Polypeptide having function of lowering cholesterol in soybean protein hydrolysate, and preparation method therefor and use thereof

Abstract

Provided are a polypeptide having the function of lowering cholesterol in a soybean protein hydrolysate, and a preparation method therefor and the use thereof. The polypeptide is derived from a peptide fragment having a binding_energy value of < -1.2 Kcal docked to a cholesterol esterase molecule, and the peptide fragment of the polypeptide subjected to molecular docking to cholesterol esterase is derived from 23 polypeptides with the biological activity score of Peptide Ranker > 0.9. Therefore, the polypeptide has a significant blood-lipid-lowering function.

Description

Polypeptide with cholesterol-lowering function in soybean protein hydrolysate, preparation method and application thereof Technical Field

[0001] The present application relates to the technical field of functional polypeptides, and in particular to a polypeptide with cholesterol-lowering function in soybean protein hydrolysate, and a preparation method and application thereof. Background Art

[0002] Dyslipidemia is associated with a variety of human diseases, and hyperlipidemia is currently becoming a threat to national health, reducing people's quality of life. Studies suggest that the lipid-lowering mechanism reduces the absorption of exogenous triglycerides by inhibiting the activity of pancreatic lipase, reduces the activity of fatty acid synthase, thereby reducing fat synthesis, and accelerates cholesterol metabolism and promotes cholesterol excretion. Cholesterol esterase is an enzyme that catalyzes the hydrolysis of cholesterol esters into cholesterol and fatty acids. Cholesterol is absorbed into the bloodstream by small intestinal epithelial cells through the formation of micelles. Excessive cholesterol in the blood can cause hyperlipidemia. Inhibiting the hydrolytic activity of cholesterol lipase can effectively lower serum cholesterol, thereby achieving the goal of lowering blood lipids.

[0003] Currently, the main medications on the market are statins and non-statins. Statins can increase blood sugar levels, leading to the risk of diabetes, while non-statins are less effective than statins. Studies have shown that protein components in natural foods can regulate dyslipidemia, with their lipid-lowering effects stemming from their peptides and amino acid residues. Developing safe and effective lipid-lowering active ingredients from natural food ingredients is of great significance to national health. Technical issues

[0004] How to provide a soy protein hydrolyzate with good cholesterol-lowering effect is the technical problem to be solved by this application. Technical Solutions

[0005] In view of this, the present application provides a polypeptide with cholesterol-lowering function in soybean protein hydrolyzate, and a preparation method and application thereof.

[0006] In the first aspect, the present application provides a polypeptide with cholesterol-lowering function in a soy protein hydrolyzate, wherein the polypeptide is derived from a peptide segment with a "binding_energy" value of less than -1.2Kcal for docking with a cholesterol esterase molecule, and the peptide segment for molecular docking with cholesterol esterase is derived from a polypeptide with a Peptide Ranker bioactivity score of >0.9.

[0007] In a second aspect, the present application provides the use of the polypeptide having cholesterol-lowering function in the soy protein hydrolyzate as described in the first aspect in the preparation of a drug for lowering hyperlipidemia, a food or a health product suitable for people with hyperlipidemia.

[0008] In a third aspect, the present application provides a method for preparing a polypeptide having a cholesterol-lowering function in a soy protein hydrolyzate, wherein the method for preparing the polypeptide having a cholesterol-lowering function in a soy protein hydrolyzate as described in the first aspect comprises the following steps:

[0009] (1) Preparation of protein hydrolysate: hydrolyzing soybean protein with protease to obtain protein hydrolysate;

[0010] (2) Cholesterol esterase inhibitory activity assay: Cholesterol esterase inhibitory activity assay;

[0011] (3) Sequence identification of soy protein hydrolysate: Soy protein hydrolysate was subjected to LC-MS / MS analysis to analyze its peptide composition;

[0012] (4) Screening of cholesterol-lowering peptides: The identified peptide fragments were first screened for potential bioactive peptides with a Peptide Ranker > 0.9, and then docked using AutoDock 4.2.6 software to screen out the most promising cholesterol-lowering peptides. Beneficial effects

[0013] The cholesterol-lowering peptide provided by this application is obtained by screening soybean protein after hydrolysis with nattokinase. The soy protein hydrolyzate obtained by hydrolysis has the activity of inhibiting cholesterol esterase. The hydrolyzate was sequenced and identified to have 2359 peptides. The -101gP values ​​of all peptides were greater than 20, with high credibility. There are 23 peptides that meet the peptide rank>0.9. After docking the 23 peptides with the cholesterol esterase molecule, the molecular docking results show that the "binding_energy" value of soybean peptide-FFFPF is below -1.2Kcal, indicating that this peptide has the best docking effect with the cholesterol esterase molecule, and has been verified by animal experiments to have a significant lipid-lowering function. The cholesterol-lowering peptide provided by this application can be developed into a lipid-lowering drug, and the active ingredient can be added to functional foods and health foods, with good market prospects in the pharmaceutical and food industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0015] FIG1 is a schematic diagram showing the interaction between polypeptide FFFPF and cholesterol esterase molecules. Modes for Carrying Out the Invention

[0016] The following is a clear and complete description of the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this invention.

[0017] The test methods used in the following examples and experimental examples are all conventional methods unless otherwise specified: Nattokinase was laboratory-made, and all other materials and reagents used were commercially available reagents and materials; the equipment used was all conventional laboratory equipment unless otherwise specified.

[0018] Example 1 Preparation of soy protein hydrolyzate

[0019] Soy protein was dissolved in pure water to create a 5% (w / v) soy protein isolate solution. Stir at 90°C for 15 minutes. The solution was cooled to 45°C and nattokinase was added at an enzyme-to-substrate ratio of 6000 IU / g. After stirring and hydrolysis for 2 hours, the solution was heated to 90°C and inactivated for 15 minutes. After stopping the hydrolysis process, the solution was centrifuged at 8000 rpm for 20 minutes. The supernatant was the soy protein hydrolyzate.

[0020] Example 2 Preparation of Soy Protein Hydrolyzate Using Alkaline Protease

[0021] Same as Example 1, except that nattokinase is replaced with alkaline protease.

[0022] Example 3 Determination of the inhibitory activity of soybean protein hydrolyzate on cholesterol esterase

[0023] In a 96-well microplate, 50 μL of the hydrolyzate from Example 1, 50 μL of the hydrolyzate from Example 2, 50 μL of a 25 μg / mL cholesterol esterase solution, and 50 μL of 10 mM p-nitrophenylbutyrate as substrate were incubated at 25°C for 5 min in a pH 7.0 phosphate buffer (containing 100 mM NaCl and 5.16 mM sodium taurocholate). The absorbance was recorded at 405 nm using a microplate reader. Simvastatin was used as a positive control, and calculations were performed according to Equation (1).

[0024] Cholesterol esterase activity inhibition rate / %= Formula (1)

[0025] In formula (1):

[0026] A: blank absorbance; B: blank control absorbance; C: sample absorbance; D: sample blank absorbance.

[0027] The test results are shown in Table 1. The hydrolyzate of Example 1 (soy protein hydrolyzed with nattokinase) exhibited higher cholesterol esterase inhibitory activity than that of Example 2 (soy protein hydrolyzed with alkaline protease), indicating that the hydrolyzate obtained in Example 1 has a greater potential for lowering cholesterol than that in Example 2. This may be due to the peptides in the hydrolyzed protein solution; Example 1 is more likely to contain cholesterol-lowering peptides.

[0028] Table 1 Inhibitory activity of hydrolyzate on cholesterol esterase

[0029]

[0030] Example 4 LC-MS / MS analysis of peptide sequences

[0031] The soy protein hydrolysate was transferred to a 10KD ultrafiltration centrifuge tube and centrifuged at 12,000 g for 10 minutes. The ultrafiltration solution was desalted using a C18 desalting column. The sample was eluted with elution buffer (0.1% FA, 60% ACN) and transferred to a new EP tube. The eluted sample was centrifuged and concentrated to dryness for mass spectrometry analysis. After centrifugation and drying, the desalted sample was re-dissolved in 100 μL of Nano-LC mobile phase A (0.1% formic acid / water) and loaded into a vial for online LCMS analysis. A 2 μL volume of the dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 Å), followed by a 20 μL wash for desalting. Liquid chromatography was performed using an Easy nLC 1200 nanoliter LC system (ThermoFisher, USA). Samples were desalted and retained on a precolumn before being separated on the analytical column, which was a C18 reversed-phase column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm 100 Å). The gradient used was from 5% to 38% mobile phase B (80% acetonitrile, 0.1% formic acid) over 30 minutes. Mass spectrometry was performed using a ThermoFisher Q Exactive system (ThermoFisher, USA) coupled with a nanospray NanoFlex ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the ion transfer tube was heated at 275°C. The mass spectrometer was scanned in data-dependent analysis (DDA) mode, with a primary mass spectrometer resolution of 70,000, a scan range of 100–1500 m / z, and a maximum injection time of 100 ms. A maximum of 20 secondary spectra with charges ranging from 1+ to 3+ were acquired during each DDA cycle, with a maximum injection time of 50 ms for secondary mass spectrometry ions. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV for all precursor ions, and dynamic exclusion was set to 6 seconds. Data from the acquired raw mass spectra were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada) using the target species protein database downloaded from Uniprot. Search parameters were as follows: primary mass spectrometry mass tolerance of 10 ppm and secondary mass spectrometry mass tolerance of 0.05 Da. Variable modifications were identified as oxidative modification (M), acetylation (protein N-term), deamidation (NQ), pyro-glu from E, and pyro-glu from Q.

[0032] Example 5 Screening of cholesterol-lowering peptides based on molecular docking technology

[0033] First, the peptides identified by LC-MS / MS were preliminarily screened using Peptide Ranker to identify peptides with predicted bioactivity scores > 0.9. These peptides were then docked with cholesterol esterase. A smaller "binding_energy" value indicated a better docking effect and a higher potential for cholesterol-lowering. Table 2 shows peptides with predicted bioactivity scores > 0.9.

[0034] Table 2

[0035]

[0036] The crystal structure of cholesterol esterase (PDB number: 1F6W) was obtained from the RCSB database (http: / / www.rcsb.org / ). The peptides with a bioactivity prediction score higher than 0.90 were docked with cholesterol esterase using AutoDock 4.2.6 software. The semi-flexible molecular docking scores are shown in Table 2.

[0037] The molecular docking scores of potential cholesterol-lowering peptides and cholesterol esterase are shown in Table 3:

[0038] Table 3

[0039]

[0040] As shown in Figure 1, the interaction between peptide FFFPF and cholesterol esterase molecules is schematically shown. Peptide-FFPF is semi-flexibly docked with cholesterol esterase. The other key amino acid residues for cholesterol esterase interaction are GLU242 and GLU278. The interaction forces include hydrogen bonds and π-C bonds. It is considered to be a potential cholesterol-lowering peptide.

[0041] Example 6 Animal Experiment on Cholesterol-Lowering Peptides

[0042] The cholesterol-lowering peptide FFFPF was prepared by Fmoc solid-phase synthesis, and the purity of the peptide was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry analysis.

[0043] To establish a hyperlipidemia animal model, 40 healthy adult male Sprague-Dawley rats weighing 200 ± 20 g were used. Ten rats served as a blank control group and were fed a standard diet. The remaining 40 rats served as a modeling group and were fed a commercially available high-fat diet (60% fat content).

[0044] After the modeling group was fed a high-fat diet for 1-2 weeks, blood was collected from the inner canthus or tail of the eye in the blank control and modeling groups without fasting. Serum was separated as soon as possible after blood collection, and serum TC, TG, LDL-C, and HDL-C levels were measured. The modeling group was randomly divided into three groups (modeling control group, cholesterol-lowering peptide group, and lovastatin control group). No significant differences in TC, TG, LDL-C, and HDL-C were found between the groups. Compared with the blank control group, the modeling groups showed significant differences in TC, TG, and LDL-C, indicating successful modeling.

[0045] The blank control group and the model control group were given an equal volume of 0.9% normal saline by gavage, and the other two groups were given sample aqueous solutions by gavage. The gavage doses are shown in Table 4.

[0046] Table 4 Oral gavage dosage for each group

[0047]

[0048] During the gavage period, the blank control group was fed a normal diet daily, while the other groups were fed a high-fat diet. All rats had free access to water and were gavaged once daily for four consecutive weeks. Body weights were measured regularly, and blood was collected from the eyeballs without fasting. Serum was separated as soon as possible after blood collection. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured.

[0049] Table 5 Changes in rat body weight

[0050]

[0051] According to the body weight results in Table 5, there was no significant difference in the body weight of rats in each group after 14 days and 28 days of administration, indicating that cholesterol-lowering peptide, lovastatin, blank control group and modeling blank group had no significant effect on the body weight of rats.

[0052] Table 6 Results of serum index detection in rats after oral administration for 4 weeks

[0053]

[0054] Note: **P<0.01 compared with the model control group;

[0055] As shown in Table 6, compared with the model control group, the cholesterol-lowering peptide group had significantly reduced serum triglycerides, total cholesterol, and low-density lipoprotein levels after four weeks of oral administration, with extremely significant differences (**P < 0.01). It also increased serum high-density lipoprotein levels, with extremely significant differences compared with the model control group (**P < 0.01). There were no significant differences in the serum parameters between the cholesterol-lowering peptide group and the lovastatin control group, but there were significant changes compared with the model control group. This indicates that the cholesterol-lowering peptides screened by the present invention can effectively reduce the levels of triglycerides, total cholesterol, and low-density lipoprotein in rats with hyperlipidemia, and the effect is consistent with that of lovastatin, showing a good lipid-lowering effect.

[0056] The above is a detailed introduction to a polypeptide with cholesterol-lowering function in a soybean protein hydrolyzate, its preparation method, and application provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method and core idea of ​​the present application. At the same time, for those skilled in the art, according to the ideas of the present application, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. Soy protein hydrolysate contains cholesterol-lowering functional polypeptides, among which: The peptide is derived from a peptide segment with a "binding_energy" value <-1.2Kcal for docking with the cholesterol esterase molecule, and the peptide segment for molecular docking with the cholesterol esterase is derived from the Peptide Ranker biological activity score> 0.9 peptide.

2. The soy protein hydrolyzate according to claim 1 contains a polypeptide having cholesterol-lowering function, wherein: The amino acid sequence of the polypeptide is at least one of FFFPF, DFPALWLL, GNWGPL, NWGPL, NNPFKF, LFLPHF, GQYPYGGF, NNNPFKFL, NNPFSF, WNPNNKPF, NNNPFKF, GSGIFGM, QQDEDEDEDEDDEDEQIPSHPPRRPSHGK, DEDEDEDEDDEDEQIPSHPPRRPSHGK, QDEDEDEDEDDEDEQIPSHPPRRPSHGK, LRPPF, FSEYPPLGRF, FEFLPPFIRR, NEPWWPKL, VFDPF, SLDVWDPFEGFPF, HFGSF and FFYNPK.

3. The polypeptide having cholesterol-lowering function in the soybean protein hydrolyzate according to claim 1 or 2, wherein: The amino acid sequence of the polypeptide is FFFPF.

4. Use of the polypeptide with cholesterol-lowering function in the soy protein hydrolyzate according to any one of claims 1 to 3 in the preparation of drugs for lowering hyperlipidemia, foods suitable for people with hyperlipidemia, or health products.

5. A method for preparing a polypeptide having cholesterol-lowering function in soybean protein hydrolyzate, wherein: The method for preparing a polypeptide having cholesterol-lowering function in a soybean protein hydrolyzate as claimed in any one of claims 1 to 3 comprises the following steps: (1) Preparing protein hydrolysate: hydrolyzing soybean protein with protease to obtain protein hydrolysate; (2) Cholesterol esterase inhibitory activity assay: cholesterol esterase inhibitory activity assay; (3) Sequence identification of soy protein hydrolysate: Soy protein hydrolysate was subjected to LC-MS / MS to analyze its peptide composition; (4) Screening of cholesterol-lowering peptides: The identified peptide fragments were first screened for potential bioactive peptides with a Peptide Ranker>0.9, and then docked using AutoDock 4.2.6 software to screen out the most promising cholesterol-lowering peptides.

6. The method for preparing a polypeptide having cholesterol-lowering function in soybean protein hydrolyzate according to claim 5, wherein: The protease is nattokinase, and the enzyme-substrate ratio is 6000 IU / g.

7. The method for preparing a polypeptide having cholesterol-lowering function in soybean protein hydrolyzate according to claim 5 or 6, wherein: The cholesterol esterase was derived from the RCSB database (PDB: 1F6W).

8. The method for preparing a polypeptide having cholesterol-lowering function in soybean protein hydrolyzate according to any one of claims 5 to 7, wherein: The following steps are involved: (1) Soy protein dissolution: Soy protein is used as the raw material and dissolved in pure water to prepare a 3-7% (W / V) soy protein isolate solution; (2) Enzymatic hydrolysis of soy protein: adding enzymes, stirring and enzymolyzing at a certain temperature to obtain a hydrolyzed protein solution; (3) Soy protein hydrolysate: inactivate the enzyme, centrifuge, and take the supernatant, which is the soy protein isolate hydrolysate; (4) Determination of cholesterol esterase inhibitory activity: Determination of cholesterol esterase inhibitory activity according to the method in the literature; (5) Identification of peptide sequences: Peptide mapping of the enzymatically hydrolyzed soy protein hydrolysate was performed using the ThermoFisher Q Exactive system; (6) Screening of cholesterol-lowering peptides: The identified peptide fragments were first screened for potential bioactive peptides with a Peptide Ranker>0.9, and then molecular docking was performed using AutoDock 4.2.6 software to screen out the most promising cholesterol-lowering peptides.

9. The method for preparing a polypeptide having cholesterol-lowering function in soybean protein hydrolyzate according to claim 8, wherein: In step (1), the soy protein isolate is dissolved by stirring at 90° C. for 15 minutes; in step (2), the temperature is 45° C. and the stirring time is 2 hours.

10. The method for preparing a polypeptide having cholesterol-lowering function in soybean protein hydrolyzate according to claim 8 or 9, wherein: In step (3), the enzyme inactivation temperature is 90° C. and the enzyme inactivation time is 15 minutes; in step (4), the centrifugal speed is 5000-10000 r / min and the centrifugal time is 20 minutes.

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