the barley protein peptide:

3) Preparing a barley peptide nano-carrier:

dispersing the barley peptide in deionized water, and performing ultrasonic treatment to prepare the barley peptide nano carrier.

5. The method for preparing a barley peptide nanocarrier according to claim 4, wherein the method for preparing barley protein in step 1) comprises: pulverizing barley, sieving, defatting, mixing defatted barley flour with 1-3% NaCl solution (w/w), stirring, and centrifuging to obtain solid residue; and then dispersing the solid residue in 70% volume fraction ethanol solution, performing water bath extraction, centrifuging, collecting precipitate, finally re-dispersing the precipitate in deionized water, adjusting the pH value to 10-12, performing extraction, centrifuging to obtain supernatant, adjusting the pH value of the supernatant to 4.4-4.6, standing at low temperature for precipitation, washing with water, and performing freeze drying to obtain the barley protein.

6. The method for preparing a barley peptide nanocarrier according to claim 4, wherein the conditions for enzymatic hydrolysis of barley protein in step 2) are as follows: the pH value of the enzymolysis environment is 7-9, the enzymolysis temperature is 30-60 ℃, and the ratio of enzyme to substrate is 1:25-100, the enzymolysis time is 0.5-4h;

in the step 2), after enzymolysis of the barley protein is finished, heating the enzymolysis product to 60-100 ℃, then sequentially cooling, adjusting the pH value to be neutral, performing rotary evaporation and concentration, and performing freeze drying to obtain the barley peptide.

7. The method for preparing barley peptide nanocarriers of claim 4, wherein in step 3), the barley protein peptides are dispersed in deionized water at a concentration of 0.1-5mg/mL, and after dissolution, the precipitates are first removed by centrifugation at 500-4000 rpm, the supernatant is collected, and then the supernatant is subjected to ultrasound.

8. The method for preparing the barley peptide nanocarriers according to claim 7, wherein in the step 3), the supernatant is placed in an ultrasonic cleaner, and the barley peptides can be orderly self-assembled to form the nanocarriers under the action of ultrasound, wherein the ultrasound power is 50-600W, and the ultrasound time is 5-45min.

9. The method for preparing a barley peptide nanocarrier according to claim 4, wherein in the step 4), after the barley peptide nanocarrier is obtained in the step 3), the separation and purification of the barley protein peptide nanocarrier in the step 4) are further performed, and the specific method in the step 4) is as follows:

separating the barley protein peptide nanoparticles by using a chromatographic column, detecting the particle size of each elution peak in the elution process by using deionized water as a mobile phase, measuring the absorbance value at 214nm, and collecting a first peak component, namely the purified barley protein peptide nano carrier.

10. The use of the barley peptide nanocarrier of claim 1 for embedding, solubilizing and stabilizing functional factors for food.