A mung bean-derived assembled peptide, its preparation method and application

Abstract

The present invention relates to a mung bean self-assembling peptide, its preparation method and application. They are named P1, P2, P3, and P4 respectively, and their amino acid sequences are Ser-Asn-Leu-Asn-Phe-Phe, Ala-Phe-Gly-Ile-Asn, Leu-Leu-Pro-His, and Glu-Ile-Pro-Thr-Glu-Val-Leu. The present invention also provides a preparation method of the self-assembling peptide. Mung bean globulin is prepared from mung beans as raw materials, and then mung bean peptides are obtained by enzymatically hydrolyzing mung bean globulin with trypsin and subtilisin. Mung bean peptide self-assembled nanoparticles are prepared by ultrasonic-assisted method, and further the mung bean peptide self-assembled nanoparticles are separated and purified by size exclusion chromatography column to obtain mung bean self-assembling peptides with self-assembling ability. The self-assembling peptides provided by the present invention can be used for encapsulation of hydrophobic functional factors, significantly improving the water solubility, stability and bioavailability of hydrophobic functional factors. Compared with the prior art, the preparation process of the self-assembling peptides provided by the present invention is simple, low in cost, free of toxic and harmful chemical solvent residues, high in safety, and has broad application prospects in the fields of food, biomedicine, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide self-assembly, and in particular relates to a mung bean self-assembled peptide, a preparation method thereof and an application thereof. Background Art

[0002] A polypeptide is a compound formed by α-amino acids linked together by peptide bonds, and has many advantages such as good biocompatibility and high safety. Self-assembled peptides are a class of polypeptides that can spontaneously form nanostructures and have important application values in the fields of biomedicine, materials, chemistry, cosmetics, food, etc. Self-assembled polypeptides originate from the self-assembly phenomenon of proteins in nature. By adjusting the structure of polypeptide molecules and changing the external environment, polypeptide molecules can self-assemble into assemblies with specific morphologies and structures spontaneously or triggered by non-covalent bond forces such as hydrogen bonds, hydrophobic interactions, and stacking interactions. Currently, an important application of self-assembled peptides is to encapsulate or load hydrophobic compounds, thereby improving the solubility and stability of hydrophobic functional components or drugs. The nano-carrier system constructed by polypeptide self-assembly has received extensive attention due to its advantages such as good biocompatibility and strong loading capacity. At present, people mainly prepare self-assembled peptides by artificial chemical synthesis methods and then prepare nanoparticles with different structures and functions for the encapsulation and loading of hydrophobic active ingredients / drugs. However, there are many problems in the artificial chemical synthesis of self-assembled peptides. For example, a large amount of organic harmful reagents are used in the chemical synthesis of self-assembled peptides, which limits their application in the fields of food, cosmetics, etc.; the cost is high. Generally, the cost of synthesizing gram-scale self-assembled peptides is often several thousand yuan, and the cost increases with the increase of the peptide chain length; it cannot be prepared on a large scale, which limits the practical application of peptide-based self-assembled nano-carriers. Therefore, there is an urgent need to find other methods to prepare safe and low-cost self-assembled peptide nano-carriers on a large scale.

[0003] Mung bean is a common food raw material. Mung bean is rich in protein, and the protein content is 22-26%, which is 2.3 times that of wheat flour, 2.7 times that of millet, 3 times that of corn flour, and 3.2 times that of rice. Currently, mung beans are mainly used to extract starch to make mung bean vermicelli and mung bean paste, etc., but its main component, mung bean protein, has not been fully utilized. Mung bean is rich in protein, so it is a potential source of self-assembled peptides. However, there is no research on preparing self-assembled peptides from mung bean protein yet. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a mung bean-derived self-assembled peptide, a preparation method thereof and an application thereof.

[0005] The present invention aims to use food - derived protein mung bean protein as a raw material, through biological enzymatic hydrolysis, and then separate and purify peptide nanoparticles to obtain self - assembling peptides, and further apply them to solubilize, stabilize hydrophobic functional components and improve their bioavailability.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] In the first aspect of the present invention, mung bean - derived self - assembling peptides are provided, selected from one or more of the following self - assembling peptides:

[0008] Self - assembling peptide P1, which is SNLNFF, and its amino acid sequence is Ser - Asn - Leu - Asn - Phe - Phe;

[0009] Self - assembling peptide P2, which is AFGIN, and its amino acid sequence is Ala - Phe - Gly - Ile - Asn;

[0010] Self - assembling peptide P3, which is LLPH, and its amino acid sequence is Leu - Leu - Pro - His;

[0011] Self - assembling peptide P4, which is EIPTEVL, and its amino acid sequence is Glu - Ile - Pro - Thr - Glu - Val - Leu.

[0012] Preferably, the self - assembling peptide is derived from mung bean globulin.

[0013] In the second aspect of the present invention, polynucleotides encoding the mung bean - derived self - assembling peptides are provided.

[0014] In the third aspect of the present invention, a preparation method of mung bean - derived self - assembling peptides is provided, which can be obtained from mung bean globulin by methods of biological enzymatic hydrolysis, separation and purification, or artificially synthesized by genetic engineering methods, or directly prepared by chemical synthesis.

[0015] Artificially synthesizing the mung bean - derived self - assembling peptides by genetic engineering methods is a technical solution that those skilled in the art can achieve. For example, it can be based on DNA recombinant technology and control the sequence synthesis of mung bean - derived self - assembling peptides through a suitable DNA template.

[0016] The method of preparation by chemical synthesis is to synthesize the mung bean - derived self - assembling peptides by using the traditional solid - phase synthesis method.

[0017] Regarding the way of obtaining from mung bean globulin by biological enzymatic hydrolysis, separation and purification, it can be: based on the amino acid sequence of the given mung bean - derived self - assembling peptides, using the conventional enzymatic hydrolysis, separation and purification methods in biological technology to obtain the mung bean - derived self - assembling peptides from mung bean globulin.

[0018] Furthermore, the present invention provides a preparation method of mung bean - derived self - assembling peptides, and this method includes the following steps:

[0019] 1) Preparation of mung bean globulin;

[0020] 2) Enzymatic hydrolysis of mung bean globulin:

[0021] 3) Preparation of mung bean globulin peptide nanoparticles:

[0022] Disperse mung bean globulin peptide in deionized water at a certain concentration, and ultrasonicate to prepare mung bean globulin peptide nanoparticles;

[0023] 4) Separation and purification of mung bean globulin self-assembled peptide:

[0024] Separate mung bean globulin peptide nanoparticles using a size exclusion chromatography column, use deionized water as the mobile phase, detect the particle size of each elution peak during the elution process, collect the particle components with a particle size of 30 - 500 nm, and obtain mung bean globulin self-assembled peptide, that is, the self-assembled peptide derived from mung bean.

[0025] In one embodiment of the present invention, in step 1), the preparation method of mung bean globulin is as follows: First, disperse defatted mung bean powder in deionized water at a solid-to-liquid ratio of 1:10, stir and extract at room temperature, and then centrifuge to collect the supernatant; then adjust the pH of the supernatant to about 4.5, let it stand for precipitation, wash with water, and freeze-dry to obtain mung bean globulin.

[0026] In one embodiment of the present invention, in step 2), the enzymatic hydrolysis conditions of mung bean globulin are as follows: The enzyme used is trypsin or subtilisin, the pH of the enzymatic hydrolysis environment is 7 - 9, the enzymatic hydrolysis temperature is 30 - 60 °C, the enzyme-to-substrate ratio is 1:25 - 100, and the enzymatic hydrolysis time is 0.5 - 4 h.

[0027] In one embodiment of the present invention, in step 3), disperse mung bean globulin peptide in deionized water at a concentration of 0.1 - 100 mg / mL; the power of ultrasonication is 100 - 500 W, and the ultrasonication time is 5 - 25 min.

[0028] In one embodiment of the present invention, in step 4), the chromatography column is a Sephadex G-75 glass chromatography column.

[0029] In the fourth aspect of the present invention, a self-assembled peptide nanoparticle or carrier containing the self-assembled peptide derived from mung bean is provided.

[0030] In the fourth aspect of the present invention, an application of the self-assembled peptide derived from mung bean in encapsulating, solubilizing hydrophobic functional factors and improving their bioavailability is provided.

[0031] Compared with the prior art, the advantages and beneficial effects of the present invention are reflected in the following aspects:

[0032] The mung bean-derived assembled peptide provided by the present invention is derived from food-derived mung bean protein, has good safety, low preparation cost, simple process, and is easy to be prepared on a large scale; the mung bean-derived assembled peptide provided by the present invention is obtained by food processing technology, without residues of toxic and harmful reagents, and the entire process flow meets food-grade requirements. In addition to being used in the biomedical field, it can also be used in the fields of food, health food, cosmetics, etc.; the mung bean-derived assembled peptide provided by the present invention can be used for the loading and embedding of hydrophobic functional factors, which can greatly increase the water solubility, stability and bioavailability of hydrophobic compounds, and has important application value. Description of the Drawings

[0033] Figure 1 Hydrolysis curves of mung bean globulin under trypsin and subtilisin;

[0034] Figure 2 Transmission electron micrograph of mung bean protein peptide self-assembled nanoparticles. A is the peptide nanocarrier obtained by trypsin digestion, and B is the peptide nanocarrier obtained by subtilisin digestion;

[0035] Figure 3 Separation map of mung bean protein peptide nanoparticles;

[0036] Figure 4 MS / MS map of mung bean protein self-assembled peptide SNLNFF;

[0037] Figure 5 MS / MS map of mung bean protein self-assembled peptide AFGIN;

[0038] Figure 6 MS / MS map of mung bean protein self-assembled peptide LLPH;

[0039] Figure 7 MS / MS map of mung bean protein self-assembled peptide EIPTEVL;

[0040] Figure 8 Transmission electron micrograph (TEM) of mung bean protein self-assembled peptide nanocarrier loaded with caffeyl phenylacetate (CAPE). A is the peptide nanocarrier obtained by trypsin digestion, and B is the peptide nanocarrier obtained by subtilisin digestion;

[0041] Figure 9 Stability of mung bean protein peptide nanocarrier loaded with caffeyl phenylacetate (CAPE) at 4°C and 25°C. A is the peptide nanocarrier obtained by trypsin digestion, B is the peptide nanocarrier obtained by subtilisin digestion, and C is free CAPE;

[0042] Figure 10 Small intestine permeability of free CAPE and mung bean protein peptide nanocarrier loaded with CAPE. Detailed Embodiment

[0043] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] Embodiment 1

[0045] The mung beans in this embodiment are derived from self-assembling peptides. The number of self-assembling peptides from which the mung beans are derived is 4, which are named self-assembling peptides P1, P2, P3, and P4 respectively. The amino acid sequence of P1 is Ser-Asn-Leu-Asn-Phe-Phe (SNLNFF), the amino acid sequence of P2 is Ala-Phe-Gly-Ile-Asn (AFGIN), the amino acid sequence of P3 is Leu-Leu-Pro-His (LLPH), and the amino acid sequence of P4 is Glu-Ile-Pro-Thr-Glu-Val-Leu (EIPTEVL).

[0046] This embodiment also provides a preparation method for the above-mentioned self-assembling peptides from which the mung beans are derived. The method includes the following steps:

[0047] I. Preparation of mung bean globulin

[0048] The defatted mung bean powder is dispersed in distilled water at a solid-liquid ratio of 1:10 (w / v), and stirred at a speed of 500 rpm at room temperature for 1 hour. Centrifuge at a speed of 4000 rpm for 15 minutes with a low-speed centrifuge for solid-liquid separation. Take the supernatant and adjust the pH to about 4.5 with 1M hydrochloric acid, and let it stand at 4°C for 30 minutes to precipitate the protein. Centrifuge at a speed of 4000 rpm for 15 minutes, take the precipitate, disperse it in deionized water at a ratio of 1:2 (v / v), adjust the pH to neutral with 1M sodium hydroxide solution, centrifuge at a speed of 4000 rpm for 15 minutes for solid-liquid separation, and then vacuum freeze-dry the precipitate to obtain mung bean globulin. The purity of the mung bean globulin obtained by this method is about 88.2%.

[0049] II. Enzymatic hydrolysis of mung bean globulin

[0050] (I) Hydrolysis of mung bean globulin by trypsin

[0051] Disperse mung bean protein powder in deionized water, adjust the pH to 8.0 with 0.5 M NaOH solution, heat it in a water bath at 37 °C with a magnetic stirrer and keep it stable. Disperse trypsin in 20 mM sodium carbonate buffer with a pH of 8.0, add the enzyme buffer to the protein solution to start enzymatic hydrolysis, and the ratio of enzyme to substrate is 4:100. During enzymatic hydrolysis, titrate with 0.5 M NaOH solution to keep the pH of the enzymatic hydrolysis system constant at 8.0, and record the volume of NaOH solution consumed. When the pH of the enzymatic hydrolysis system does not change within 5 minutes or changes less than 0.01 within 15 minutes, it is considered that the enzymatic hydrolysis is complete. Boil the enzymatic hydrolysate for 5 minutes to inactivate the enzyme to stop enzymatic hydrolysis, adjust the pH to neutral, centrifuge at 1000 rpm for 10 minutes with a low-speed centrifuge to remove some insoluble substances or large particles, and freeze-dry the supernatant to obtain trypsin-hydrolyzed mung bean protein peptide powder.

[0052] (II) Hydrolysis of mung bean globulin by subtilisin

[0053] Disperse mung bean protein powder in deionized water, adjust the pH to 8.0 with 0.5 M NaOH solution, heat it in a water bath at 50 °C with a magnetic stirrer and keep it stable. Pipette the subtilisin solution, mix it evenly with 20 mM sodium carbonate buffer with a pH of 8.0, and add the enzyme buffer to the protein solution to start enzymatic hydrolysis. The mass fraction of the enzyme in the subtilisin solution is 9%, and the solution density is 1.25 g / mL. Therefore, the ratio of enzyme to substrate is approximately 2:100. During enzymatic hydrolysis, titrate with 0.5 M NaOH solution to keep the pH of the enzymatic hydrolysis system constant at 8.0, and record the volume of NaOH solution consumed. When the pH of the enzymatic hydrolysis system does not change within 5 minutes or changes less than 0.01 within 15 minutes, it is considered that the enzymatic hydrolysis is complete. Boil the enzymatic hydrolysate for 5 minutes to inactivate the enzyme to stop enzymatic hydrolysis, adjust the pH to neutral, centrifuge at 1000 rpm for 10 minutes with a low-speed centrifuge to remove some insoluble substances or large particles, and freeze-dry the supernatant to obtain subtilisin-hydrolyzed mung bean protein peptide powder.

[0054] Calculate the degree of protein hydrolysis based on the volume of 0.5 M NaOH solution consumed recorded in the protein hydrolysis experiment. Record the volume of the cumulative consumed 0.5 M NaOH solution at 10, 30, 60, 90, 120, 150, 180, 210, and 240 minutes of enzymatic hydrolysis time. Calculate the degree of hydrolysis using the pH-stat method, and plot a hydrolysis curve with the enzymatic hydrolysis time as the abscissa and the degree of hydrolysis as the ordinate.

[0055] Figure 1 is the hydrolysis curve of mung bean globulin. Among them, subtilisin has a wide range of hydrolysis sites, so the final degree of hydrolysis of mung bean globulin exceeds 20% and reaches 22%; trypsin specifically hydrolyzes hydrophilic amino acids and has strong specificity, and the final degree of hydrolysis of mung bean globulin is 7%.

[0056] III. Preparation of Mung Bean Globulin Peptide Nanoparticles

[0057] Disperse mung bean globulin peptides prepared by different proteases and with different degrees of hydrolysis at a concentration of 1 mg / mL in deionized water, and sonicate for 10 min under the condition of an ultrasonic power of 300 W to accelerate the dissolution of the peptides and promote the ordered self-assembly of the peptides. After completion, use a dynamic light scattering instrument to measure the self-assembled peptide nano size and polydispersity index (PDI); observe the morphology of the self-assembled peptide nanoparticles by transmission electron microscopy.

[0058] Table 1 shows the sizes and PDI of peptide nanoparticles formed by the self-assembly of mung bean protein peptides hydrolyzed by different proteases and with different degrees of hydrolysis. The results show that the protease type and degree of hydrolysis affect the size and dispersibility of the formed peptide nanoparticles. The size of the self-assembled nanoparticles of mung bean protein peptides first decreases and then increases with the degree of hydrolysis. Therefore, the size of the peptide nanocarrier can be regulated by controlling the degree of hydrolysis. The polydispersity index (PDI) of the mung bean peptide nanocarriers prepared by hydrolysis with subtilisin is smaller than that of the peptide nanocarriers prepared by the other two enzymes. This may be because peptides with weak amphiphilicity tend to form the hydrophobic core of micelles or adsorb hydrophilically on the peptide surface, while peptides with strong amphiphilicity tend to self-assemble to form micelles. These research results have guiding significance for the preparation of other plant protein peptide nanocarriers.

[0059] Table 1 Peptide Nanoparticles Formed by the Self-Assembly of Mung Bean Protein Peptides Hydrolyzed by Different Proteases and with Different Degrees of Hydrolysis

[0060]

[0061] DH represents the degree of hydrolysis, and different letters indicate significant differences, p < 0.05.

[0062] Figure 2 Figure is the transmission electron microscopy (TEM) image of the self-assembled nanoparticles of mung bean protein peptides prepared. A is the peptide nanocarrier obtained by enzymatic hydrolysis with trypsin, and B is the peptide nanocarrier obtained by enzymatic hydrolysis with subtilisin. The mung bean globulin peptide nanoparticles have regular shapes and good stability in aqueous solution, with a clear and transparent appearance and are not prone to precipitation during storage, overcoming the disadvantages that simple mung bean globulin nanoparticles are prone to instability and aggregation. In addition, research reports show that size significantly affects the uptake of nanoparticles by cells, and nanoparticles with a size of less than about 100 nm are more easily taken up. Therefore, the mung bean protein peptide nanoparticles prepared in this patent will be easily absorbed by the body.

[0063] IV. Isolation and Purification of Self-Assembled Peptides of Mung Bean Globulin

[0064] The mung bean globulin self-assembled peptide nanoparticles were separated by size exclusion chromatography. In this example, a Sephadex G-75 glass chromatography column was used. 20 mg of the mung bean globulin peptide nanoparticles prepared in the above steps was dissolved in 2 mL of deionized water, and after passing through a 0.45 μm filter membrane, it was loaded onto the column. Deionized water was used as the mobile phase, and the nanoparticles were eluted at a flow rate of 1 mL / min. The eluate was detected with a UV detector at a wavelength of 214 nm, and the size exclusion chromatogram was recorded by a chromatogram analyzer. An elution tube was collected every 5 minutes, and after dilution by a certain multiple, the particle size of the components of each elution peak was measured. The fraction containing the mung bean globulin peptide nanoparticles was the mung bean globulin self-assembled peptide.

[0065] The separation chromatogram of the mung bean globulin peptide nanoparticles is shown in Figure 3 . The purpose of separation by size exclusion chromatography is to remove some peptides without self-assembly ability and some impurities to purify the sample in order to obtain the self-assembled peptide. Combining the size exclusion chromatogram and particle size analysis, it can be found that most of the self-assembled nanocarriers cannot enter the pores of the gel particles (less than 10 nm) and are quickly eluted from the gaps between the gel particles. The first elution peak is the nanoparticles formed by the self-assembly of the peptides with self-assembly ability. The substances eluted after a long time are hydrophilic peptides that have not undergone self-assembly, or the bound pigments and some salts released during proteolysis.

[0066] Example 2 Analysis and Identification of Mung Bean Globulin Self-Assembled Peptide

[0067] The prepared mung bean-derived self-assembled peptide was dissolved in an aqueous formic acid solution, and its sequence was identified by mass spectrometry. A reversed-phase chromatography column was used for separation. Mobile phase A was 0.1% aqueous formic acid solution, and mobile phase B was 0.1% formic acid / 80% acetonitrile solution. The separation gradient was: 4% B (0 min) - 8% B (2 min); 8% B (2 min) - 40% B (45 min); 40% B (45 min) - 60% B (55 min); 60% B (55 min) - 95% B (56 min); 95% B (56 min) - 95% B (66 min). The ion source type was electrospray ionization source (ESI), positive ion scan mode, spray voltage 2200 V, and capillary temperature 270 °C. The primary mass spectrometry parameter settings were: scan range 100 - 2000 m / z, maximum resolution 70000, and automatic gain parameter 3000000. The secondary mass spectrometry parameter settings were: scan range 50 - 2000 m / z.

[0068] The self-assembled peptides prepared from trypsin showed two main ion peaks in mass spectrometry analysis, with m / z = 741.35, carrying 1 charge; and m / z = 521.26, carrying 1 charge. The self-assembled peptides prepared from subtilisin also showed two main ion peaks in mass spectrometry analysis, namely m / z = 479.29, carrying 1 charge; and m / z = 800.44, carrying 1 charge. These four ion peaks were further analyzed by tandem mass spectrometry, and the tandem mass spectrometry spectra are shown in Figures 4 to 7 . Through database matching and manual parsing and calculation, the amino acid sequences of the ions with m / z = 741.35, 521.26, 479.29, and 800.44 were identified as Ser-Asn-Leu-Asn-Phe-Phe (SNLNFF), Ala-Phe-Gly-Ile-Asn (AFGIN), Leu-Leu-Pro-His (LLPH), and Glu-Ile-Pro-Thr-Glu-Val-Leu (EIPTEVL), respectively.

[0069] Vicilin is mainly 8S vicilin, and its amino acid sequence is shown below. It can be seen that the four identified self-assembled peptides are all present in mung bean 8S vicilin.

[0070] The amino acid sequence of mung bean 8S vicilin is shown in SEQ ID NO.1 (single letter, Uniprot protein accession number: Q198W3), as follows:

[0071] MVRARVQLLLGILFLASLSVSFGIVHREHQESQEESDSRGQNNPFYFNSDRRFHTLFKNQYGHLRVIHRFDQRSKQIQNLENYRVVEFKSKPNTLLLPHHADADFLLVVLNGRAILTLVNPDGRDSYILEQGHAQKIPAGTTFFLVNPNDNDNLRIIKLAIPVNNPHRFQNFFLSSTEAQQSYLRGFSKNILEASFDSDFKEIDRVLFGEERQQQHGEESQEEGVIVELKREQIRELIKHAKSSSRKELSSQDEPFNLRNSNPIYSNKFGRWYEITPEKNPQLKDLDVFISSVDMKEGGLLLPHYNSKAIVILVINEGEAKIELVGPSDQQQQDESLEVQRYRAELSEDDVFVIPAAYPVAINATSNLNFFAFGINAENNQRNFLAGEKDNVMSEIPTEVLDVSFPASGNKVEKLIKKQSESHFVDAQPEQQQREEGHKGRKGSLSSILGSLY

[0072] Among them, SNLNFF is located at positions 366 - 371, AFGIN is located at positions 372 - 376, LLPH is located at positions 96 - 99 or 301 - 304, and EIPTEVL is located at positions 395 - 401.

[0073] Example 3 Verification of the self - assembly ability of mung bean globulin self - assembly peptides

[0074] Four peptides, Ser - Asn - Leu - Asn - Phe - Phe (SNLNFF), Ala - Phe - Gly - Ile - Asn (AFGIN), Leu - Leu - Pro - His (LLPH), and Glu - Ile - Pro - Thr - Glu - Val - Leu (EIPTEVL), were commissioned to Zhejiang Hongtuo Technology Co., Ltd. for synthesis by peptide solid - phase synthesis method. The purity of the synthesized peptides was verified to be greater than 95% by high - performance liquid method and mass spectrometry technology. The four peptides were dispersed in deionized water at a concentration of 1 mg / mL and sonicated for 10 min under the condition of an ultrasonic power of 300 W to accelerate the dissolution of the peptides and promote the ordered self - assembly of the peptides. After that, a dynamic light scattering instrument was used to measure the nano - size and polydispersity index (PDI) of the self - assembled peptides. The results are shown in Table 2. It can be seen from the results that the four identified peptides all have the ability of self - assembly and can self - assemble into peptide particles with a size of 20 - 400 nm.

[0075] Table 2 Verification of the self - assembly ability of four peptides

[0076]

[0077] Example 4 Encapsulation of caffeic acid phenethyl ester (CAPE) by mung bean globulin self - assembly peptides

[0078] Dissolve caffeic acid phenethyl ester (CAPE) in ethanol to prepare a stock solution. Weigh the self-assembled peptide of mung bean globulin and disperse it in deionized water. After adding the CAPE ethanol solution, sonicate it in a sonicator cell disruptor at a power of 240 W for 5 minutes. Centrifuge at 1000×g for 2 minutes and repeat 5 times to precipitate the unencapsulated CAPE and remove the foam on the solution. The supernatant is the CAPE-peptide nanocarrier solution. Take 100 μL of the nanocarrier solution and make up to 1 mL with ethanol. Prepare a standard curve with CAPE ethanol stock solution at concentrations of 1, 5, 10, 15, and 20 μg / mL. The determination of CAPE content uses HPLC method. Use a 250 mm×4.6 μm C18 reverse-phase chromatographic column, and use 80% methanol solution (methanol: water: formic acid = 80:20:0.1, v:v:v) as the mobile phase for isocratic elution for 10 minutes. The detection wavelength is 323 nm, the column temperature is 35 °C, and the mobile phase flow rate is 1 mL / min. The sample solution passes through a 0.45 μm organic phase filter membrane, and 15 μL is injected each time. Add 100 μL of the CAPE ethanol stock solution to 10 mL of deionized water and sonicate it. Centrifuge at 1000×g to remove the insoluble components to prepare a saturated aqueous solution of CAPE, which is used as a control. The calculation formulas for the encapsulation efficiency and drug loading are as follows:

[0079]

[0080]

[0081] As a small molecule active substance, CAPE has a variety of bioactive functions, such as antioxidant, anticancer, antiviral, and anti-inflammatory effects, and has important applications in the food and pharmaceutical fields. However, CAPE has poor water solubility and is easily oxidized by light, oxygen, etc., which greatly limits the application of CAPE. As shown in Table 3, the solubility of CAPE in water is 1.80 μg / mL, but the self-assembled peptide of mung bean globulin can well encapsulate CAPE, greatly enhancing the solubility of CAPE. The self-assembled peptide of mung bean prepared with trypsin and subtilisin can encapsulate CAPE, increasing the solubility of CAPE to 177.8 and 164.8 μg / mL respectively, which are increased by 98.6 times and 91.4 times respectively. Figure 8 It is the transmission electron microscopy (TEM) image of the peptide nanocarrier loaded with caffeic acid phenylacetate (CAPE). These results indicate that the self-assembled peptide of mung bean globulin can be used to increase the solubility of hydrophobic active substances.

[0082] Table 3 Results of mung bean self-assembled peptide loaded with CAPE

[0083]

[0084] CAPE-Tps and CAPE-AP respectively represent the encapsulation of CAPE by mung bean self-assembled peptides prepared with trypsin and subtilisin

[0085] Example 5: Mung bean globulin self-assembled peptides enhance the stability of CAPE

[0086] Prepare fresh CAPE-peptide nanocarriers, centrifuge to remove unencapsulated CAPE, sterilize through a 0.45 μm filter membrane, and store sealed at 4 °C and 25 °C. At the same time, store an aqueous solution of CAPE at room temperature and refrigerated conditions to evaluate the storage stability of CAPE-peptide nanocarriers. Take a tube of aliquoted samples at days 0, 3, 7, 14, 21, and 28 respectively to determine the content of CAPE. Take 100 μL of the solution and dilute it to 1 mL with ethanol, and measure its absorbance at 323 nm using a microplate reader. The standard curve is plotted by measuring CAPE ethanol solutions at concentrations of 1, 5, 10, 15, and 20 μg / mL. The retention rate of CAPE is calculated by the following formula:

[0087]

[0088] The results are as Figure 9 shown. CAPE is easily oxidized in water, and the retention rate is 22% after storage at 4 °C and 25 °C for 28 days. The retention rates of CAPE loaded with mung bean self-assembled peptides prepared with trypsin and subtilisin as carriers are 29% and 32% after storage at room temperature for 28 days, while the retention rates are 89% and 69% after storage at 4 °C. Mung bean globulin peptides as carriers have a good protective effect on CAPE during the entire storage process. Mung bean self-assembled peptides can competitively inhibit the oxidation of CAPE, and the oxidation rate of the peptides decreases at low temperatures, so CAPE has a high retention rate.

[0089] Example 6: Mung bean globulin self-assembled peptides enhance the bioavailability of CAPE

[0090] Prepare fresh CAPE-peptide nanocarriers and determine the content of CAPE. Take a small segment of empty ileum, tie one end with a cotton thread, and use a blunt syringe to inject K-B solution to check for leaks. Inject 0.5 mL of freshly prepared CAPE-peptide nanocarrier solution into the small segment of empty ileum, tie it tightly, and immerse it in 5 mL of K-B solution. Conduct an in vitro small intestine permeability experiment by shaking in a water bath at 37 °C. Take 1 mL of the outer K-B solution at 30, 60, 120, 180, and 240 minutes, and supplement 1 mL of fresh K-B solution. Use the intestinal segment filled with K-B solution as a reference control for permeability, and measure the absorbance at a wavelength of 323 nm. Dilute the CAPE stock solution with K-B to prepare concentrations of 1, 5, 10, 15, and 20 μg / mL to make a standard curve. Plot the cumulative permeability with time as the abscissa and the cumulative permeability as the ordinate. The cumulative permeability is calculated by the following formula:

[0091]

[0092] The results are as Figure 10 shown. After 4 h of absorption, the cumulative permeability of CAPE embedded in mung bean self-assembled peptides prepared with trypsin and subtilisin was significantly higher than that of unembedded CAPE. This indicates that mung bean peptide embedding can improve the absorption of CAPE in the small intestine and enhance the bioavailability of CAPE.

[0093] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. The mung bean is derived from an assembled peptide, characterized in that, It is SNLNFF, and the amino acid sequence is Ser-Asn-Leu-Asn-Phe-Phe.

2. A polynucleotide encoding the mung bean-derived self-assembling peptide as claimed in claim 1.

3. The preparation method of mung beans derived from assembled peptides according to claim 1, characterized in that, It is obtained from mung bean globulin by methods of biological enzymolysis, separation and purification, or artificially synthesized by genetic engineering methods, or directly prepared by chemical synthesis; the enzyme used in biological enzymolysis is trypsin or subtilisin.

4. The preparation method according to claim 3, characterized in that, It is obtained from mung bean globulin by methods of biological enzymolysis, separation and purification, specifically including the following steps: 1) Preparation of mung bean globulin; 2) Enzymolysis of mung bean globulin: 3) Preparation of mung bean globulin peptide nanoparticles: Disperse mung bean globulin peptide in deionized water at a certain concentration, and perform ultrasonic treatment to prepare peptide nanoparticles; 4) Separation and purification of mung bean globulin self-assembling peptide: Separate mung bean globulin peptide nanoparticles using a size exclusion chromatography column, use deionized water as the mobile phase, detect the particle size of each elution peak during the elution process, collect the particle components with a particle size of 30 - 300 nm, and obtain the mung bean globulin self-assembling peptide, that is, the mung bean-derived self-assembling peptide as claimed in claim 1.

5. The preparation method according to claim 4, characterized in that, In step 1), the preparation method of mung bean globulin is: first disperse defatted mung bean powder in deionized water at a solid-liquid ratio of 1:10, stir and extract at room temperature, and then centrifuge to collect the supernatant; then adjust the pH of the supernatant to about 4.5, let it stand for precipitation, wash with water, and freeze-dry to obtain mung bean globulin.

6. The preparation method according to claim 4, characterized in that, In step 2), the enzymolysis conditions of mung bean globulin are: the enzyme used is trypsin or subtilisin, the pH of the enzymolysis environment is 7 - 9, the enzymolysis temperature is 30 - 60 °C, the ratio of enzyme to substrate is 1:25 - 100, and the enzymolysis time is 0.5 - 4 h.

7. The preparation method according to claim 4, characterized in that, In step 3), disperse mung bean globulin peptide in deionized water at a concentration of 0.1 - 100 mg / mL; the power of ultrasonic treatment is 100 - 500 W, and the ultrasonic time is 5 - 25 min.

8. The preparation method according to claim 4, characterized in that, In step 4), the chromatography column is a Sephadex G-75 glass chromatography column.

9. A self-assembling peptide nanoparticle or carrier containing the mung bean-derived self-assembling peptide as claimed in claim 1.

10. Use of the mung bean-derived self-assembling peptide as claimed in claim 1 in embedding, solubilizing hydrophobic functional factors and improving their bioavailability.

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