Cholesterol ester transfer protein inhibitory peptide as well as preparation method and application thereof

Abstract

The invention provides a cholesterol ester transfer protein inhibitory peptide as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The cholesteryl ester transfer protein inhibitory peptide provided by the invention can inhibit the effect of cholesteryl ester transfer protein so as to achieve the purpose of controlling hyperlipidemia, and the mullet fish skin as an inhibitory peptide source is easy to obtain, the treatment process is simple, industrial production is easy, the fish skin as leftovers is comprehensively utilized, and the additional value can be increased.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a cholesterol ester transfer protein inhibitory peptide and a preparation method and application thereof. Background Art

[0002] Hyperlipidemia, also known as dyslipidemia, is a chronic disease caused by increased total cholesterol, triglycerides, and low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol in plasma. Low-density lipoprotein cholesterol will spread throughout the body and cause hyperlipidemia, while high-density lipoprotein can transfer cholesterol esters to the liver for decomposition, thereby reducing blood lipid levels in the body. Cholesterol ester transfer protein, as a key enzyme in hyperlipidemia, causes cholesterol esters to transfer from high-density lipoprotein to low-density lipoprotein or very low-density lipoprotein, thereby increasing the risk of hyperlipidemia. Therefore, cholesterol ester transfer protein is one of the important molecular targets for the development of drugs for the treatment of hyperlipidemia. The Chinese invention patent with publication number CN113332287A and name "Cholesterol ester transfer protein inhibitors and pharmaceutical compositions containing the inhibitors for the treatment or prevention of cardiovascular diseases" discloses cholesterol ester transfer protein inhibitors and pharmaceutical compositions containing the inhibitors, which proves that they have significant inhibitory effects on hyperlipidemia.

[0003] At present, several inhibitory drugs for cholesterol ester transfer protein have been developed, but most of them are developed through chemical modification, and the resulting inhibitors also have the risk of toxicity and inadaptability. Therefore, bioactive peptides have become one of the new research directions for cholesterol ester transfer protein inhibitors. The research on seafood-derived lipid-lowering peptides mainly uses shellfish as raw materials, and the processing process is cumbersome and the yield is low. There are no reports in China on the preparation of lipid-lowering peptides from marine low-value fish protein.

[0004] Fish skin protein is a high-quality source of bioactive peptides. Many fish skin protein-derived peptides have been shown to have the effects of lowering blood pressure, lowering blood lipids, lowering blood sugar and anti-cancer. Pike, also known as golden pike or pike, is a common economic edible fish. It is rich in unsaturated fatty acids, which is beneficial to blood circulation and is a good food for cardiovascular patients. As a natural lipid-lowering food, its fish skin is selected as the raw material for screening lipid-lowering peptides. It is economical and easy to obtain. The preparation methods of peptides can be divided into direct extraction method, artificial synthesis method and protein degradation method. The process of protein hydrolysis by protease is simple to operate and easy to control, and the active peptides produced are highly safe and have no side effects. It is the most commonly used.

[0005] Therefore, the technical problem to be solved by the present invention is to provide a cholesterol ester transfer protein inhibitory peptide to inhibit the action of cholesterol ester transfer protein so as to control and treat hyperlipidemia. Summary of the invention

[0006] The purpose of the present invention is to provide a cholesterol ester transfer protein inhibitory peptide to inhibit the action of cholesterol ester transfer protein so as to achieve the purpose of controlling and treating hyperlipidemia.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a cholesterol ester transfer protein inhibitory peptide, the amino acid sequence of which is Gly-Ala-Pro-Gly-Phe-Pro-Gly-Pro-Arg.

[0009] The present invention also provides a method for preparing the inhibitory peptide, comprising the following steps:

[0010] (1) treating fish skin with a NaOH solution and a n-butanol solution in sequence, and drying at room temperature to obtain a first product;

[0011] (2) placing the first product in acetic acid, adding pepsin, inactivating the enzyme at low temperature, and removing impurities by centrifugation to obtain a second product;

[0012] (3) salting out the second product, centrifuging to obtain a precipitate, dissolving the precipitate in acetic acid and dialyzing it, and freeze-drying it to obtain a collagen product;

[0013] (4) hydrolyzing the collagen product with alkaline protease to obtain the cholesterol ester transfer protein inhibitory peptide.

[0014] Preferably, in step (1), the mass volume ratio of the fish skin to the NaOH solution is 1:15-25, the concentration of the NaOH solution is 0.05-0.15 mol / L, and the treatment is to mix the fish skin and the NaOH solution and stir for 20-28 hours, and change the solution every 5-7 hours.

[0015] Preferably, in step (1), the mass volume ratio of the fish skin to n-butanol is 1:25-35, the mass fraction of the n-butanol is 8%-12%, and the treatment is to mix the fish skin and the n-butanol solution and stir for 20-28h, change the solution every 5-7h, and then air-dry.

[0016] Preferably, in step (2), the mass volume ratio of the first product to acetic acid is 1:40-50, the concentration of the acetic acid is 0.4-0.6 mol / L, the amount of pepsin added is 1-3% of the mass of the first product, the low temperature effect is maintained at 3-5°C for 12-18h, the enzyme is inactivated by adjusting the pH to 7.5-8.5 with 4-6 mol / L NaOH solution, the centrifugal speed is 7000-9000 r / min, and the centrifugal time is 9-11 min.

[0017] Preferably, the salting out in step (3) is performed by mixing a 2.5-3.5 mol / L NaCl solution with the second product, the volume ratio of the second product to the NaCl solution is 5:2-3, and the centrifugation is performed at 3-5°C and a speed of 9000-11000 r / min for 15-25 min.

[0018] Preferably, the mass volume ratio of the precipitate to acetic acid in step (3) is 1 g:30 mL, and the dialysis is performed using an 8 KDa dialysis bag and a pure water system of 1:90-100 for 48-72 hours, and the retentate is freeze-dried.

[0019] Preferably, the enzymatic hydrolysis conditions in step (4) are pH 8-12, temperature 40-60° C., and enzyme dosage 1000-5000 U / g.

[0020] The present invention also provides the use of the cholesterol ester transfer protein inhibitory peptide or the cholesterol ester transfer protein inhibitory peptide obtained by the preparation method in the preparation of anti-hyperlipidemic drugs or functional foods.

[0021] In this study, pike skin was used as a raw material to inhibit the activity of cholesterol ester transfer protein and thus reduce hyperlipidemia, and the pike skin collagen was characterized. The enzymatic hydrolysis of collagen by alkaline protease was optimized through single-factor experiments and orthogonal experiments, and the enzymatic hydrolysis products were subjected to ultrafiltration treatment and LC-MS identification. A nonapeptide GAPGFPGPR with significant inhibitory activity on cholesterol ester transfer protein was screened out, and the role of nonapeptide GAPGFPGPR and cholesterol ester transfer protein was clarified. As a functional ingredient, nonapeptide GAPGFPGPR can be used in the research and development of drugs or functional foods with the effect of treating hyperlipidemia, so as to achieve the purpose of controlling and treating hyperlipidemia. In addition, pike skin is easy to obtain as a source of inhibitory peptides, the processing process is simple, and it is easy to industrialize. Fish skin can be comprehensively utilized as a waste material to increase added value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a characterization of the pike skin collagen in Example 1; wherein Figure A is an SDS-PAGE electrophoresis diagram, Figure B is an ultraviolet-visible spectrum diagram, Figure C is a Fourier transform infrared spectrum diagram, and Figures D and E are microstructure diagrams;

[0023] Figure 2 The results of the single factor test and orthogonal test in Example 2 are analyzed; wherein Figure A is a diagram showing the influence of pH on the degree of hydrolysis, Figure B is a diagram showing the influence of temperature on the degree of hydrolysis, Figure C is a diagram showing the influence of enzyme dosage on the degree of hydrolysis, Figure D and Figure d are diagrams showing the influence of pH and temperature on the degree of hydrolysis, Figure E and Figure e are diagrams showing the influence of pH and enzyme dosage on the degree of hydrolysis, and Figure F and Figure f are diagrams showing the influence of temperature and enzyme dosage on the degree of hydrolysis;

[0024] Figure 3 The plasma flow chart of the collagen peptide obtained under the optimal enzymatic hydrolysis conditions in Example 2, which was screened by a protein ultrafiltration instrument and identified by LC-MS of polypeptides less than or equal to 1 KDa;

[0025] Figure 4 In Example 3, after the biological activity, water solubility, toxicity, ADMET and molecular docking screening of the polypeptides, the best performance was the nonapeptide GAPGFPGPR; wherein Figure A is the secondary mass spectrum of the nonapeptide GAPGFPGPR, Figure B is the 3D diagram of the docking result of the nonapeptide GAPGFPGPR and the cholesterol ester transfer protein, and Figure C is the 2D diagram of the docking result of the nonapeptide GAPGFPGPR and the cholesterol ester transfer protein;

[0026] Figure 5 The results of the in vitro inhibition of nonapeptide GAPGFPGPR and cholesterol ester transfer protein in Example 4 are shown in Figure A; Figure A is a synchronous fluorescence spectrum of the binding of nonapeptide GAPGFPGPR to cholesterol ester transfer protein, and Figure B is a graph showing the inhibitory effect of nonapeptide GAPGFPGPR on cholesterol ester transfer protein;

[0027] Figure 6 The figure is a diagram of the optimal cholesterol addition amount for constructing a zebrafish hyperlipidemia model in Example 5; Figure A is a diagram showing the effect of different cholesterol addition amounts on zebrafish survival rate, and Figure B is a diagram showing the effect of different cholesterol addition amounts on the content changes of total cholesterol, triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in zebrafish;

[0028] Figure 7 This is a model diagram of the effect of the nonapeptide GAPGFPGPR on zebrafish in Example 5. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] Preparation and characterization of collagen from pike skin

[0032] The fish skin was pretreated by mixing the fish skin (g) and 0.1 mol / L NaOH (mL) in a ratio of 1:20, stirring at a constant speed of 400 r / min for 24 h, changing the solution every 6 h, taking out the fish skin and mixing it with a 10% n-butanol solution in a ratio of 1 g:30 mL, stirring at a constant speed of 400 r / min for 24 h, changing the solution every 6 h, and drying to dryness and toughness;

[0033] The dried fish skin was mixed with 0.5 mol / L acetic acid at a ratio of 1:45 of sample (g): acetic acid (mL), and 2% pepsin of the sample mass was added. After 15 h of low temperature at 4°C, the solution was taken out, the pH value was adjusted to 8 with 5 mol / L NaOH solution to inactivate the enzyme, and the impurities were removed by centrifugation at 8000 r / min for 10 min.

[0034] The purified collagen solution was salted out with 3 mol / L NaCl solution at a volume ratio of 5:2, and the precipitate was centrifuged at 4°C and 10000 r / min for 20 min to obtain the precipitate, which was dissolved in 0.5 mol / L acetic acid at a mass volume ratio of 1:30 and dialyzed with an 8KDa dialysis bag for 3 days. The retentate was freeze-dried, and the chamber vacuum was set to 10 Pa, the cold trap temperature was set to -80°C, and the sample temperature was set to -80°C to obtain the collagen product;

[0035] The collagen product was dissolved in water to prepare a 1 mg / mL collagen solution. The molecular weight distribution of the solution was analyzed by SDS-PAGE electrophoresis, which mainly showed α1, α2 and β chains. The molecular weights of α1 and α2 chains were greater than 100 KDa, of which the molecular weight and proportion of α1 chain were greater than that of α2 chain. The molecular weight of β chain was about 230 KDa, which was a typical type I collagen (such as Figure 1 A);

[0036] The collagen product was mixed with 0.5 mol / L acetic acid solution to form a 1.0 mg / mL collagen solution, and a UV-visible light scan was performed using a UV spectrophotometer within a wavelength range of 190-400 nm. The results showed that the highest absorption value was at 239.8 nm, which was presented by the addition of carboxyl (-COOH), amide (-CONH2) and carbonyl (-C=O) chromophores under UV scanning. It can be determined that the desired fish skin collagen belongs to typical type I collagen (such as Figure 1 B);

[0037] The collagen product was mixed with potassium bromide powder in a mass ratio of 1:100, ground, dried and pressed into tablets, and the sample was analyzed using a Fourier transform infrared spectrometer at 4000-400 cm -1 Scanning is performed within the range of 32 times with a resolution of 4 cm -1 The absorption peaks in the infrared spectrum of type I collagen usually appear in five amide bands: amide A, B, I, II and III. The amide A band of fish skin collagen is at 3441 cm -1 This is due to the absorption peak caused by the vibration of the NH bond in the amide molecule. The amide B band is at 2926.01 cm -1 This is due to the stretching vibration of CN and CH2. The amide I band is at 1637.56 cm -1The amide I band is involved in the formation and maintenance of the triple helix structure of collagen, which mainly corresponds to the stretching vibration of C=O. The amide II band appears at 1558.48 cm -1 The absorption peak is related to the bending vibration of heterogeneous NH and the stretching vibration of CN. The amide III band is at 1415.75 cm -1 The five amide bands are located at 3441 cm -1 、2926.01cm -1 、1637.56cm -1 、1558.48cm -1 and 1415.75cm -1 This indicates that the collagen retains a complete triple helical structure and has typical protein structural characteristics (such as Figure 1 C);

[0038] The amino acid composition of collagen was detected by liquid chromatography. Weigh 100 mg of uniform collagen product in a sealed bottle, add 10 mL of 6 mol / L hydrochloric acid, fill with nitrogen for 1 min, seal the bottle, and hydrolyze at 110 ° C for 22 h. Take out and cool, dilute with water to 50 mL, take 1 mL of nitrogen at 95 ° C, blow dry, accurately add 1 mL of 0.01 mol HCl to dissolve, pass through a 0.22 μm filter membrane for determination, and then perform gradient elution on the sample. The results are shown in Table 1. In 1 g of pike skin collagen sample, the glycine content is as high as 83.359 mg / g, which is the highest content among the 17 amino acids tested, and meets the standard of collagen amino acid composition characteristics. Secondly, the content of alanine (36.722 mg / g) and proline (36.113 mg / g) is relatively high. Studies have shown that the content of arginine and hydrophobic amino acids is related to high biological activity, and pike skin collagen contains a large amount of hydrophobic amino acids and arginine, making pike skin collagen peptide one of the choices for inhibitory peptides of cholesterol ester transfer protein;

[0039] Table 1 Amino acid content of pike skin collagen

[0040]

[0041] Scanning electron microscopy (SEM) was used to study the changes in the microstructure and properties of collagen. The freeze-dried pike skin collagen product was fixed on a conductive glue, and the sample was sprayed with gold and then placed in a field emission scanning electron microscope for scanning. The image was magnified 500 times (3KV acceleration voltage) Figure 1 D) and 1000 times ( Figure 1E) imaging, it can be seen that the microscopic morphology of collagen has a fiber structure, a sheet structure, a multi-layered aggregate structure, and is loose and porous. Collagen has a relatively uniformly distributed fiber structure and a denser network structure, which is honeycomb-like and porous, with interconnected filaments of different thicknesses, indicating that pike skin collagen has good moisturizing properties.

[0042] Example 2

[0043] Optimization of enzymatic hydrolysis conditions and ultrafiltration identification of collagen peptides

[0044] The degree of hydrolysis of collagen was calculated using the following formula: 36% neutral formaldehyde and -NH 3+ The reaction fixes its alkalinity to make -COOH- show acidity, and then titrates with a standard 0.05 mol / L NaOH solution, and uses a pH meter to determine the endpoint of the final titration.

[0045] Take 10mL of the reaction solution, add 6 times the volume of water, place it on a magnetic stirrer for uniform stirring, titrate it with a standard 0.05mol / L sodium hydroxide solution to a pH of 8.2, record the volume of sodium hydroxide consumed, then add 10mL of formaldehyde and mix well, continue titrating to a pH of 9.2, and after the value stabilizes, record the volume of NaOH added and perform a blank experiment. Substitute the recorded data into the following formula for calculation:

[0046]

[0047] Wherein, V is the volume of NaOH consumed after adding formaldehyde (mL); V0 is the volume of NaOH consumed in the blank experiment (mL); C is the concentration of NaOH (mol / L); V1 is the constant volume of the sample diluent (mL); 0.014 is the mass of nitrogen equivalent to 1.00mL of sodium hydroxide standard titration solution [C(NaOH)=1.000mol / L] (g); M is the mass of the sample (g); V2 is the amount of sample diluent taken (mL);

[0048] Each time, 0.2g of collagen was used to prepare a collagen solution with water at a ratio of 1g:100mL, and alkaline protease was added for enzymolysis. The degree of hydrolysis was used as an indicator for the determination of the enzymolysis effect. pH values ​​of 8, 9, 10, 11, and 12, temperatures of 40°C, 45°C, 50°C, 55°C, and 60°C, and enzyme dosages of 1000U / g, 2000U / g, 3000U / g, 4000U / g, and 5000U / g were set up for single-factor experiments to determine the best enzymolysis conditions (such as Figure 2 A, B and C);

[0049] According to the results of the single-factor experiment, three factors, pH (A), temperature (B) and enzyme dosage (C), were selected. The optimal variable (optimal enzymatic hydrolysis condition) and two nearby variables were selected to form three levels, forming a three-factor three-level orthogonal experimental design and conducting experiments to calculate the degree of hydrolysis. The Design-Expert 13 software was used for experimental design and SPSS software was used for analysis. The results are shown in Tables 2 and 3. With the increase of the levels of the three factors, pH, temperature and enzyme dosage, the enzymatic hydrolysis effect of fish skin collagen showed a trend of first increasing and then decreasing. The sensitivity of the three factors to the response value was in the order of temperature>enzyme dosage>pH. The openings of each response surface were all facing downward, indicating that the experimental results contained a maximum value within the experimental area. Therefore, it can be determined that pH 10, enzyme addition amount of 3000U / g, and temperature of 50°C are the optimal enzymatic hydrolysis conditions. Under these conditions, the yield of collagen polypeptides is 26.03% (such as Figure 2 Dd, Ee and Ff);

[0050] Table 2 Orthogonal experimental design and results

[0051]

[0052] Table 3 Hydrolysis degree variance analysis results

[0053]

[0054]

[0055] in, * represents significant difference (p<0.05), ** Represents extremely significant difference (p<0.01).

[0056] According to the results of the orthogonal test, the optimal enzymatic hydrolysis conditions are pH = 10, 50°C, and 3000U / g. A large amount of pike skin collagen was enzymatically hydrolyzed, and the collagen peptides after enzymatic hydrolysis were screened with an ultrafiltration instrument. A 1KDa ultrafiltration membrane was selected for assembly. The main pump and the auxiliary pump were first rinsed with pure water for 3 times, and the enzymatically hydrolyzed collagen peptide sample was added thereto. The pressure was controlled at about 0.4, the discharge valve was opened and collected with a collection bag, and the sample was rinsed with water 3 times after the end. The obtained ultrafiltration solution was evaporated and concentrated with a rotary evaporator, and the concentrated pike skin collagen peptides with a molecular weight of less than 1KDa were freeze-dried and detected by LC-MS. The results showed that ions were generated at 9 minutes, and the relative abundance reached the highest at 17 minutes. A total of 220 pike skin collagen peptides (such as Figure 3 ).

[0057] Example 3

[0058] Virtual screening of collagen peptides

[0059] PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), pepcalc (https: / / pepcalc.com / ppc.php), ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php) and ADMETLAB3.0 (https: / / admetlab3.scbdd.com / server / evaluationCal) online tools were used to predict biological activity, water solubility, toxicity and ADMET performance, and 9 peptides with non-toxicity, good water solubility, strong biological activity and good ADMET performance were screened. The results are shown in Table 4;

[0060] Table 4 Screening of various peptide indicators

[0061]

[0062] The selected peptides were molecularly docked with cholesterol ester transfer protein using Discovery Studio 2017 software, and their binding energies (-kcal / mol) were all above 60. The inhibitory peptide with the best comprehensive performance was GAPGFPGPR (SEQ ID NO.1), which was sent to Shanghai Biotechnology Co., Ltd. for synthesis (such as Figure 4 ).

[0063] Example 4

[0064] In vitro inhibition effect verification

[0065] Cholesterol ester transfer protein was combined with nonapeptide GAPGFPGPR, reacted at 37°C in the dark for 30 minutes, and synchronous fluorescence spectroscopy was performed. The interval between the excitation wavelength and the emission wavelength was set to Δλ=15nm, the initial excitation wavelength was 200nm, and the emission wavelength was 230nm. The spectral data in the wavelength range of 250-500nm were recorded and a spectrum was drawn. The control group was cholesterol ester transfer protein, and the experimental group was the nonapeptide GAPGFPGPR and cholesterol ester transfer protein interaction group. The absorption wavelength of cholesterol ester transfer protein was 339.4nm and the fluorescence intensity was 811.9. The addition of nonapeptide GAPGFPGPR reduced the fluorescence intensity of cholesterol ester transfer protein. After adding nonapeptide GAPGFPGPR, its fluorescence intensity dropped from 811.9 to 391.1, and its absorption wavelength was red-shifted from 339.4nm to 366.2nm (such as Figure 5 A);

[0066] Using zebrafish serum samples as substrates, cholesterol ester transfer protein and nonapeptide GAPGFPGPR were combined and incubated in serum samples for 3 hours. High-density lipoprotein cholesterol was used as a detection index to verify the inhibitory effect of nonapeptide GAPGFPGPR on cholesterol ester transfer protein. A control group, an experimental group, and an inhibitory peptide group were set up, which were zebrafish serum samples and pure water, zebrafish serum samples and cholesterol ester transfer protein co-incubated, and zebrafish serum samples, cholesterol ester transfer protein and nonapeptide GAPGFPGPR co-incubated. The volumes of pure water, cholesterol ester transfer protein solution, and cholesterol ester transfer protein and nonapeptide GAPGFPGPR mixed solution were kept consistent. The results showed that the addition of cholesterol ester transfer protein to zebrafish serum samples would reduce the content of high-density lipoprotein cholesterol, while the experimental group with the addition of GAPGFPGPR polypeptide had a higher content of high-density lipoprotein cholesterol than the experimental group. This is because the polypeptide inhibited the active site of cholesterol ester transfer protein, which weakened its effect, indicating that nonapeptide GAPGFPGPR has a good effect on cholesterol ester transfer protein (such as Figure 5 B).

[0067] Example 5

[0068] Verification of lipid-lowering effect in vivo

[0069] Feed preparation: Select zebrafish aged 3-4 months under specific conditions, add food-grade cholesterol to ordinary feed to make it into 5%, 10%, 15%, and 20% cholesterol feed, mix them evenly and use anhydrous ethanol as a co-solvent to allow cholesterol to adhere to the feed, mix for 30 minutes, and evaporate the ethanol from the mixed feed in a fume hood to prevent ethanol from affecting the experiment. Five groups of experiments were designed to feed zebrafish, namely blank control group (no cholesterol added), 5% cholesterol group, 10% cholesterol group, 15% cholesterol group, and 20% cholesterol group, with 10 zebrafish in each group, fed twice in the morning and evening for 20 days, and the survival rate of zebrafish was observed. The results showed that the survival rate of zebrafish in the 15% cholesterol group and the 20% cholesterol group was reduced, indicating that too high cholesterol content in the feed will reduce the survival rate of zebrafish (such as Figure 6 A);

[0070] Index detection: The blank control group (no cholesterol added), 5% cholesterol group, 10% cholesterol group, 15% cholesterol group and 20% cholesterol group were tested for indicators after 14 days of feeding. Six zebrafish were randomly selected from each group to measure the total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels. The average value of three parallel experiments was taken for each sample to observe whether there were significant changes between the indicators to determine the optimal cholesterol ratio. The results showed that the total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels in zebrafish fed with feeds with different cholesterol contents changed significantly. It can be seen from various indicators that the effect of the feed with 20% cholesterol content is the most obvious, but zebrafish died during the feeding process of 15% and 20% cholesterol feeds, so the 10% cholesterol feed was used as the best added amount feed (such as Figure 6 B);

[0071] Model construction: Zebrafish were divided into a blank control group, a hyperlipidemia model group, and an inhibitory peptide experimental group, with 10 in each group. During the experiment, zebrafish were fed twice a day, once in the morning and once in the evening. Each time they were fed, the feed had to be consumed within 5 minutes. The water temperature of the culture system was 26-28°C, and the fish tank was cleaned regularly to maintain a good living environment. The blank control group was fed with normal feed for 28 days. The hyperlipidemia model group was fed with a high-fat diet containing 10% cholesterol for 14 days, and then fed with normal feed for 14 days. After the inhibitory peptide experimental group was fed with a high-fat diet containing 10% cholesterol for 14 days, the zebrafish were transferred to an 8μg / mL inhibitory peptide solution, the solution was changed every 12 hours, and the normal feed was fed for 14 days. The results of the body shape index are shown in Table 5. There was no significant difference in the body length characteristics. The zebrafish fed with a high-fat diet grew slightly, but there was no significant difference (p>0.05). For body width characteristics, compared with the blank control group, the inhibitory peptide experimental group had no significant difference (p>0.05), the hyperlipidemia model group had a very significant difference in body width (p<0.01), and the inhibitory peptide experimental group had a very significant difference from the hyperlipidemia model group (p<0.01). For body weight characteristics, compared with the blank control group and the hyperlipidemia experimental group, the inhibitory peptide experimental group had significant differences (p<0.05), and there were very significant differences between the blank control group and the hyperlipidemia experimental group (p<0.01). The effect of the nonapeptide GAPGFPGPR on zebrafish is shown in Figure 7 , A is the hyperlipidemia model group, B is the inhibitory peptide experimental group, and C is the blank control group. It was found that the zebrafish fed with high-fat diet were relatively wide, followed by the inhibitory peptide experimental group, and the blank control group was the most slender;

[0072] Table 5 Changes in body length, body width and weight of zebrafish in each group

[0073]

[0074] symbol * and Symbol ** Respectively indicate significant difference (p<0.05) and extremely significant difference (p<0.01) compared with the blank control group. # and Symbol ## They respectively indicate significant differences (p<0.05) and extremely significant differences (p<0.01) compared with the hyperlipidemia model group.

[0075] The measured total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol index data were expressed in the format of mean ± standard deviation (X ± SD), and the four index data were processed and analyzed using IBM SPSS Statistics data software. The different indexes were compared by one-way analysis of variance, and the same index was tested by LSD method. The results are shown in Table 6. Compared with the blank control group, the total cholesterol, triglyceride and high-density lipoprotein cholesterol levels in the inhibitory peptide experimental group were significantly different (p < 0.05), and the low-density lipoprotein cholesterol content was not significantly different. The total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels in the hyperlipidemia model group were extremely significantly different (p < 0.01).

[0076] Compared with the hyperlipidemia model group, the inhibitory peptide experimental group had significant differences in triglyceride and low-density lipoprotein cholesterol levels (p<0.05), and extremely significant differences in total cholesterol and high-density lipoprotein cholesterol levels (p<0.01), and the blank control group had extremely significant differences (p<0.01).

[0077] Table 6 Various indicators of zebrafish in different groups

[0078]

[0079] symbol * and Symbol ** Respectively indicate significant difference (p<0.05) and extremely significant difference (p<0.01) compared with the blank control group. # and Symbol ## They respectively indicate significant differences (p<0.05) and extremely significant differences (p<0.01) compared with the hyperlipidemia model group.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cholesterol ester transfer protein inhibitory peptide, characterized in that The amino acid sequence of the cholesterol ester transfer protein inhibitory peptide is Gly-Ala-Pro-Gly-Phe-Pro-Gly-Pro-Arg.

2. The method for preparing the inhibitory peptide according to claim 1, characterized in that: The following steps are involved: (1) treating fish skin with a NaOH solution and a n-butanol solution in sequence, and drying the fish skin to obtain a first product; (2) placing the first product in acetic acid, adding pepsin, inactivating the enzyme at low temperature, and removing impurities by centrifugation to obtain a second product; (3) salting out the second product, centrifuging to obtain a precipitate, dissolving the precipitate in acetic acid and dialyzing it, and freeze-drying it to obtain a collagen product; (4) hydrolyzing the collagen product with alkaline protease to obtain an enzymatic solution containing the cholesterol ester transfer protein inhibitory peptide, and separating and purifying to obtain the inhibitory peptide.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the fish skin and the NaOH solution in step (1) is 1 g:15-25 mL, the concentration of the NaOH solution is 0.05-0.15 mol / L, and the treatment is to mix the fish skin and the NaOH solution and stir for 20-28 hours, and change the solution every 5-7 hours.

4. The preparation method according to claim 3, characterized in that: The mass volume ratio of the fish skin and n-butanol in step (1) is 1g:25-35mL, the mass fraction of the n-butanol is 8%-12%, and the treatment comprises mixing the fish skin and the n-butanol solution and stirring for 20-28h, changing the solution every 5-7h, and then drying.

5. The preparation method according to claim 4, characterized in that: In step (2), the mass volume ratio of the first product and acetic acid is 1 g: 40-50 mL, the concentration of the acetic acid is 0.4-0.6 mol / L, the amount of pepsin added is 1-3% of the mass of the first product, the low temperature is maintained at 3-5° C. for 12-18 h, the enzyme is inactivated by adjusting the pH to 7.5-8.5 with 4-6 mol / L NaOH solution, the centrifugal speed is 7000-9000 r / min, and the centrifugal time is 9-11 min.

6. The preparation method according to claim 5, characterized in that: The salting out in step (3) is performed by mixing a 2.5-3.5 mol / L NaCl solution with the second product, wherein the volume ratio of the second product to the NaCl solution is 5:2-3, and the centrifugation is performed at a speed of 9000-11000 r / min at 3-5°C for 15-25 min.

7. The preparation method according to claim 6, characterized in that: In step (3), the mass volume ratio of the precipitate to acetic acid is 1 g: 28-32 mL, and the dialysis is performed using an 8 KDa dialysis bag and a pure water system of 1:90-100 for 48-72 hours, and the retentate is freeze-dried.

8. The preparation method according to claim 7, characterized in that: The conditions for the enzymatic hydrolysis in step (4) are as follows: pH 8-12, temperature 40-60° C., and enzyme dosage 1000-5000 U / g.

9. Use of the cholesterol ester transfer protein inhibitory peptide according to claim 1 or the cholesterol ester transfer protein inhibitory peptide obtained by the preparation method according to any one of claims 2 to 8 in the preparation of anti-hyperlipidemic functional food.

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