Soybean-derived cholesterol-lowering polypeptide as well as preparation method and application thereof

Abstract

The invention discloses soybean-derived cholesterol-lowering polypeptide as well as a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the soybean-derived cholesterol-lowering polypeptide is shown as any one of SEQ ID NO.1-2. The invention further discloses a preparation method of the soybean-derived cholesterol-lowering polypeptide. The two kinds of functional polypeptides obtained through screening are derived from natural plant soybeans and have a definite cholesterol lowering effect, the action mechanism of the polypeptides is that the activity of cholesterol esterase is specifically inhibited, hydrolysis and absorption of dietary cholesterol ester are reduced, then the cholesterol level in the body is reduced, and the cholesterol lowering effect is achieved. The action is doubly verified through a molecular docking technology and an in-vitro enzyme activity inhibition experiment, the mechanism is clear, and the action target is definite. In view of the characteristics, the two polypeptides as natural cholesterol-lowering active components can be directly used as functional factors to develop functional food and products in the related medicine field, which are beneficial to maintaining the health level of blood fat (cholesterol), and have remarkable application value and wide market prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a soybean-derived cholesterol-lowering polypeptide and a preparation method and application thereof. Background Art

[0002] High cholesterol levels have become a significant risk factor for human health, significantly associated with the development and progression of numerous chronic diseases, including coronary heart disease, malignant tumors, metabolic syndrome, and type 2 diabetes. Cholesterol esterase (CE), a key enzyme regulating cholesterol metabolism, is secreted from the pancreas into the duodenum. It catalyzes the hydrolysis of cholesterol esters into free cholesterol and fatty acids, which are readily absorbed by the intestinal mucosa and induce hypercholesterolemia. Therefore, inhibiting CE activity to reduce dietary cholesterol absorption has become a new strategy for prevention and treatment.

[0003] Although statins achieve lipid-lowering effects by inhibiting cholesterol synthesis, their target of action is limited to the liver and they cannot block the intestinal cholesterol absorption pathway. Long-term use also has side effects such as myalgia and abnormal liver enzymes. In addition, the high cost of treatment makes it difficult to meet the large-scale chronic disease prevention needs. At the same time, consumers' preference for functional ingredients of natural origin is growing, and promoting nutritional intervention and functional food research and development has become an important direction for regulating cholesterol metabolism. Bioactive peptides derived from plant proteins have attracted much attention due to their safety and metabolic regulation potential, especially the active peptides released after enzymatic hydrolysis of soy protein, which can participate in lipid metabolism regulation through multi-target mechanisms. However, existing studies have mostly focused on whole proteins or random screening, and lack systematic exploration of functional peptides in specific digestion products. In particular, there is still a research gap in the precise screening technology and mechanism verification combined with CE inhibitory activity.

[0004] Based on this, the present invention intends to systematically screen and identify functional peptides with CE inhibitory activity after digestion of soybean peptide gel loaded with plant sterols through high-performance liquid chromatography-mass spectrometry sequencing, peptidomics analysis, molecular docking and other methods, evaluate their cholesterol-lowering efficacy, provide a scientific basis for the development of new functional foods that help maintain healthy blood lipid (cholesterol) levels, and fill the research gap in the exploration and verification of specific cholesterol-lowering peptides from the digestion products of soybean peptide nanogels. Summary of the Invention

[0005] The present invention aims to provide a soybean-derived cholesterol-lowering polypeptide, its preparation method, and its application to address the aforementioned problems of the prior art. The soybean-derived cholesterol-lowering polypeptide provided by the present invention inhibits cholesterol esterase activity and can be directly added as a functional factor to functional foods. The polypeptide has significant application value and broad market prospects in the development of functional foods and related medical fields that help maintain healthy blood lipid (cholesterol) levels.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a soybean-derived cholesterol-lowering polypeptide, the amino acid sequence of the polypeptide being shown in any one of SEQ ID NOs. 1-2.

[0008] The present invention also provides a method for preparing the soybean-derived cholesterol-lowering polypeptide, comprising the step of preparing the soybean-derived cholesterol-lowering polypeptide by solid-phase synthesis.

[0009] The present invention also provides the use of the above polypeptide in the preparation of cholesterol-lowering drugs.

[0010] The present invention also provides a cholesterol-lowering drug, the active ingredient of which includes the soybean-derived cholesterol-lowering polypeptide.

[0011] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the auxiliary materials include fillers, excipients, binders, disintegrants, emulsifiers or preservatives.

[0013] The present invention also provides the use of the soybean-derived cholesterol-lowering polypeptide in the preparation of functional foods that help maintain healthy blood lipid levels.

[0014] The present invention also provides a functional food that helps maintain healthy blood lipid levels, the active ingredient of which includes the above-mentioned soybean-derived cholesterol-lowering polypeptide.

[0015] The present invention also provides the use of the soybean-derived cholesterol-lowering polypeptide in the preparation of a cholesterol esterase inhibitor.

[0016] The present invention also provides a cholesterol esterase inhibitor, the active ingredient of which includes the above-mentioned soybean-derived cholesterol-lowering polypeptide.

[0017] The present invention discloses the following technical effects:

[0018] This study systematically investigated the digestive and metabolic processes of soy peptide nanogels, successfully screening and preparing functional peptides QFPFPRPPQQ and DHHDPIMPY with highly effective cholesterol esterase (CE) inhibitory activity. These peptides specifically inhibit cholesterol esterase activity, blocking the hydrolysis of dietary cholesterol esters and significantly reducing intestinal absorption, thereby effectively lowering cholesterol levels in the body. This mechanism of action, validated by both molecular docking simulations and in vitro enzyme inhibition experiments, demonstrates a clear mechanism and targeted action.

[0019] Given the above characteristics, these two polypeptides, as natural cholesterol-lowering active ingredients, can be directly used as functional factors to develop functional foods and related medical products that help maintain healthy blood lipid (cholesterol) levels. They have significant application value and broad market prospects.

[0020] In addition, the present invention has established a multi-dimensional screening system guided by cholesterol esterase inhibitory activity, integrating mass spectrometry sequencing, molecular docking and biological activity verification technologies, breaking through the bottleneck of low efficiency and vague targets of traditional functional peptide screening, and providing a standardized technical path for the precise development of natural cholesterol-lowering peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The molecular docking diagram of simvastatin and CE; A is the relative overall structure diagram of simvastatin and CE; B is the two-dimensional interaction diagram of simvastatin and CE; C is the force type diagram of simvastatin and CE;

[0023] Figure 2 is the molecular docking diagram of QQ and CE; A is the relative overall structure diagram of QQ and CE; B is the two-dimensional interaction diagram of QQ and CE; C is the force type diagram of QQ and CE;

[0024] Figure 3 is the molecular docking diagram of DY and CE; A is the relative overall structure diagram of DY and CE; B is the two-dimensional interaction diagram of DY and CE; C is the force type diagram of DY and CE;

[0025] Figure 4 This is the relationship diagram between QQ peptide concentration and cholesterol esterase inhibition effect;

[0026] Figure 5 This is the relationship diagram between DY peptide concentration and cholesterol esterase inhibition effect. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Materials and Methods

[0034] 1.1 Materials

[0035] Soy protein isolate (protein content ≥95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China); phytosterols were purchased from Shaanxi Haisifu Bioengineering Co., Ltd. (Shaanxi, China); alkaline protease (400,000 U / g) was purchased from Angel Enzyme Co., Ltd. (Yichang, China); pepsin (4000 U / mL) and trypsin (200 U / mL) were purchased from Xiaodong Yijian Instrument Co., Ltd. (Suzhou, China); all other reagents were of analytical grade.

[0036] 1.2 Nanogel preparation and digestion

[0037] Alkaline protease (enzyme to substrate mass ratio of 1:10) was added to a 5wt% aqueous soy protein solution and hydrolyzed at 55°C for 6 hours to produce soy peptide with a degree of hydrolysis of 29.06%. The enzyme was inactivated by heating at 100°C for 15 minutes, and the supernatant was collected by centrifugation at 8000 rpm for 15 minutes. Using emulsion evaporation, 22.5 mg of phytosterols were encapsulated in 50 mL of soy peptide to produce nanogel HSPN.

[0038] Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the method of 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”.

[0039] Gastric digestion: adjust the pH of HSPN to 2.0, mix with an equal amount of SGF, shake at 37°C for 2 h, heat at 100°C for 10 min to inactivate the enzyme, centrifuge at 4000 rpm for 10 min, and collect the supernatant to obtain the gastric digestion product.

[0040] Intestinal digestion: Adjust the pH of the gastric digestion product to 7.0 with 0.1 mol / L NaOH, add equal amounts of SIF, bile salts and pancreatic enzymes, shake at 37°C for 3 hours, heat at 100°C for 10 minutes to terminate the reaction, centrifuge at 4000 rpm for 15 minutes, and collect the supernatant, which is the intestinal digestion product.

[0041] 1.3 Peptide identification and screening

[0042] The intestinal digestion product was desalted by C18 column, and the amino acid sequence was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The high frequency peptides were obtained by searching the UniProt Glycine max database.

[0043] 1.4 Molecular docking

[0044] The three-dimensional structures of the selected peptides were constructed in ChemBioDraw Ultra 22.0 and docked with CE (PDB ID: 1F6W) using Autodock Vina 1.5.6. The conformation with the lowest binding energy was selected as the optimal conformation. The interaction forces were analyzed and visualized using Discovery Studio 4.5 Client.

[0045] 1.5 Peptide synthesis and inhibition experiments

[0046] The screened peptides were synthesized using solid-phase synthesis as follows:

[0047] First, weigh 0.5g of 2-chlorotrityl resin and swell it in DCM for 30 minutes. Then, add a 3-fold molar excess of Fmoc-Asn(Trt)-OH and a 10-fold molar excess of DIEA. Dissolve it in a small amount of DMF and shake for 1 hour to connect the first amino acid. Then, wash it six times with alternating DMF and DCM. Deprotection is then performed with 20% piperidine in DMF (first for 5 minutes, then for 15 minutes). After removing the solution, the resin is washed with ethanol and tested positive with solutions such as ninhydrin. Washing is then repeated with DMF, methanol, and DMF. Then, add a 3-fold molar excess of a protected amino acid (such as Fmoc-Ala-OH) and HBTU, and condense it with a 10-fold molar excess of DIEA for 40 minutes. After washing, repeat the deprotection, detection, washing, and condensation steps to connect all amino acids in the sequence from right to left. Finally, the resin tests negative and is washed with methanol. Next, a cutting solution was prepared using TFA, TIS, EDT, and H2O in appropriate proportions. The mixture was mixed with the resin at a ratio of 10 mL / g and shaken at a constant temperature for 2 hours. The cleavage solution was dried with nitrogen, chromatographed and washed six times with ether, and evaporated to dryness at room temperature to obtain the crude peptide. 200 mg of the crude product was then dissolved in 50% acetonitrile aqueous solution, sonicated, and filtered. The product was analyzed by analytical HPLC, prepared by preparative HPLC, and verified for purity by MS. The purified solution was lyophilized to obtain the finished product, which was then sealed and stored at -20°C.

[0048] The product purity is determined to be ≥95% by reverse phase high performance liquid chromatography and can be used for cholesterol esterase inhibition experiments.

[0049] Cholesterol esterase inhibition assay: The reaction system contained 50 μL of 10 mM p-nitrophenylbutyrate (dissolved in 5.16 mM sodium taurocholate and 100 mM NaCl phosphate buffer), 50 μL of peptide solutions of different concentrations, 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.

[0050] CE inhibition rate = [1-(CD) / (AB)] × 100%;

[0051] Among them, A is the absorbance value with enzyme but no sample, B is the absorbance value with enzyme but no sample, C is the absorbance value with enzyme and sample, and D is the absorbance value with enzyme but no enzyme.

[0052] Dose effect verification: Prepare peptide solutions of different concentrations and determine the inhibition rate according to the above cholesterol esterase inhibition experimental method.

[0053] 1.6 Statistical analysis

[0054] The experiment was repeated three times and the results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Duncan test were used for analysis, and p < 0.05 was considered significant.

[0055] 2. Experimental Results

[0056] 2.1 Peptide screening results

[0057] The intestinal digestion products were desalted, analyzed by HPLC-MS / MS, and database searched to obtain the peptides QFPFPRPPQQ (named QQ; SEQ ID NO. 1) and DHHDPIMPY (named DY; SEQ ID NO. 2).

[0058] As shown in Table 1, the bioactivity of QQ was evaluated using PeptideRanker, with a score of 0.837248 and a p-value of 0.0007242 for the probability of interaction with CE, indicating high activity. DY had a PeptideRanker score of 0.659541 and a p-value of 0.02699 for the probability of interaction with CE, indicating it is a potentially active peptide.

[0059] 2.2 Molecular docking experimental results

[0060] The binding energy of QQ docking with CE (PDB ID: 1F6W) is -8.1 kcal / mol. Figure 2 As shown, QQ embeds into the hydrophobic pocket of the enzyme through conventional hydrogen bonding and alkyl interactions. A glutamine residue in QQ forms a hydrogen bond with GLN71, and a proline residue forms an alkyl interaction with ALA108. These interactions facilitate QQ binding to CE. By occupying part of the substrate-binding region, QQ interferes with the catalytic function of CE.

[0061] The binding energy of DY docking with CE (PDB ID: 1F6W) is -8.4 kcal / mol, e.g. Figure 3 As shown, DY binds via hydrogen bonds and hydrophobic interactions. The carbon-hydrogen bonds between VAL391 and TRP522 help maintain the overall conformation of the peptidase complex, enabling DY to stably bind to CE. Furthermore, the π-electron cloud formed near HIS283 creates favorable charge interactions, which enhances the specificity of peptide-enzyme binding. Its binding site partially overlaps with that of simvastatin (LEU224, TRP522, etc.; see for details). Figure 1 ), these overlapping sites are important areas for CE to bind to substrates. The binding of DY will hinder the substrate from entering the active center, thereby inhibiting the catalytic activity of CE.

[0062] Table 1 Bioactivity prediction and molecular docking results of key peptides

[0063]

[0064]

[0065] 2.3 Inhibition experiment results

[0066] The results of the cholesterol esterase inhibition test showed that the inhibition rate of 10 mg / mL QQ solution on CE was 42.08%, and the inhibition rate of 20 mg / mL LDY solution on CE was 38.78%.

[0067] Prepare 0.375-20 mg / mL QQ solution and measure it according to the cholesterol esterase inhibition test method. The results are shown in Figure 4 .Depend on Figure 4 It can be seen that in the peptide content range of 0.375-10 mg / mL, the inhibition rate gradually increased with the increase of concentration, from nearly 2.19% to 42.08%; when the concentration exceeded 10 mg / mL, the inhibition rate growth gradually slowed down and entered a plateau period, maintaining at around 42%.

[0068] Prepare 0.75-40 mg / mL DY solution and measure it according to the cholesterol esterase inhibition test method. The results are shown in Figure 5 As shown in Figure 5, in the concentration range of 0.75-20 mg / mL of peptide content, the inhibition rate gradually increased with the increase of concentration, from 2.95% to about 38.78%; when the concentration exceeded 20 mg / mL, the inhibition rate growth rate became smaller, gradually approached a platform, and maintained at about 38%.

[0069] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A soybean-derived cholesterol-lowering polypeptide, characterized in that: The amino acid sequence of the soybean-derived cholesterol-lowering polypeptide is shown in any one of SEQ ID NOs. 1-2.

2. A method for preparing the soybean-derived cholesterol-lowering polypeptide according to claim 1, characterized in that: The method comprises the steps of preparing the soybean-derived cholesterol-lowering polypeptide by a solid phase synthesis method.

3. Use of the soybean-derived cholesterol-lowering polypeptide according to claim 1 in the preparation of cholesterol-lowering drugs.

4. A cholesterol-lowering drug, characterized in that: The active ingredient comprises the soybean-derived cholesterol-lowering polypeptide according to claim 1.

5. The cholesterol-lowering drug according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The cholesterol-lowering drug according to claim 5, characterized in that The auxiliary materials include fillers, excipients, binders, disintegrants, emulsifiers or preservatives.

7. Use of the soybean-derived cholesterol-lowering polypeptide according to claim 1 in preparing functional foods that help maintain healthy blood lipid levels.

8. A functional food that helps maintain healthy blood lipid levels, characterized in that: The active ingredient comprises the soybean-derived cholesterol-lowering polypeptide according to claim 1.

9. Use of the soybean-derived cholesterol-lowering polypeptide according to claim 1 in the preparation of a cholesterol esterase inhibitor.

10. A cholesterol esterase inhibitor, characterized in that The active ingredient comprises the soybean-derived cholesterol-lowering polypeptide according to claim 1.

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