Soybean peptide-phytosterol nanogel system with cholesterol lowering function as well as preparation method and application of soybean peptide-phytosterol nanogel system

Abstract

The invention discloses a soybean peptide-phytosterol nanogel system with a cholesterol lowering function as well as a preparation method and application thereof, and relates to the technical field of biology. The preparation method comprises the following steps: carrying out encapsulation treatment on phytosterol by using a soybean peptide solution by adopting an emulsification evaporation method to prepare a soybean peptide-phytosterol nanogel system; the preparation method of the soybean peptide solution comprises the following steps: performing enzymolysis treatment on soybean protein by using alkaline protease, and performing centrifugal separation to obtain the soybean peptide solution. According to the soybean peptide-phytosterol nanogel system provided by the invention, the embedding delivery efficiency and stability of phytosterol can be remarkably improved, cholesterol absorption can be competitively inhibited through PS, meanwhile, soybean peptide promotes bile acid excretion, a dual regulation and control mechanism is formed, and a better cholesterol lowering effect is synergistically achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a soybean peptide-phytosterol nanogel system with cholesterol-lowering function, a preparation method and application thereof. BACKGROUND

[0002] High cholesterol levels are a core risk factor for inducing cardiovascular disease. Long-term reliance on drug control of cholesterol levels, its side effects and limitations are increasingly prominent. In this context, natural cholesterol-lowering factors have become a research hotspot.

[0003] Phytosterols (PS) as a natural bioactive compound derived from plants, can effectively reduce blood cholesterol levels by competitively inhibiting the absorption of cholesterol in the intestine, and play an important role in the prevention of cardiovascular disease. However, its strong hydrophobicity leads to low bioavailability, which seriously restricts its large-scale application in the food industry.

[0004] In recent years, proteins and their hydrolysates have been widely used as delivery wall materials for hydrophobic active substances due to their amphiphilic properties and excellent binding performance. Among the many protein species, soy protein is one of the most thoroughly studied proteins in the delivery field. Whether used alone or in combination with other materials, it has shown good application potential. Compared with intact proteins, soy protein hydrolysates not only have smaller molecular size, stronger environmental tolerance, but also better delivery capacity. In addition, the peptides in the hydrolysates also have health-promoting properties, which are expected to produce a synergistic effect with hydrophobic active substances. Using suitable soy peptide (SPP) to prepare nanogels to load PS can fully exert its dual functions as a delivery carrier and cholesterol-lowering.

[0005] It is worth noting that the degree of hydrolysis (DH) is a key control parameter in this study. Peptides of different molecular weights exhibit different properties: on the one hand, DH has a significant impact on the assembly process of hydrolysis products, which directly affects the encapsulation effect of PS. For example, it has been found that different degrees of hydrolysis treatment of wheat gluten protein will produce different assembly behaviors. At the same time, the hydrophobic groups exposed during protein hydrolysis increase the binding sites with active substances and improve the binding stability. Studies have shown that the hydrolysis of whey protein (WPI) promotes the formation of intermolecular hydrogen bonds between WPI and PS, stabilizing the network structure of the complex. As the degree of hydrolysis deepens, the hydrogen bonding sites of Lys are exposed, which is more conducive to the combination of WPI and PS through hydrogen bonds. On the other hand, DH also significantly affects the biological activity of protein hydrolysis products. Studies have shown that the antioxidant and ACE inhibitory activity of whey protein hydrolysate will increase with the increase of DH. However, how to accurately control DH to ensure the effective release of PS and its high cholesterol-lowering ability still needs to be studied. Based on this, the present application intends to prepare a soybean peptide nanogel loaded with phytosterol to achieve efficient delivery of phytosterol and improve its cholesterol-lowering ability. SUMMARY

[0006] The purpose of the present application is to provide a soybean peptide-phytosterol nanogel system with cholesterol-lowering function and its preparation method and application, in order to solve the problems existing in the prior art. The soybean peptide-phytosterol nanogel system can significantly improve the embedding delivery efficiency and stability of phytosterol, not only can compete with cholesterol absorption through PS, but also can promote bile acid excretion through soybean peptides, forming a double regulation mechanism, and synergistically achieving a better cholesterol-lowering effect.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] The present application provides a preparation method of a soybean peptide-phytosterol nanogel system with cholesterol-lowering function, comprising the following steps: using an emulsion evaporation method, using a soybean peptide solution to encapsulate phytosterol to prepare the soybean peptide-phytosterol nanogel system.

[0009] The preparation method of the soybean peptide solution comprises the following steps:

[0010] The soybean protein is subjected to enzymatic treatment by using alkaline protease, and the soybean peptide solution is obtained by centrifugal separation;

[0011] The mass ratio of the alkaline protease to the soybean protein is 1:10; the temperature of the enzymatic treatment is 55℃, the pH is 9.0, and the time is 3h.

[0012] Further, the specific steps of the emulsion evaporation method include: dissolving the phytosterol in an organic solvent, then adding the soybean peptide solution dropwise, after ultrasonic and homogenization treatment, evaporating the organic solvent by drying treatment to obtain the soybean peptide-phytosterol nanogel system.

[0013] Further, the organic solvent is ethanol.

[0014] Further, the ratio of the phytosterol, the organic solvent and the soybean peptide solution is 22.5 mg:5 mL:50 mL.

[0015] The application also provides a soybean peptide-phytosterol nanogel system prepared by the preparation method.

[0016] The application also provides the use of the above-mentioned soybean peptide-phytosterol nanogel system in the preparation of a cholesterol-lowering drug.

[0017] The application also provides a cholesterol-lowering drug, and the active ingredient includes the above-mentioned soybean peptide-phytosterol nanogel system.

[0018] Further, the drug also includes a pharmaceutically acceptable excipient.

[0019] The application also provides the use of the above-mentioned soybean peptide-phytosterol nanogel system in the preparation of a functional food that helps maintain a healthy level of blood lipids.

[0020] The application also provides a functional food that helps maintain a healthy level of blood lipids, and the active ingredient includes the above-mentioned soybean peptide-phytosterol nanogel system.

[0021] The application discloses the following technical effects:

[0022] The application innovatively constructs a soybean peptide-phytosterol nanogel system to solve the problem of low bioavailability of phytosterol (PS) due to poor water solubility and insufficient stability. The soybean peptide-phytosterol nanogel system can significantly improve the embedding delivery efficiency and stability of PS, and the dynamic complex interface formed between the soybean peptide and PS is confirmed by structural characterization, which can effectively delay the degradation of PS. Functional evaluation shows that the soybean peptide-phytosterol nanogel system not only competes with PS to inhibit cholesterol absorption, but also promotes the excretion of bile acids through soybean peptides, forming a double regulation mechanism to achieve a better cholesterol-lowering effect. This technology breaks through the bottleneck of low bioavailability of existing PS delivery systems, solves the industry problem of unclear synergistic mechanism of soybean peptide DH regulation and activity, provides an innovative technical path for developing functional foods and related medical field products that help maintain a healthy level of blood lipids (cholesterol) with high drug loading, structural stability and functional synergy, and has significant clinical application value and industrialization prospect. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Statistical graphs showing the encapsulation efficiency (EE) and intestinal release rate (IRE) of PS nanogels under different DH conditions;

[0025] Figure 2 Statistical graphs of average particle size and PDI of soybean peptides and nanogels under different DH conditions;

[0026] Figure 3 Figure 1 shows the detection results of the surface hydrophobicity of soybean peptides and nanogels under different DH conditions;

[0027] Figure 4 Fourier transform infrared (FT-IR) spectra of soybean peptides and nanogels under different DH values;

[0028] Figure 5 The diagram shows the secondary structure composition of soybean peptides and nanogels under different DH conditions;

[0029] Figure 6 Fluorescence spectra of soybean peptides and nanogels under different DH values;

[0030] Figure 7 X-ray diffraction patterns of soybean peptides and nanogels under different DH values;

[0031] Figure 8 The graph shows the detection results of cholesterol micelle inhibition rate (CMI) of soybean peptides and nanogels under different DH conditions;

[0032] Figure 9 The graph shows the results of detecting the bile acid binding capacity (BBA) of soybean peptides and nanogels under different DH conditions.

[0033] Figure 10 This is a statistical graph showing the inhibition rate of peptides on cholesterol esterase. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] 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 present application. Additionally, for a range of values of, for example, a parameter, an individual value from the range can be expressly disclosed herein to mean that each and every intermediate value of the range is also expressly disclosed. All intermediate values of the range are explicitly included herein. The terms "comprises", "comprising", "includes", "including", "having", "have" and / or "contains", "containing" and / or "consists" and / or "consisting" and / or "consists essentially" and / or "consisting essentially of" used herein specify the presence of stated features, integers, steps, operations, elements, and / or components but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The scope of the present application is not intended to be limited to specific illustrative embodiments but is intended to cover all embodiments consistent with the principles of the present application. The disclosure of the present application is intended to be taken by way of example only and to illustrate the spirit and scope of the present application. Modifications of structure, method of use or process steps are intended to be within the scope of the present application.

[0036] 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application.

[0037] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the disclosure herein. The description and examples are illustrative only and are not intended to limit the scope of the application.

[0038] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" or "contain", "containing" are open-ended and do not exclude additional, unrecited elements or method steps.

[0039] Plant sterols have poor water solubility, poor stability and low bioavailability. Therefore, it is urgent to improve the bioavailability of plant sterols. Constructing a nanogel delivery system is an effective means to deliver plant sterols and improve the bioavailability of plant sterols. Although the prior art knows that the DH of soy protein affects the assembly of hydrolysis products, and in turn affects the encapsulation of hydrophobic active substances, there are the following problems: lack of methods to control DH to achieve ideal encapsulation rate. This is mainly because the hydrolysis process of soy protein is complex, and the secondary and tertiary structures of soy peptides with different DH have not been fully understood. The effects of DH of soy peptides on the encapsulation and release of plant sterols, structural changes and cholesterol-lowering effects have not been clearly defined. Therefore, three kinds of soy peptides with different DH were prepared, and the corresponding PS-loaded nanogels were prepared. The EE and IRE of PS were evaluated, the delivery effect and structural modification of the nanogel were characterized, and the CMI and BBA were used to evaluate the cholesterol-lowering activity. The results show that the nanogel not only can effectively improve the embedding delivery efficiency and bioavailability of plant sterols, but also the soy peptides in it can synergize with PS to reduce cholesterol.

[0040] Example 1

[0041] 1. Materials and methods

[0042] 1.1 Experimental materials

[0043] Soybean protein isolate (protein content ≥ 95%) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China); phytosterol was purchased from Shaanxi Haishifu Biological Engineering Co., Ltd. (Shaanxi, China); alkaline protease (400000 U / g) was purchased from Angel Se Enzyme Preparation Co., Ltd. (Yichang, China); pepsin (4000 U / mL) and trypsin (200 U / mL) were purchased from Xiaodong Yijian Instrument Co., Ltd. (Suzhou, China); other reagents were all analytical grade.

[0044] 1.2 Preparation of nanogel (SPN)

[0045] A 5 wt% aqueous solution of soy protein was adjusted to pH 9.0 and preheated to 55°C. Alkaline protease was added (enzyme to substrate mass ratio of 1:10), and the solution was continuously stirred for 1, 3, and 6 hours, respectively. The reaction was terminated by heating in a boiling water bath at 100°C for 15 minutes. After cooling, the solution was centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected to obtain low (5.21%), medium (15.96%), and high (29.06%) degree of hydrolysis soy peptide solutions, designated as LSPP, MSPP, and HSPP, respectively.

[0046] Nanogels LSPN, MSPN, and HSPN were prepared by emulsification evaporation using LSPP, MSPP, and HSPP, respectively, according to the following method: 22.5 mg of phytosterol was dissolved in 5 mL of anhydrous ethanol, and then added dropwise to 50 mL of a soy peptide solution (LSPP, MSPP, and HSPP). The resulting mixture was ultrasonically treated at 50°C for 30 minutes, and then magnetically stirred at the same temperature for 2 hours. The ethanol was removed in a vacuum drying oven at 50°C for 30 minutes to obtain the nanogel.

[0047] 1.3 Nanogel encapsulation efficiency (EE) detection

[0048] The prepared 1 mL nanogel solution was mixed with 4 mL n-hexane, centrifuged at 4000 rpm for 10 min, and the unencapsulated PS was extracted, and the supernatant was transferred to a 50 mL centrifuge tube. The extraction was repeated 3 times with n-hexane, and the combined extract was blown with nitrogen. 100 μL of derivatization reagent (1-methylimidazole: MSHFBA, 5:95, v / v) was added, and the sample was derivatized at 75°C for 30 min. The supernatant was fixed to 1 mL with n-hexane and placed in a liquid injection bottle for testing. The Agilent GC-MS 5973 determination parameters were as follows: DB-5 chromatographic column (30 m x 0.25 mm x 0.25 μm), carrier gas was helium, sample size was 1 μL, split ratio was 20:1, carrier gas flow rate was 1 mL / min, and sample injection temperature was 290°C. The initial temperature was 100°C, which was increased to 290°C at a rate of 40°C / min and maintained for 25 min. The ionization mode was EI, the ion source temperature was 230°C, the ionization energy was 70 eV, the mass spectrometry quadrupole temperature was 150°C, and the detection was performed in ion selection mode. EE was calculated according to the following formula:

[0049] EE (%) = m e / m t x 100 %;

[0050] m t is the total amount of phytosterols, and m e is the encapsulation amount of phytosterols.

[0051] 1.4 Particle size and polydispersity index detection

[0052] After the NG was diluted 100 times, the average particle size and polydispersity index (PDI) were measured using a dynamic light scattering (DLS) analyzer (Malvern Pro, Instruments Inc., UK).

[0053] 1.5 Surface hydrophobicity (H0)

[0054] The nanogel was dissolved in 0.01 mol / L H3PO4 buffer with a pH of 7.0, and the sample mass concentration was 0-10 mg / mL. 2 mL of sample solution was added with 20 μL of 8 mmol / L ANS, and after rotation, it was placed for 3 min. A 96-well microplate was added, and the fluorescence intensity was measured using a multifunctional microplate reader (Spectramax i3x, Molecular Devices, Bioinsight Consulting, LLC). The excitation wavelength was 390 nm, the emission wavelength was 470 nm, and the slit correction was 5 nm. The slope of the linear regression curve representing the fluorescence intensity and the protein concentration was used to characterize H0.

[0055] 1.6 Fourier transform infrared spectroscopy

[0056] After freeze-drying the samples, the secondary structure was analyzed using an FT-IR spectrophotometer (Japan-shimadzu i-IR Tracer 100) in the 400-4000 cm -1 range with a resolution of 4 cm -1 -1, scanning 32 times, according to the KBr tablet method. The infrared spectral data of the samples were collected.

[0057] 1.7 Fluorescence spectra

[0058] The nanogels were diluted to 1 mg / mL and the fluorescence spectra of the samples were obtained using a fluorescence spectrophotometer (LS45, PerkinElmer, Waltham, MA, USA) to analyze the tertiary structure. At an excitation wavelength of 280 nm, the emission spectrum was determined in the range of 300-450 nm. The excitation slit width was 5 nm, the emission slit width was 10 nm, the scan speed was 240 nm / min, and the accelerating voltage was 400 V. The excitation slit width was 5 nm and the emission slit width was 10 nm.

[0059] 1.8 XRD

[0060] The crystal structure of the nanogels was studied using x-ray diffraction (XRD) (SmartLab 9 kW, Rigaku, Japan). The 2 theta was in the range of 3°-30°, with a step of 0.05°, the generator voltage was 40 kV, and the tube current was 30 mA.

[0061] 1.9 Simulated gastrointestinal digestion

[0062] Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were first prepared according to the literature“Gao, Y., Chen, L., Chi, H., Li, L., & Teng, F. (2024). Insights into the soybean protein isolate hydrolysates: Performance characterization, emulsion construction and in vitro digestive behavior. International Journal of Biological Macromolecules, 279(3), 135372”. The pH of the nanogel was adjusted to 2.0 for gastric digestion, 5 mL of nanogel was mixed with an equal amount of SGF, and shaken at 37°C for 2h, then heated at 100°C for 10 min to inhibit pepsin activity, and centrifuged at 4000 rpm for 10 min to separate the supernatant. Before intestinal digestion, the sample after SGF digestion was adjusted to pH 7.0 with 0.1 mol / L NaOH, then an equal amount of SIF, bile salts and pancreatin were added to the digested sample, and shaken in a 37°C water bath for 3h. Finally, heat to 100°C for 10 min to terminate digestion, and centrifuge at 4000 rpm for 15 min to separate the supernatant.

[0063] 1.10 Intestinal release rate of phytosterols (IRE)

[0064] 1 mL of the nanogel intestinal digestion solution was taken and centrifuged for 10 min to remove unencapsulated PS from the SPN. The method for measuring the amount of PS released was the same as 1.3. The intestinal release efficiency (IRE) of SPN was calculated as follows:

[0065] IRE (%) = m i / m e x 100%;

[0066] where m i is the amount of PS released from the intestine after in vitro digestion, and m e is the amount of PS encapsulated.

[0067] 1.11 Cholesterol micelle inhibition rate (CMI) and bile acid binding capacity determination (BBA)

[0068] Cholesterol solution contained 1 mM oleic acid, 0.4 mM cholesterol, 132 mM NaCl, 10 mM sodium taurocholate, and 15 mM phosphate buffer (pH 7.4). The solution was emulsified by sonication for 20 min. Subsequently, 5 mg / mL nanogel was added to the micellar solution, and incubated at 37 °C for 24 h to allow the formation of cholesterol micelles. After incubation, the solution was centrifuged at 4000 rpm for 10 min. 25 μL of the supernatant was mixed with 75 μL acetic acid and 100 μL ferric alum reagent, and incubated at 37 °C for 30 min. The absorbance of the resulting solution was measured at 560 nm using a multifunctional microplate reader. The inhibition rate of cholesterol micelles in the supernatant was calculated according to the standard curve Y = 0.0645x - 0.0471, R = 0.9962 (where Y is the mass concentration of cholesterol, unit: mmol / L, and x is the OD value). 2 -1 -1 Digest (1 mL) and 0.01 mol / L HC1 (0.5 mL) were incubated at 37 °C for 1 h. Subsequently, 5 mL of a bile salt solution containing sodium cholate and sodium deoxycholate (1 mg / mL) was added and mixed. After incubation at 37 °C for 1 h, the solution was precipitated with 4 volumes of 95% ethanol and centrifuged at 1000 g for 10 min. The final bile acid content in the supernatant, representing the total amount of bile acids, was determined using a total bile acid assay kit (E003-2-1, Nanjing Jiancheng Bioengineering, Nanjing, China). The bile acids were measured using a microplate spectrophotometer at a primary wavelength of 405 nm and a secondary wavelength of 630 nm. The final absorbance value was obtained by subtracting the absorbance at 630 nm from that at 405 nm. The relative bile acid binding capacity was calculated.

[0070] 1.11 Statistical analysis

[0071] The experimental results were repeated at least three times, and expressed as the mean standard deviation. One-way analysis of variance (ANOVA) with Duncan's test was used for data analysis, and a p-value < 0.05 was considered statistically significant.

[0072] 2. Experimental results

[0073] 2.1 EE and IRE of phytosterols

[0074] ​With the increase of soybean protein hydrolysis degree, the EE of PS showed a trend of first rising and then falling, and the MSPN reached the highest (80.17%), and the HSPN was the lowest (42.63%). This was because moderate hydrolysis made the flexibility of soybean peptide chain increased, and the dense gel network structure was formed by hydrophobic interaction, which provided a stable hydrophobic core environment for PS. The LSPN was due to the too strong rigidity of the peptide chain, which reduced the binding sites of PS. The IRE trend was consistent with the EE, and the MSPN (57.59%) was significantly higher than the LSPN (33.79%) and the HSPN (20.85%). The LSPN had complex peptide chain conformation, and the protein aggregation and the binding capacity of PS were not strong, which was released too early in the stomach digestion. The MSPN formed a balance between hydrophilic and hydrophobic and a dense cross-linking structure, which had strong resistance to pepsin degradation and ensured the release of PS in the intestine to a certain extent. The embedding structure of HSPN was loose and easy to be damaged, which caused the early release of PS. In summary, the MSPN showed the synergistic advantages of encapsulation and release, which proved that moderate hydrolysis could meet the "encapsulation stability" and "intestinal targeted release" at the same time.

[0075] 2.2 Morphological characterization of nanogels

[0076] The size and morphology of nanogels are shown in Figure 2 The average particle sizes of LSPP, MSPP and HSPP were 363.72 ± 10.17 nm, 349.98 ± 32.48 nm and 307.20 ± 6.45 nm, respectively, and the PDI values of MSPP and HSPP were lower than that of LSPP. Due to the encapsulation of PS, the particle size and PDI of SPN increased, and the trends of particle size and PDI with DH were similar in nanogels. The particle sizes of MSPN (595.54 nm) and HSPN (557.52 nm) were smaller than that of LSPN (875.43 nm). Smaller particle size and lower PDI were more conducive to the dispersion and absorption of SPN. Therefore, the nanogels prepared by medium-high DH of soybean peptides had more uniform particle size distribution and more suitable size range, which showed higher absorption performance in the intestinal delivery process, and laid a good carrier foundation for improving the bioavailability of phytosterols.

[0077] The surface hydrophobicity H0 reflects the protein conformation and functional characteristics. As Figure 3As shown, compared with SPI, H0 of soybean protein hydrolysate increased significantly. After the H0 of MSPP reached the peak, the degree of hydrolysis continued to increase, and H0 decreased instead. This may be because hydrolysis exposes the internal hydrophobic groups of SPI, and moderate hydrolysis increases H0, while excessive hydrolysis causes the hydrophobic groups to re-aggregate and hide. Higher H0 is more conducive to providing binding sites for hydrophobic active substances and improving loading capacity, so MSPP has stronger loading capacity for PS, which is consistent with the trend of EE data. After PS embedding, H0 decreased overall, PS embedded in the hydrophobic aggregate and combined to improve the functional properties; the H0 of MSPN decreased the most (61.44%), because PS occupied more hydrophobic binding sites of MSPP, forming a more stable SPN, reducing the AN binding sites, and reducing H0.

[0078] The potential interaction between protein peptides and PS particles was further understood by FT-IR spectrum. Figure 4 The amide A band of nanogel showed a slight shift in peak position (3288 cm -1 →3304 cm -1 ), and the intensity decreased with the increase of DH, indicating that the intermolecular hydrogen bond was destroyed by hydrolysis. After embedding PS, the amide A band intensity was significantly restored, and the peak value was stable at 3292-3298 cm -1 , indicating that new hydrogen bonds were formed between PS and SPP.

[0079] The percentage distribution of secondary structure is shown in Figure 5 . The hydrolysis and encapsulation of PS significantly changed the composition of soybean peptide secondary structure, and β-sheet and α-helix cooperatively maintained the rigid structure of protein. LSPP maintained a relatively rigid and complete structure, which also limited the embedding of PS to a certain extent. Compared with LSPP, the content of α-helix and β-sheet in MSPP and HSPP was lower, while the content of random coil was higher, which may be due to the gradual exposure of protein side chain hydrophobic groups during hydrolysis, which destroyed the hydrogen bond. Hydrolysis destroyed the ordered structure of protein, indicating that high DH led to peptide chain breakage, and the structure was mainly flexible and loose. After PS encapsulation, the percentage of α-helix and β-sheet in LSPN and MSPN increased, and the percentage of random coil decreased compared with their respective soybean peptides, and MSPN showed a trend of random coil to other three structures. That is, the addition of PS makes soybean peptides change from disordered structure to ordered structure. This may be due to the synergistic effect of hydrogen bond recombination (C=O combined with sterol hydroxyl) and hydrophobic interaction to restore the order of secondary structure. However, there was no significant difference in the structural change of HSPN compared with HSPP, which may be that the highly hydrolyzed soybean peptides have been hydrolyzed into relatively small fragments, and the embedding of phytosterol is difficult to interfere with the conformation of the entire peptide chain.

[0080] According to the fluorescence spectrum, the tertiary structure changes of SPP and SPN with different degrees of hydrolysis can be analyzed by the response of aromatic amino acids (Trp, Tyr, Phe) to the polar environment. As shown in Figure 6 After hydrolysis of SPI, the maximum wavelength of fluorescence peak (λmax) moves from 345 nm, and the intensity significantly increases: the λmax of LSPP is 360 nm, and that of MSPP and HSPP further moves to 365 nm, indicating that the hydrolysis enhances the polarity of the environment around the fluorescent group. Among them, the fluorescence intensity of MSPP is significantly higher than that of LSPP and HSPP, because the degree of hydrolysis of LSPP is low, and the internal fluorescent groups (such as Trp / Tyr) are not completely released, while the HSPP leads to the rearrangement of the hydrophobic microregion due to the excessive breakage of the peptide chain, and the light-emitting group is buried. MSPP can fully expose aromatic residues and enhance endogenous fluorescence. After loading PS, the fluorescence intensity of SPN decreases sharply, which is speculated to be due to the interaction between PS and exposed hydrophobic residues, with these residues re-embedded in the internal hydrophobic region of the protein, achieving stable combination of PS and soybean peptides and promoting the formation of a hydrophobic core structure of SPN. Among them, the fluorescence intensity of MSPN decreases the most, and the λmax remains stable at 365 nm, which may benefit from the formation of a coordinated and stable protein network structure through hydrogen bonding and hydrophobic interaction, which has a weak shielding effect on Trp / Tyr fluorescence, confirming the structural advantage of the medium hydrolysis system in PS embedding.

[0081] As shown in Figure 7 XRD shows that in the nanogel, the characteristic peaks of PS in the characteristic peaks of LSPN, MSPN and HSPN almost completely disappear, indicating the successful embedding of PS.

[0082] 2.3 Analysis of cholesterol-lowering effect

[0083] The inhibition rate of cholesterol micelles and the binding capacity of bile acids can reflect the cholesterol-lowering capacity to some extent, and the results are shown in Figure 8 and Figure 9 Among LSPP, MSPP and HSPP groups, the two indicators are low because they are not loaded with phytosterols; in SPN, MSPN is significantly superior, and in CMI, MSPN (73.22%) is higher than LSPN (60.90%) and HSPN (65.81%), and MSPN has stronger inhibition on the formation of cholesterol micelles, which can reduce the absorption of cholesterol in the intestine; in terms of BBA, MSPN (66.14%) is superior to LSPN (59.29%) and HSPN (60.89%), and MSPN can bind more bile acids to promote cholesterol metabolism. In terms of reducing cholesterol absorption and improving cholesterol metabolism, MSPN is superior to LSPN and HSPN, and its delivery system has better cholesterol-lowering efficacy, providing a high-quality carrier for the application of phytosterols.

[0084] Example 2

[0085] 1. Experimental method

[0086] 1.1 Polypeptide identification and screening

[0087] Intestinal digestion products of LSPN, MSPN and HSPN of Example 1 were obtained after simulated gastrointestinal fluid digestion, desalted by C18 column, and analyzed for amino acid sequence by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). High frequency peptides were obtained by searching the UniProt Glycine max database. The biological activity of the polypeptides was evaluated using PeptideRanker, and the interaction probability with cholesterol esterase (CE, PDBID: 1F6W) was predicted by PepSite2, and high potential activity peptides were screened.

[0088] 1.2 Peptide synthesis and inhibition experiment

[0089] The screened peptides were synthesized by solid phase synthesis, and the product purity was determined by reverse phase high performance liquid chromatography to be ≥95%, which could be used for cholesterol esterase inhibition experiment.

[0090] Cholesterol esterase inhibition experiment: the reaction system contained 50 μL of 10 mM p-nitrophenyl butyrate (dissolved in 5.16 mM sodium taurocholate and 100 mM NaCl phosphate buffer), 50 μL of 4 mg / mL polypeptide solution, and 50 μL of 25 μg / mL CE solution. After incubation at 37°C for 5 min, the absorbance was measured at 405 nm and the inhibition rate was calculated.

[0091] CE inhibition rate = [1-(C-D) / (A-B)] x 100%;

[0092] Wherein, A is the absorbance value of no sample with enzyme, B is the absorbance value of no sample without enzyme, C is the absorbance value of sample with enzyme, and D is the absorbance value of sample without enzyme.

[0093] 1.3 Statistical analysis

[0094] The experiment was repeated 3 times, and the results were expressed as mean ± standard deviation. Single factor analysis of variance (ANOVA) and Duncan test were used, and p<0.05 was considered significant.

[0095] 2. Experimental results

[0096] 2.1 Polypeptide identification and screening results

[0097] The intestinal digestion products were desalted, analyzed by HPLC-MS / MS, and searched in a database to obtain polypeptides HLPSYSPYPQ (named HQ; SEQ ID NO. 1), SSPDIFNPQ (named SQ; SEQ ID NO. 2), QFPFPRPPQQ (named QQ; SEQ ID NO. 3), and DHHDPIMPY (named DY; SEQ ID NO. 4).

[0098] The biological activity prediction and molecular docking results of the screened polypeptides are shown in Table 1.

[0099] Table 1 Biological activity prediction and molecular docking results of polypeptides

[0100]

[0101] 2.2 Inhibition experiment results

[0102] The detection results of the cholesterol esterase inhibition experiment are shown in Table 2. Figure 10 The results show that the inhibition rate of 4 mg / mL HQ solution on CE is 7.48%; the inhibition rate of 4 mg / mL SQ solution on CE is 28.22%; the inhibition rate of 4 mg / mL QQ solution on CE is 21.55%; and the inhibition rate of 4 mg / mL DY solution on CE is 15.48%.

[0103] In summary, the digestion products of MSPN contain various functional polypeptides with cholesterol esterase inhibition activity, which explains the cholesterol-lowering function of MSPN from the side.

[0104] The above-described embodiments are merely preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A method for preparing a soybean peptide-phytosterol nanogel system with cholesterol-lowering function, characterized in that, The process includes the steps of encapsulating phytosterols with soybean peptide solution using an emulsification evaporation method to prepare the soybean peptide-phytosterol nanogel system. The preparation method of the soybean peptide solution includes the following steps: Soybean protein was enzymatically hydrolyzed using alkaline protease, and the resulting soybean peptide solution was obtained by centrifugation. The mass ratio of the alkaline protease to the soybean protein is 1:10; the enzymatic hydrolysis treatment is performed at a temperature of 55°C, a pH of 9.0, and a time of 3 hours.

2. The preparation method according to claim 1, characterized in that, The specific steps of the emulsification evaporation method include: dissolving the phytosterol in an organic solvent, then adding it dropwise to a soybean peptide solution, subjecting it to ultrasonic and homogenization treatment, and then evaporating the organic solvent through drying treatment to obtain the soybean peptide-phytosterol nanogel system.

3. The preparation method according to claim 2, characterized in that, The organic solvent is ethanol.

4. The preparation method according to claim 2, characterized in that, The ratio of the phytosterol, the organic solvent, and the soybean peptide solution is 22.5 mg: 5 mL: 50 mL.

5. A soybean peptide-phytosterol nanogel system prepared by the preparation method according to any one of claims 1-4.

6. The application of the soybean peptide-phytosterol nanogel system as described in claim 5 in the preparation of cholesterol-lowering drugs.

7. A cholesterol-lowering drug, characterized in that, The active ingredient includes the soybean peptide-phytosterol nanogel system as described in claim 5.

8. The cholesterol-lowering drug according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application of the soybean peptide-phytosterol nanogel system as described in claim 5 in the preparation of functional foods that help maintain healthy blood lipid levels.

10. A functional food that helps maintain healthy blood lipid levels, characterized in that, The active ingredient includes the soybean peptide-phytosterol nanogel system as described in claim 5.

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