CN118303635A

CN118303635A - TGase-plant protein peptide nanogel delivery system loaded with fat-soluble active substances

Info

Publication number: CN118303635A
Application number: CN202410452713.5A
Authority: CN
Inventors: 方梦雪; 张良晓; 李培武; 马飞; 喻理; 王督
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2024-04-16
Filing date: 2024-04-16
Publication date: 2024-07-09

Abstract

The invention discloses a TGase-plant protein peptide nanogel delivery system for loading fat-soluble active substances, which belongs to the technical field of nanogels. Once ingested, the nanogel degrades in the gastrointestinal tract, ensuring a stable release of the active agent. At the same time, peptides or amino acids produced after digestion are also biologically active, possibly acting synergistically with the biologically active compounds.

Description

TGase-plant protein peptide nanogel delivery system loaded with fat-soluble active substances

Technical Field

The invention belongs to the technical field of nanogels, and particularly relates to a TGase-plant protein peptide nanogel delivery system loaded with a fat-soluble active substance.

Background

In recent years, high fat diets have raised a number of health problems including cardiovascular disease (CVD), diabetes, obesity, non-alcoholic fatty liver disease, and the like. Total cholesterol is a major risk factor for cardiovascular disease. An effective way to control cholesterol levels is to reduce cholesterol absorption and synthesis. In view of the side effects of cholesterol-lowering drugs such as traditional statins, food-derived bioactive compounds have received increased attention for their safety and acceptability.

Resveratrol (3, 5,4' -trihydroxystilbene) is a non-flavonoid polyphenol organic compound, and is antitoxin produced by various plants such as grape, blueberry, raspberry, mulberry, peanut, desmodium and the like. Numerous studies have reported health promoting effects of resveratrol, particularly cholesterol lowering effects. Phytosterol is a functional component which exists in a free state or in a state of being combined with fatty acid, sugar and the like, and is widely present in cell membranes of various plants such as vegetables, fruits and the like. Plant sterols are an active ingredient in plants and have many benefits to human health. The research shows that the phytosterol has the effects of reducing blood cholesterol, preventing and treating prostatic hyperplasia, inhibiting tumor, inhibiting hyperplasia of mammary glands, regulating immunity and the like. Resveratrol and phytosterol are fat-soluble active substances, are fast in metabolism and low in solubility in water, so that bioavailability is low, and application of resveratrol and phytosterol is limited. In order to achieve nutritional supplementation or preventive treatment value, the protein/peptide is currently used as a preferred packaging material for preparing the nanogel, and can improve the solubility and stability of bioactive substances. Considering the disadvantages of animal proteins/polypeptides, such as the unsustainable high price and low acceptance of vegetarian consumers, vegetable proteins/polypeptides have many advantages in terms of sustainability, low price, strong targeting and environmental friendliness, and have great potential as a delivery system wall material. However, hydrolysis of the protein can adversely affect gel formation.

From the above, it is clear that the lipid-soluble active substance has poor water solubility, poor stability and low absorption rate, and thus has extremely low bioavailability, and thus improvement of the bioavailability is strongly desired. There have been multiple difficulties in the delivery of hydrophobic actives, and the construction of self-assembled gel delivery systems is an effective means of increasing the bioavailability of fat-soluble actives. Food-source proteins and polypeptides are widely used for the preparation of hydrophobic active delivery systems due to their good safety and gelling properties. Soy protein, oat protein, pea protein, whey protein, etc. have been used to construct liposoluble active delivery systems, although to some extent the bioavailability and bioavailability of liposoluble actives has been improved. However, the following problems still remain: 1. although many vegetable proteins are inexpensive and safe, they are poorly water soluble, especially soy proteins, and therefore result in undesirable water-soluble effects of the delivery system. 2. The vegetable protein has compact structure and is not easy to digest, so that the delivery system is not easy to degrade after being digested by intestinal tracts, and the release rate of the fat-soluble active substances is not high.

Disclosure of Invention

In order to solve the technical problems, the invention provides a TGase-plant protein peptide nanogel delivery system loaded with a fat-soluble active substance, which not only can improve the bioavailability of the fat-soluble active substance, but also can effectively guide the construction of the nanogel delivery system, thereby providing theoretical basis and technical guidance for the efficient delivery of the hydrophobic active substance and the construction of the peptidyl nanogel delivery system.

In order to achieve the above purpose, the present invention provides the following technical solutions:

The invention provides a preparation method of a TGase-plant protein peptide nanogel delivery system loaded with a fat-soluble active substance, which comprises the following steps:

Dissolving the plant protein peptide in water, stirring until the plant protein peptide is completely dissolved, and refrigerating overnight to obtain a plant protein peptide solution;

dissolving fat-soluble active substances in ethanol to obtain fat-soluble active substance solution;

dissolving TGase (transglutaminase) in water, and stirring until the TGase is completely dissolved to obtain TGase solution;

mixing the plant protein peptide solution, the fat-soluble active substance solution and the TGase solution, heating for reaction, and cooling to obtain the resveratrol loaded TGase-plant protein peptide nanogel delivery system.

Further, the plant protein peptide comprises soybean peptide (SPP), rapeseed peptide or peanut peptide; the mass concentration of the plant protein peptide solution is 0.1-0.2%; further preferred is a soy peptide.

Further, the fat-soluble active substance comprises resveratrol or phytosterol; the concentration of the fat-soluble active substance solution is 6-12 mg/mL; further preferred is resveratrol.

Further, the mass concentration of the TGase solution is 0.15-3%.

Further, after mixing the plant protein peptide solution, the fat-soluble active substance solution and the TGase solution, the concentration of TGase is 90-900U/g protein. Taking 90U/g protein as an example, the concentration of TGase was calculated as follows: the activity of TGase is 3000U/g, if the mass concentration of the TGase solution is 0.15%, i.e. 0.15g (450U) of TGase is dissolved in 100mL of water, 200. Mu.L of TGase (0.9U) solution is taken and added into 10mL of plant protein peptide solution (the mass concentration of the plant protein peptide solution is 0.1%, i.e. 10mL of plant protein peptide solution contains 0.01g of protein), the ratio of TGase to protein is 0.9U/0.01g of protein, i.e. 90U/g of protein.

Further, the heating reaction is as follows: heating at 50deg.C for 2h, and then heating at 90deg.C for 30min.

Further, the preparation method of the TGase-plant protein peptide nanogel delivery system loaded with the fat-soluble active substances comprises the following steps:

100-200 mg of SPP is dissolved in 100mL of deionized water, stirred until the SPP is completely dissolved, and refrigerated overnight at 4 ℃ to obtain SPP solution;

Dissolving 60-120 mg of fat-soluble active substance in 10mL of ethanol to obtain fat-soluble active substance solution;

dissolving 0.15-3 g of TGase in 10mL of deionized water, and stirring until the TGase is completely dissolved to obtain a TGase solution;

200 mu L of the fat-soluble active substance solution and 200 mu L of the TGase solution are added into 10mL of the SPP solution, after vortex mixing, the mixture is heated for 2 hours at 45-55 ℃, then heated for 30 minutes at 90-95 ℃, and the TGase-plant protein peptide nanogel delivery system loaded with the fat-soluble active substance is obtained after cooling, wherein the final concentration of the TGase is about 90-900U/g protein.

Preferably, the soybean peptide is embedded with resveratrol. Compared with protein, the polypeptide has the advantages of gelling capability and various biological activities, and is an ideal wall material for embedding resveratrol. Therefore, the resveratrol loaded nanogel prepared by reasonably utilizing the soybean peptide can not only meet the effective encapsulation of resveratrol, but also meet the effective release of resveratrol. Once ingested, the nanogel degrades in the gastrointestinal tract, ensuring a stable release of the active agent. At the same time, peptides or amino acids produced after digestion are also biologically active, possibly acting synergistically with the biologically active compounds. To avoid this problem, the present invention uses SPP to prepare a nanogel delivery system, since hydrolysis of the protein can adversely affect gel formation. Hydrophobic attractive forces between SPPs can re-embed hydrophobic groups in larger agglomerates, ultimately leading to improved stability of the nanogel. TGase can induce the acyl transfer between glutamine residue and lysine residue, and is a cross-linking agent for forming network structure by protein microcapsule wall material. Compared with soybean protein, the hydrolysate, namely soybean peptide, has the following advantages: 1. the soybean protein has better water solubility, the rigid structure is destroyed after the soybean protein is hydrolyzed, the molecular weight is reduced, the water solubility is enhanced, and the water solubility of a delivery system can be improved. 2. The soybean peptide is easier to digest, can be degraded and released in intestinal tracts, and improves the resveratrol release rate. 3. The soybean peptide also has gel capability, and can effectively embed resveratrol. 4. The soybean peptide has multiple physiological functions and can play a synergistic effect with resveratrol. However, soybean peptide also has a problem that stability is poor and an embedding effect may be not ideal, so that TGase needs to be added. TGase is an enzyme capable of catalyzing lysine and glutamine side chains to generate isopeptide crosslinking, so that the nanogel is not easy to degrade and damage in the gastric digestion process, and the biological accessibility of resveratrol is improved; the crosslinked structure of the nano gel can be enhanced, and the embedding rate and stability of resveratrol are improved. In addition, TGase has a protective effect on the active peptide in the soybean peptide, can maintain the functional activity of the active peptide, and can induce more active peptides in the digestion process. Therefore, the soybean peptide and the TGase are jointly used for constructing the resveratrol loaded nanogel delivery system, the bioavailability and the bioavailability of the resveratrol can be effectively improved, and meanwhile, the resveratrol and the bioactive peptide can still be released after the crosslinked peptide nanogel (namely the resveratrol loaded TGase-soybean peptide nanogel delivery system) is digested in a complex matrix.

The invention also provides a TGase-plant protein peptide nanogel delivery system loaded with the fat-soluble active substances, which is obtained by the preparation method.

The invention also provides application of the TGase-plant protein peptide nanogel delivery system loaded with the fat-soluble active substances in improving the bioavailability of resveratrol.

The invention also provides an application of the TGase-plant protein peptide nanogel delivery system loaded with the fat-soluble active substances in preparation of medicines for reducing cholesterol.

Compared with the prior art, the invention has the following advantages and technical effects:

According to the invention, the vegetable protein peptide and the TGase are utilized to prepare the nanogel delivery system for loading the fat-soluble active substances, preferably, the soybean peptide and the TGase are utilized to prepare the nanogel delivery system for loading the resveratrol, so that the bioavailability of the resveratrol can be effectively improved, and substances with cholesterol-reducing activity can be generated after gastrointestinal digestion, and the resveratrol is assisted to play a role in reducing cholesterol together.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

FIG. 1 is a graph showing the encapsulation and release rates of SPP-NG in comparative example 1, SPP-TGNG in example 1, and SPI-NG in comparative example 2;

FIG. 2 is the size and morphology of SPP-NG in comparative example 1, SPP-TGNG in example 1, and SPI-NG in comparative example 2, where A is an AFM image of SPI-NG, B is an AFM image of SPP-NG, C is an AFM image of SPP-TGNG, D is a TEM image of SPI-NG, E is a TEM image of SPP-NG, and F is a TEM image of SPP-TGNG;

FIG. 3 is a microstructure of SPP-NG in comparative example 1, SPP-TGNG in example 1, and SPI-NG in comparative example 2, wherein A is an SEM image of SPI-NG, B is an SEM image of SPP-NG, and C is an SEM image of SPP-TGNG;

FIG. 4 is the bile acid binding capacity of the SPI-NG digest of comparative example 1, SPP-TGNG of example 1, and comparative example 2;

FIG. 5 is a schematic diagram showing the docking of cholesterol lowering peptide to CE molecules, wherein A is a schematic diagram showing the docking hydrogen bond between orlistat (control) and CE molecules, B is a schematic diagram showing the docking two-dimensional interaction between orlistat (control) and CE molecules, and C is a schematic diagram showing the docking of orlistat (control) and CE molecules Schematic of residues in the range, D is schematic of the hydrogen bond between peptide PDCW and CE molecule, E is schematic of the two-dimensional interaction between peptide PDCW and CE molecule, F is schematic of the docking between peptide PDCW and CE moleculeIn-range residue schematic diagram, G is schematic diagram of peptide SL and CE molecule butt joint hydrogen bond, H is schematic diagram of peptide SL and CE molecule butt joint two-dimensional interaction, I is peptide SL and CE molecule butt jointSchematic representation of residues within the range;

FIG. 6 shows the effect of soy peptide nanogels of different TGase concentrations on resveratrol encapsulation efficiency in examples 2-3, comparative examples 3-4;

FIG. 7 shows the effect of soy peptide nanogels of different TGase concentrations on resveratrol release rates in examples 2-3, comparative examples 3-4;

FIG. 8 is AFM image of soybean peptide nanogels of different TGase concentrations in examples 2-3, comparative examples 3-4, where A is comparative example 3, B is comparative example 4, C is example 2, and D is example 3;

FIG. 9 shows CE inhibition ratios of soybean peptide nanogels of different TGase concentrations in examples 2 to 3 and comparative examples 3 to 4;

Fig. 10 is a schematic diagram of the docking of a cholesterol lowering peptide to a CE molecule, wherein a is a schematic diagram of the docking of a peptide LASLLPWV to a CE molecule, B is a schematic diagram of the docking of a peptide LASLLPWV to a CE molecule, C is a schematic diagram of the docking two-dimensional interaction of a peptide LASLLPWV to a CE molecule, D is a schematic diagram of the docking of a peptide SNGPFQLTKN to a CE molecule, E is a schematic diagram of the docking of a peptide SNGPFQLTKN to a CE molecule, and F is a schematic diagram of the docking two-dimensional interaction of a peptide SNGPFQLTKN to a CE molecule.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The invention discloses a TGase-plant protein peptide nanogel delivery system loaded with a fat-soluble active substance. The following examples illustrate resveratrol and soy protein in detail.

In the examples of the present invention, each of the raw materials used was commercially available. Typically, but not by way of limitation, soy Protein (SPI) and resveratrol are purchased from Shanghai source leaf biotechnology limited (Shanghai, china). Soy peptide (SPP) was purchased from cantonese peptide biotechnology limited (cultuce celebration). Transglutaminase (TGase, activity: 3000U/g) was purchased from Kinetika Biotechnical Company (Lugano, switzerland). Pepsin (EC 3.4.21.4), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) were purchased from Sigma-Aldrich (St.Louis, MO, USA). Chromatographic grade acetonitrile and methanol were purchased from Thermo FISHER SCIENTIFIC (Rockford, ill., USA). The other reagents are all analytical grade reagents.

In an embodiment of the invention, the characterization method is as follows:

(1) Calculation of Encapsulation Efficiency (EE):

Firstly, mixing 0.5mL of sample with 9.5mL of ethanol, centrifuging for 10min, and removing unencapsulated resveratrol in the nanogel; the concentration of free resveratrol in the supernatant was then assessed by uv absorbance at 306nm (Liu, qin, jiang, chen, & Zhang, 2022) and the concentration of resveratrol was determined according to the resveratrol standard curve.

EE is calculated as follows:

EE(％)＝m_e/m_t×100％

Wherein m _t is the total resveratrol addition amount, and m _e is the resveratrol encapsulation amount.

(2) Atomic Force Microscope (AFM)

To investigate the morphology of the nanogels, 5 μl of the samples were deposited on a mica plate and then air dried. The mica plate is then placed on the console of an AFM (Dimension Icon, BRUKER, germany) and imaged with a cantilever probe scan.

(3) Transmission Electron Microscope (TEM)

A 0.5 μl sample of the nanogel was dropped onto a carbon-coated copper mesh and dried completely at room temperature. Then, TEM images were observed with TEM (JEM-F200, JEOL, japan) at an accelerating voltage of 200kV (Wen et al 2023).

(4) Scanning Electron Microscope (SEM)

To observe the network structure of the nanogel, the lyophilized nanogel was attached to an aluminum strip with a double-sided adhesive carbon tape, and coated with gold, then observed by scanning electron microscopy (Quanta 250feg, fei, usa) (Wang et al, 2022).

(5) In vitro digestion of nanogels

Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) were prepared according to previous studies (Fang et al, 2022; chen et al 2020). For gastric digestion, the pH of the nanogel was adjusted to 2.0, then 5mL of the nanogel was placed in 5mL SGF, shaken in a 37℃water bath for 2h, heated to 100℃to stop pepsin activity for 10min, centrifuged at 4,000g for 15min, and the supernatant was isolated. Intestinal digestion the pH of the gastric sample was adjusted to 7.0 with saturated NaHCO ₃ and 1M NaOH, 10mL SIF was added, incubated for 4h in a 37℃water bath with shaking, digestion stopped by heating at 100℃for 10min,4000g centrifuged for 15min, and the supernatant was isolated.

(6) Calculation of the Release Rate (RE) of nanogels after digestion

0.5ML of the nanogel intestinal digest was mixed with 9.5mL of ethanol and centrifuged for 10min to remove unencapsulated resveratrol from the nanogel. The concentration of free resveratrol in the supernatant was determined using the 306nm ultraviolet absorbance. And determining the concentration of resveratrol according to the resveratrol standard curve. RE of the nanogel was calculated according to the following formula.

RE(％)＝m_r/m_e×100％

M _e is resveratrol encapsulation amount, and m _r is resveratrol release amount after in vitro digestion.

(7) Determination of the inhibition Capacity of nanogel digests CE (cholesterol esterase)

The digestions were incubated with 50. Mu.L of 5mM p-nitrobenzoate substrate in 100mM sodium phosphate buffer and 100mM NaCl in 96-well plates. 50. Mu.L of 5. Mu.g/mL porcine pancreatic cholesterol esterase was added to each well, incubated at 37℃for 30min, quenched by the addition of 1M HCl, and absorbance was measured at 405 nm. CE suppression capability was calculated as follows.

CE inhibition (%) = (OD _c-OD_d)/(OD_a-OD_b) ×100%

OD _a is absorbance with cholesterol esterase and without sample; OD _b is the absorbance without cholesterol esterase and without sample; OD _c is absorbance with cholesterol esterase and sample; OD _d is absorbance with cholesterol esterase and without sample.

(8) Determination of bile acid binding Capacity of nanogel digests

The digestions (1 mL) and 0.01mol/L HCl (0.5 mL) were incubated at 37℃for 1h. Then 5mL (1 mg/mL) of a bile salt solution containing sodium cholate and sodium deoxycholate was added and mixed. After incubation for 1h at 37℃precipitation with 4 volumes of 95% ethanol, centrifugation at 1000g for 10min. The final bile acid content in the supernatant was determined using a total bile acid assay kit (E003-2-1, nanjing's established bioengineering, nanjing, china) and represented the total bile acid content. Bile acids were measured using a microplate spectrophotometer at a dominant wavelength of 405nm and a minor wavelength of 630 nm. The 630nm absorbance was subtracted from the 405nm absorbance to give the final absorbance value. The relative bile acid binding capacity was calculated using cholestyramine as a control.

(9) Post-digestion peptide fragment analysis

Polypeptides in the in vitro digests were identified using LC-MS/MS (AB SCIEX, canada) with some modifications (Fang, xiong, hu, yin, & You, 2019). MS scans were performed from 100 to 1500 μm with a time span of 250 milliseconds. For MS/MS analysis, each scan cycle includes one full scan mass spectrum (m/z ranging from 100 to 1500, charge state from 2 to 5), followed by 40 MS/MS events. The threshold count is set to 120 to activate MS/MS accumulation and the pre-target ion exclusion is set to 18s. The data is retrieved in UniProt Glycine max database (http:// www.UniProt.org /). Peptide sequences with high confidence (> 95%) were considered identified peptides and analyzed in this study. The spatial structure of the peptide was demonstrated with VMD 1.9.3 (Humphrey, dalke, & Schulten, 1996). The potential of peptide sequences to exert biological effects associated with peptides was assessed using an in-line tool PEPTIDERANKER (http:// discoldeep. Ucd. Ie/PEPTIDERANKER /).

(10) Molecular docking

The docking software used in the present invention is Autodock vina.1.2. According to the method of Silva et al (2021), HMG-CoA reductase (PDB ID:1HW 9) was used as protein receptor for peptides with potential HMG CoA reductase binding peptides. The grid points on each side of XYZ are set asThe connection time was set to 10. The resting conformation with the lowest binding energy is selected as the optimal conformation.

According to the method of Ajayi et al (2021), for peptides with potential CE reductase binding peptides, the docked protein receptor was selected as CE (PDB ID:1F 6W), and the three-dimensional structure was downloaded from the Protein Database (PDB). Orlistat was used as control. The grid points on each side of XYZ are set asThe connection time was set to 10. The resting conformation with the lowest binding energy is selected as the optimal conformation.

Data analysis: the experimental results were repeated at least three times and expressed as the mean standard deviation. Data were statistically significant using one-way analysis of variance (ANOVA) with a p-value <0.05 for differences using the Duncan test. Statistical analysis and data were performed using GRAPHPAD PRISM (GraphPad Software, san Diego, CA, USA).

The technical scheme of the invention is further described by the following examples.

Example 1

100Mg of SPP is dissolved in 100mL of deionized water, stirred magnetically until the SPP is completely dissolved, and refrigerated overnight at 4 ℃ to obtain 0.1wt% of SPP solution;

dissolving 62.5mg of resveratrol in 10mL of ethanol to obtain a resveratrol solution of 6.25 mg/mL;

170mg of TGase is dissolved in 10mL of deionized water, and stirred until the TGase is completely dissolved, so as to obtain TGase solution;

200 mu L of resveratrol solution and 200 mu L of TGase solution are added into 10mL of SPP solution, vortex mixing is carried out, heating is carried out for 2 hours at 45 ℃, heating is carried out for 30 minutes at 90 ℃, and the mixture is placed into an ice bath for cooling, so that the TGase-soybean peptide nanogel delivery system loaded with resveratrol is obtained, wherein the final concentration of TGase is 100U/g protein and is named as SPP-TGNG.

Example 2

200Mg of SPP is dissolved in 100mL of deionized water, stirred magnetically until the SPP is completely dissolved, and refrigerated overnight at 4 ℃ to obtain 0.2wt% of SPP solution;

dissolving 100mg of resveratrol in 10mL of ethanol to obtain 10mg/mL of resveratrol solution;

Dissolving 300mg of TGase in 10mL of deionized water, and stirring until the TGase is completely dissolved to obtain TGase solution;

200 mu L of resveratrol solution and 200 mu L of TGase solution are added into 10mL of SPP solution, vortex mixing is carried out, heating is carried out for 2 hours at 55 ℃, heating is carried out for 30 minutes at 95 ℃, and the mixture is placed into an ice bath for cooling, so that the TGase-soybean peptide nanogel delivery system loaded with resveratrol is obtained, wherein the final concentration of TGase is 90U/g protein and is named as P90.

Example 3

The difference from example 2 is that the amount of TGase added is 3g, i.e. the final concentration of TGase in this comparative example is 900U/g protein, and the prepared resveratrol loaded TGase-soy peptide nanogel delivery system is designated as P900.

Comparative example 1

The procedure is as in example 1, except that no TGase is added and the resulting nanogel is designated SPP-NG.

Comparative example 2

The difference from example 1 is only that SPP is replaced by an equal amount of SPI, and the resulting nanogel is designated SPI-NG.

Comparative example 3

The difference was only that TGase was not added and the prepared nanogel was designated as P0 as in example 2.

Comparative example 4

The difference from example 2 was that the amount of TGase added was 0.03g, i.e., the final concentration of TGase was 9U/g protein in this comparative example, and the prepared nanogel was designated as P9.

Encapsulation and release rates are important indicators for evaluating the efficacy of nanogel administration. The encapsulation and release rates of SPP-NG in comparative example 1, SPP-TGNG in example 1, and SPI-NG in comparative example 2 are shown in FIG. 1, and the encapsulation rate of SPP-NG is lower than that of SPI-NG, probably due to the looser structure formed by the hydrolyzed peptides (see FIG. 3 for details). The higher release rate of SPP-NG also indicates that the structure is not compact enough, and is easy to degrade in the digestion process, so that more resveratrol is released. Thus, to enhance EE of the nanogel, TGase was incorporated into SPP to form intramolecular and intermolecular isopeptide crosslinks. The strengthening of the network structure is beneficial to improving the EE of resveratrol. There is no significant difference between the encapsulation efficiency of SPP-TGNG and SPI-NG, and the encapsulation efficiency is relatively high; there is no significant difference between the release rate of SPP-TGNG and SPP-NG, and the release rate is relatively high; the SPP-TGNG effect is best by comprehensively considering the encapsulation efficiency and the release rate. Therefore, the resveratrol loaded TGase-soybean peptide nanogel delivery system has the strongest resveratrol encapsulation rate and release rate.

The dimensions and morphology of SPP-NG in comparative example 1, SPP-TGNG in example 1, and SPI-NG in comparative example 2 are shown in FIG. 2. As can be seen from FIG. 2, SPI-NG is the largest size, about 32.5nm, but may be related to protein aggregation, with an irregular SPI-NG shape. SPP-NG is the smallest in size (about 9.5 nm) and is more irregular in shape than SPI-NG, probably due to the degradation of the protein into very small fragments, thus affecting the morphology after gelation. After treatment with TGase, SPP-TGNG was in the form of regular spheres and increased in size to around 12.8nm, probably due to cross-linking of the polypeptide. The microstructure observed by SEM (fig. 3) demonstrates the improvement of TGase. The SPI-NG structure is coarse and the pores are larger, which may be related to the self-assembly ability and aggregation characteristics of SPI. After hydrolysis to SPP, the number and density of pores in the SPP-NG become greater and the microstructure becomes disordered and disordered, resembling the packing of fragments. The presence of TGase enhances gelation of SPP, and SPP-TGNG has smoother surface, denser network, and significantly reduced and smaller pores. Morphological characterization results indicate that hydrolysis of SPI to SPP reduces the size and network structure of the nanogel, while addition of TGase can enhance structure by inducing formation of isopeptide bonds. The improvement of the internal reticular structure of the nano gel ensures that the matrix structure is more uniform and compact, and is beneficial to the encapsulation of resveratrol. Enzymatic hydrolysis may be an efficient method of manufacturing protein nanoparticles because of the small size favoring solubility and biological accessibility. Therefore, the resveratrol loaded TGase-soybean peptide nanogel delivery system has a compact network structure and smaller size, and is beneficial to encapsulating resveratrol.

The bile acid binding capacity can reflect to some extent the cholesterol lowering capacity, and the SPP-NG in comparative example 1, SPP-TGNG in example 1 and SPI-NG digest bile acid binding capacity in comparative example 2 are shown in FIG. 4. Compared with SPI-NG and SPP-NG, SPP-TGNG has the strongest bile acid binding capacity of 50.92%. Therefore, the digest of the resveratrol loaded TGase-soy peptide nanogel delivery system has the strongest cholesterol lowering capacity.

Peptide PDCW and peptide SL in SPP-TGNG digests were predicted to have cholesterol lowering ability by inhibiting CE activity by analysis and prediction of peptide fragments in digests using PEPTIDE RANKER and Pepsite 2. CE is a member of the alpha/beta hydrolase family, a bile salt-activated lipase that catalyzes the hydrolysis of dietary cholesterol esters to free cholesterol in the small intestine. Thus, inhibition of CE activity can reduce serum cholesterol levels at a reasonable rate. The intrinsic mechanism of CE inhibition by molecular docking pair PDCW and SL was analyzed in depth and the results are shown in figure 5. The results show that PDCW and SL have good binding capacities with respect to CE binding energies of-8.7 and-5.3 kcal/mol, respectively. Therefore, the resveratrol loaded TGase-soybean peptide nanogel delivery system can generate cholesterol-lowering peptide and improve the cholesterol-lowering capacity of the resveratrol-loaded TGase-soybean peptide nanogel delivery system.

The effect of the soy peptide nanogels of different TGase concentrations in examples 2-3, comparative examples 3-4 on resveratrol encapsulation efficiency is shown in fig. 6, and the effect of the soy peptide nanogels of different TGase concentrations in examples 2-3, comparative examples 3-4 on resveratrol release efficiency is shown in fig. 7. It can be known that the encapsulation efficiency of the nanogel is gradually increased along with the increase of the TGase concentration, and the release rate is not changed significantly, which indicates that the TGase concentration of 90 to 900U/g protein is beneficial to improving the encapsulation efficiency and the release rate of the soybean peptide on resveratrol.

AFM images of soybean peptide nanogels of different TGase concentrations in examples 2-3, comparative examples 3-4 are shown in FIG. 8, which shows that the nanogel size increases gradually from 3.5nm to 59.1nm as the TGase concentration increases. Thus, TGase induced cross-linking increases the size of the nanogel.

The CE inhibition ratios of the soybean peptide nanogels of examples 2 to 3 and comparative examples 3 to 4 at different TGase concentrations are shown in fig. 9, and it is understood that the CE inhibition ratio of the nanogels gradually increases with increasing TGase concentration, but there is no significant difference between P90 and P900. Thus, a TGase concentration of 90 to 900U/g protein is beneficial to increase the cholesterol lowering capacity of the soy peptide nanogel digests.

Peptide fragments in the digestate were analyzed and predicted using PEPTIDE RANKER and Pepsite2, and peptides LASLLPWV and SNGPFQLTKN in the P900 digestate were presumed to have cholesterol-lowering ability by inhibiting CE activity. CE is a member of the alpha/beta hydrolase family, a bile salt-activated lipase that catalyzes the hydrolysis of dietary cholesterol esters to free cholesterol in the small intestine. Thus, inhibition of CE activity can reduce serum cholesterol levels at a reasonable rate. The intrinsic mechanism of CE inhibition was analyzed in depth using molecular docking pairs LASLLPWV and SNGPFQLTKN, and the results are shown in fig. 10. The results show that LASLLPWV and SNGPFQLTKN have good binding capacity to CE. Therefore, the TGase concentration of 900U/g protein can enable the soybean peptide nanogel to generate cholesterol-reducing peptide, and improve the cholesterol-reducing capability of the soybean peptide nanogel.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a TGase-plant protein peptide nanogel delivery system loaded with a fat-soluble active substance, characterized in that it comprises the following steps:

Dissolve the plant protein peptide in water, stir until completely dissolved, and refrigerate overnight to obtain a plant protein peptide solution;

dissolving the fat-soluble active substance in ethanol to obtain a fat-soluble active substance solution;

Dissolve TGase in water and stir until completely dissolved to obtain a TGase solution;

The plant protein peptide solution, the fat-soluble active substance solution and the TGase solution are mixed, heated for reaction, and cooled to obtain a TGase-plant protein peptide nanogel delivery system loaded with the fat-soluble active substance.

2. The method for preparing the TGase-plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 1, characterized in that the plant protein peptide comprises soybean peptide, rapeseed peptide or peanut peptide; and the mass concentration of the plant protein peptide solution is 0.1-0.2%.

3. The method for preparing the TGase-plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 1, characterized in that the fat-soluble active substances include resveratrol or phytosterols; and the concentration of the fat-soluble active substance solution is 6 to 12 mg/mL.

4. The method for preparing the TGase-plant protein peptide nanogel delivery system loaded with a fat-soluble active substance according to claim 2 or 3, characterized in that the plant protein peptide is a soybean peptide; and the fat-soluble active substance is resveratrol.

5. The method for preparing the TGase-plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 1, characterized in that the mass concentration of the TGase solution is 0.15-1.5%.

6. The method for preparing the TGase-soybean plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 1, characterized in that after mixing the plant protein peptide solution, the fat-soluble active substance solution and the TGase solution, the concentration of TGase is 90 to 900 U/g protein.

7. The method for preparing the TGase-soybean plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 1, characterized in that the heating reaction is: first heating at 45-55°C for 2h, and then heating at 90-95°C for 30min.

8. A TGase-plant protein peptide nanogel delivery system loaded with fat-soluble active substances, characterized in that it is prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the TGase-soybean plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 8 in improving the bioavailability of resveratrol.

10. Use of the TGase-soybean plant protein peptide nanogel delivery system loaded with fat-soluble active substances according to claim 8 in the preparation of a cholesterol-lowering drug.