Schizochytrium limacinum polypeptide with lipid metabolism regulating effect as well as separation and purification method and application of schizochytrium limacinum polypeptide

Abstract

The invention discloses schizochytrium limacinum polypeptide with a lipid metabolism regulating effect as well as a separation and purification method and application. The separation and purification method comprises the following steps: S1, preparing a schizochytrium limacinum protein suspension; s2, adjusting the schizochytrium limacinum protein suspension obtained in the step S1 to be neutral, adding neutral protease for hydrolysis, cooling after boiling, and hydrolyzing to obtain schizochytrium limacinum protein hydrolysate; s3, the schizochytrium limacinum protein enzymatic hydrolysate obtained in the step S2 is filtered through a membrane, polypeptide with the molecular weight being smaller than 3 kDa and the amino acid sequence being SEQ ID NO.1 is obtained, and the schizochytrium limacinum polypeptide with the lipid metabolism adjusting effect is obtained. According to the invention, accumulation of triglyceride (TG) and cholesterol (TC) in cells is effectively reduced, lipotoxicity stress is relieved, and efficient and low-toxicity candidate active molecules are provided for development of drugs for resisting fatty liver, reducing blood fat or treating metabolic syndrome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioactive peptides, and particularly relates to a Schizochytrium sp. polypeptide with a lipid metabolism regulating effect and a separation and purification method and application thereof. BACKGROUND

[0002] With the development of economy and the improvement of living standards, some unhealthy living habits have also emerged, such as high-energy diet, overnutrition and lack of physical exercise. Further, these conditions can cause metabolic disorders and body function disorders, leading to obesity, hypertension and hyperlipidemia and other metabolic syndromes. About 17 million people die of cardiovascular diseases worldwide each year due to hyperlipidemia, accounting for 30% of global deaths, so timely prevention and treatment of hyperlipidemia is crucial. At present, many bioactive substances in food have significant lipid-lowering activity.

[0003] Schizochytrium sp. belongs to marine fungi and contains a large amount of bioactive substances beneficial to humans, such as unsaturated fatty acids, pigments and proteins. Schizochytrium sp. is an oil-producing microorganism and is widely reported to contain a large amount of docosahexaenoic acid (DHA), a long-chain omega-3 polyunsaturated fatty acid, which exists in the developing brain. A large number of researchers have focused on the industrial production of docosahexaenoic acid of Schizochytrium sp. The protein content in the algal residue left after extracting unsaturated fatty acids from Schizochytrium sp. is as high as more than 40% of the dry mass, which is currently mainly used as animal feed to provide nutrition for animals and is also used as fertilizer to promote plant growth. Bioactive peptides prepared from Schizochytrium sp. are increasingly becoming the focus of scientific research due to their excellent antibacterial, cell proliferation promoting and anti-inflammatory biological activities.

[0004] Ultrafiltration is widely used in the separation and purification of macromolecular proteins and other substances due to its high filtration efficiency, stable chemical properties, high filtration precision, easy cleaning and maintenance, energy saving and environmental protection and wide application. With ultrafiltration tubes of various specifications, macromolecular substances are screened out, and polypeptide components with high purity and moderate molecular weight are effectively enriched to meet the research and application needs in different fields. At the same time, liquid chromatography-mass spectrometry is commonly used for the identification of the structure of bioactive peptides. Generally, protein hydrolysate samples include several peptide segments, and it is time-consuming and laborious to rely solely on experiments for screening. In this regard, in-silico analysis of identified polypeptides is a promising method that can screen all identified sequences without involving experimental data, and the combination of molecular docking and other technologies is more helpful to determine whether the peptides have biological activity.

[0005] Constructing in vitro liver cell lipid metabolism model is conducive to efficiently screening functional food ingredients with significant effects on improving lipid metabolism, and provides more safe and effective treatment options for patients with hyperlipidemia. In our daily dietary habits, palmitic acid (PA) as an important member of saturated fatty acids and oleic acid (OA) as a representative of monounsaturated fatty acids have particularly prominent content in various foods, and become the main source of fatty acids in our daily intake. When studying the lipid-lowering effect of drugs or natural active ingredients, a mixture of OA (oleic acid) and PA (palmitic acid) is often used as free fatty acid to simulate a high-fat environment and induce a high-fat injury model. This model can effectively simulate the physiological state that may occur in the human body under a high-fat diet, and then evaluate the potential effect of different drugs or natural active ingredients in reducing blood lipid levels. SUMMARY

[0006] Therefore, the present application aims to provide a Schizochytrium polypeptide with lipid metabolism regulating effect, and a separation and purification method and application thereof, so as to solve at least one technical problem in the background art.

[0007] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0008] A Schizochytrium polypeptide with lipid metabolism regulating effect, wherein the amino acid composition of the Schizochytrium polypeptide is SEQ ID NO. 1.

[0009] SEQ ID NO. 1 is Val-Ile-Glu-Ile-Pro-Asp-Asp-Val-Ala-Glu-Lys.

[0010] Further, the molecular weight of the Schizochytrium polypeptide is less than 3 kDa.

[0011] Further, the use concentration of the polypeptide is 50-200 μM.

[0012] The preparation method of the above-mentioned Schizochytrium polypeptide with lipid metabolism regulating effect comprises the following steps:

[0013] S1: preparing a Schizochytrium protein suspension;

[0014] S2: adjusting the Schizochytrium protein suspension obtained in step S1 to neutral, adding neutral protease for hydrolysis, boiling and cooling, and obtaining a Schizochytrium protein hydrolysate;

[0015] S3: filtering the Schizochytrium protein hydrolysate obtained in step S2 through a membrane to obtain a polypeptide with a molecular weight of less than 3 kDa and an amino acid sequence of SEQ ID NO. 1, thereby obtaining a Schizochytrium polypeptide with lipid metabolism regulating effect.

[0016] Further, the step S1 of preparing the Schizochytrium protein suspension includes formulating the Schizochytrium protein and the phosphate buffer into the Schizochytrium protein suspension.

[0017] The mass concentration of the Schizochytrium protein suspension is 3.50%-4.30%.

[0018] Further, the preparation method of the Schizochytrium protein includes the following steps:

[0019] A1: weighing the Schizochytrium powder, mixing the Schizochytrium powder and n-hexane to obtain a mixture, stirring the mixture for defatting, and air-drying to obtain defatted Schizochytrium powder;

[0020] A2: mixing the defatted Schizochytrium powder with deionized water, adjusting to alkaline, stirring and centrifuging, collecting the supernatant, adjusting to acid, standing and centrifuging, collecting the precipitate, dialyzing the precipitate, and drying and freezing to obtain the Schizochytrium protein.

[0021] Further, the mass-volume ratio of the Schizochytrium powder and n-hexane in step A1 is 1:4-6.

[0022] The step A1 of stirring the mixture for defatting includes stirring the mixture in a fume hood at room temperature for 11-13 h for defatting.

[0023] The mass-volume ratio of the defatted Schizochytrium powder and deionized water in step A2 is 1:8-12.

[0024] The step A2 of adjusting to alkaline includes adjusting the pH to 9.5-10.5 using NaOH.

[0025] The step A2 of stirring and centrifuging includes stirring at 35-45°C for 3.5-4.5 h, and then centrifuging at 3-5°C and 9000-11000 rpm for 18-22 min using a centrifuge.

[0026] The step A2 of adjusting to acid includes adjusting the pH of the collected supernatant to 4.0-4.4 using HCl.

[0027] The step A2 of standing and centrifuging includes standing for 0.3-0.8 h, and then centrifuging at 3-5°C and 9000-11000 rpm for 12-18 min using a centrifuge.

[0028] The step A2 of dialyzing the precipitate and drying and freezing to obtain the Schizochytrium protein includes placing the precipitate in a dialysis bag, and then dialyzing with distilled water for 46-50 h, replacing the outer dialysate every 7.5-8.5 h. After pre-freezing the protein in the dialysis bag, it is placed in a vacuum freeze dryer for vacuum freeze drying, and the Schizochytrium protein is obtained.

[0029] Further, the neutral protease of step S2 is purchased from Nanning Pangbo Biological Engineering Co., Ltd., and the model is 200,000 u / g; the mass fraction of the neutral protease is 9.60%;

[0030] The temperature of the neutral protease hydrolysis of step S2 is 40-50℃, and the hydrolysis time is 3.5-4.5h;

[0031] The cooling time after boiling in step S2 is 8-12min;

[0032] Further, the membrane filtration of the obtained Schizochytrium sp. protease hydrolysate of step S2 in step S3 includes passing through a 0.45μm microporous filter membrane, and then sequentially separating through 10kDa and 3kDa ultrafiltration tubes to obtain polypeptides with a molecular weight less than 3kDa, and then screening polypeptides with the amino acid sequence of SEQ ID NO.1.

[0033] The Schizochytrium sp. polypeptide prepared by the above-mentioned preparation method of a Schizochytrium sp. polypeptide with a lipid metabolism regulating effect is applied to a medicine with a lipid metabolism regulating effect, and is applied to an anti-fatty liver medicine, a blood lipid-lowering medicine or a metabolic syndrome treatment medicine.

[0034] Compared with the prior art, the Schizochytrium sp. polypeptide with a lipid metabolism regulating effect, the preparation method and the application thereof have the following advantages:

[0035] The Schizochytrium sp. polypeptide (VIEIPDDVAEK) prepared in the present application can improve the lipid accumulation of high-fat HepG2 cells induced by FFAs by inhibiting cell lipid synthesis, promoting fatty acid oxidation and lipid decomposition process, and inhibiting the uptake and promoting the transport rate of free fatty acids. This synergistic effect of multiple mechanisms effectively reduces the accumulation of intracellular triglycerides (TG) and cholesterol (TC), relieves lipid toxicity stress, and provides a high-efficiency and low-toxicity candidate active molecule for the development of anti-fatty liver, blood lipid-lowering or metabolic syndrome treatment medicines. BRIEF DESCRIPTION OF DRAWINGS

[0036] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the schematic embodiments of the present application and the descriptions thereof are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0037] Figure 1 It is a comparison diagram of the pancreatic lipase inhibition ability curves and IC50 values of the three polypeptides (A is the pancreatic lipase inhibition ability curve of SEQ ID NO.15, B is the pancreatic lipase inhibition ability curve of SEQ ID NO.1, C is the pancreatic lipase inhibition ability curve of SEQ ID NO.2, and D is the comparison diagram of the IC50 values of the pancreatic lipase inhibition ability of the three polypeptides);

[0038] Figure 2 Figure 8 shows the toxicity of VIE on HepG2 cells (Note: "*" indicates the significance of the difference between each group and the control group);

[0039] Figure 3 Figure 9 shows the effect of VIE-FFAs on lipid accumulation in HepG2 cells (A: control group; B: 500 μM FFAs; C: 500 μM FFAs + 50 μM VIE; D: 500 μM FFAs + 100 μM VIE; E: 500 μM FFAs + 200 μM VIE; F: 500 μM FFAs + 10 μM Simvastatin, magnification 400x);

[0040] Figure 4 Figure 10 shows the effect of VIE on the levels of TG, LDL-C and HDL-C in cells (A: effect of VIE on the level of TG in cells, B: effect of VIE on the level of LDL-C in cells, C: effect of VIE on the level of HDL-C in cells);

[0041] Figure 5 Figure 11 shows the expression of lipid synthesis-related genes (SREBP1c, FAS) (A: expression of SREBP1c, B: expression of FAS);

[0042] Figure 6 Figure 12 shows the expression of lipid breakdown (ATGL) and fatty acid oxidation-related genes (PPARα and CPT1A) (A: expression of ATGL, B: expression of PPARα, C: expression of CPT1A);

[0043] Figure 7 Figure 13 shows the expression of lipid output-related genes (MTTP and APOB) and lipid uptake-related genes (CD36) (A: expression of MTTP, B: expression of APOB, C: expression of CD36);

[0044] Figure 8 Figure 14 shows the effect of VIE on the expression levels of related proteins in HepG2 (A: expression of SREBP1c, B: expression of FAS, C: expression of ATGL, D: expression of CPT1A, E: expression of PPARα, F: expression of CD36). DETAILED DESCRIPTION

[0045] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0046] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0047] The neutral protease was purchased from Nanning Pangbo Biological Engineering Co., Ltd., with a model number of 200,000 u / g; the mass fraction of the neutral protease was 9.60%;

[0048] Embodiment 1

[0049] A Schizochytrium polypeptide VIEIPDDVAEK having a lipid metabolism regulating effect, characterized in that the VIEIPDDVAEK has a molecular weight of 1227.36 Da and an amino acid composition of Val-Ile-Glu-Ile-Pro-Asp-Asp-Val-Ala-Glu-Lys, and a secondary structure in which an alpha-helix accounts for 29.1% and random coil accounts for 33.3%.

[0050] A separation and purification method of a Schizochytrium polypeptide having a lipid metabolism regulating effect, comprising the following steps:

[0051] (1) A proper amount of Schizochytrium powder was weighed, and n-hexane was added at a ratio of Schizochytrium powder:n-hexane = 1:5 (w / v), and the mixture was stirred in a fume hood at room temperature for 12 h for degreasing, and then air-dried in the fume hood to obtain degreased Schizochytrium powder.

[0052] (2) A proper amount of the degreased algal powder was weighed, deionized water was added at a ratio of algal powder:deionized water = 1:10 (w / v), 1M NaOH was used to adjust the pH to 10.0, and stirring was performed at 40°C for 4 h, and then a centrifuge was used to centrifuge at 4°C and 10,000 rpm for 20 min, and the supernatant was collected. The collected supernatant was adjusted to pH 4.2 with 0.5M HCl, and then stood for 0.5 h, and then a centrifuge was used to centrifuge at 4°C and 10,000 rpm for 15 min to collect the precipitate. The precipitate was placed in a dialysis bag, and then dialyzed with distilled water for 48 h, and the outer dialysis liquid was replaced every 8 h. The proteins in the dialysis bag were pre-frozen, and then placed in a vacuum freeze dryer for vacuum freeze drying to obtain Schizochytrium protein.

[0053] (3) The Schizochytrium protein was prepared into a Schizochytrium protein suspension with a concentration of 3.90% using 0.01M phosphate buffer, and the pH was adjusted to 7.0, and 9.60% neutral protease was added, and hydrolysis was performed in a 45°C water bath for 4 h. After the enzymolysis was completed, boiling was performed in a boiling water bath for 10 min, and then cooled to room temperature. The Schizochytrium protein hydrolysate obtained by hydrolysis was filtered through a 0.45μm microporous filter membrane (water system). Two ultrafiltration tubes with pore sizes of 10 kDa and 3 kDa were selected for step-by-step separation to obtain three groups of components F1 (>10 kDa), F2 (3-10 kDa), and F3 (<3 kDa). Taking the pancreatic lipase (PL) inhibition rate as an index, it was found that the PL inhibition activity of the component with a molecular weight of <3 kDa was optimal, being 57.38%.

[0054] (4) The sample with a molecular weight <3 kDa was identified by liquid chromatography-mass spectrometry, containing 26 polypeptides. According to the relative abundance, 15 effective peptide segments were determined, and the results are shown in Table 1. Combined with PeptideRanker score, combined with its sequence, the number of amino acids, molecular weight, abundance and parent protein, the results are shown in Table 2, and 5 kinds of polypeptides (SEREQSNKPEAGDRRIFMDPASFV, STSFGWFVPEVFAAQAGLDNGRKSEP, SFGWFVPEVFAAQAGLDNGRKSEPWFIEK, ILHGPV, VIEIPDDVAEK) were screened for subsequent analysis.

[0055] Table 1, 15 effective peptide segment identification results statistics table;

[0056]

[0057]

[0058] Table 2, basic properties of 15 kinds of polypeptides identified by LC-MS / MS;

[0059]

[0060] Table 3, 5 kinds of polypeptides to be selected and PPL binding energy and their physicochemical properties

[0061]

[0062]

[0063] Combined with molecular docking, other prediction software (ToxinPred, AllerTOP v.2.0 and Innovagen), it is concluded that they are combined with pancreatic lipase (PPL), and the content of hydrophobic amino acids, isoelectric point, solubility and allergenicity, and the results are shown in Table 3. SEQ ID NO. 15, SEQ ID NO. 2: and SEQ ID NO. 1 three kinds of polypeptides were screened and synthesized for subsequent analysis. Further, the inhibitory effect of the three polypeptides on pancreatic lipase was determined by in vitro experiment, and the inhibition curve is shown in Figure 1 The results show that they can all inhibit the activity of pancreatic lipase, and because of the difference in relative molecular mass, the IC50 values of SEQ ID NO. 15, SEQ ID NO. 2: and SEQ ID NO. 1 for inhibiting pancreatic lipase activity are 8.46, 5.10 and 5.26 μM, respectively. Combined with the cost, the Eustigmatos polyphemis polypeptide VIEIPDDVAEK (hereinafter referred to as VIE) with the function of regulating lipid metabolism is selected as the subsequent research material.

[0064] The model of VIE regulating free fatty acid-induced lipid accumulation in HepG2 cells was constructed. The effects of VIE on the activity and lipid accumulation of HepG2 cells were analyzed to preliminarily study the mechanism of VIE alleviating free fatty acid-induced lipid accumulation in HepG2 cells. The expression of key proteins and genes in the FXR / PPARa lipid metabolism-related pathway was detected by Western blot and qRT-PCR experiments to clarify the molecular mechanism of the Schizochytrium sp. polypeptide improving free fatty acid-induced lipid accumulation in HepG2 cells.

[0065] Effects of VIE on the activity of HepG2 cells

[0066] The polypeptide VIE IPDDVAEK (hereinafter referred to as VIE) was prepared into a 1 mM stock solution with 10% complete medium. The stock solution was diluted into different concentration gradients (500 μM, 250 μM, 100 μM, 50 μM, 25 μM, 10 μM). After 24 h of starvation and 48 h of treatment, the safe concentration range of cell proliferation was selected by the CCK-8 method.

[0067] As can be seen from Figure 2 , within the concentration range of 0-250 μM VIE, the cell viability remained stable with the increase of the polypeptide concentration, and there was no significant difference with the control group (p>0.05). When the VIE treatment concentration was 500 μM, VIE had a significant inhibitory effect on cell proliferation activity (p<0.05). In this study, VIE was added as a cell-protective substance, so the selected VIE concentration should be in the safe range of no toxicity or low toxicity to cells.

[0068] Effects of VIE-FFAs on cell lipid accumulation

[0069] Cells in the logarithmic growth phase were inoculated in a 6-well plate, 2 mL was added to each well, and the cells were cultured in a cell incubator for 24 h. After 80%-90% of the HepG2 cells adhered, the supernatant was discarded, and the cells were grouped according to 4.2.2. The control group was added with 10% complete medium, and the experimental group was added with FFAs and different doses of VIE. After 48 h of culture, the old culture medium containing the drug was discarded, the cells were washed twice with PBS, 4% paraformaldehyde was added, and the samples were fixed at room temperature for 30 min. The above washing steps were repeated, the staining solution was prepared according to the kit instructions, and oil red O staining was performed. The oil red O staining solution needs to be prepared immediately before use. After 30 min of staining, the 6-well plate can be shaken every 10 min to ensure that the staining solution fully contacts the cells. 1 mL of 60% isopropyl alcohol was added for 1-2 min of washing. The cells were washed twice with PBS until the background was clean. After staining, the cells were observed and photographed under an inverted microscope. The neutral fat was red, the phospholipid was pink, and the cell nucleus was blue or blue-violet. The staining results are shown in Figure 3As shown. The normal control group showed less lipid deposition around HepG2 cells (…). Figure 3 A), the control group, i.e., the high-lipid model group, showed a large number of aggregated red lipid droplets, with obvious lipid deposition and a large amount of fatty acids accumulating between cells, indicating that the high-lipid model of HepG2 cells induced by FFAs was successfully constructed. Figure 3 B). Compared with the model group, different concentrations of VIE treatment ( Figure 3 The number of red lipid droplets in CE cells was significantly reduced, and lipid accumulation was significantly improved.

[0070] After 48 hours of culture, the old culture medium was discarded, and freshly prepared cell lysis buffer (RIPA:PMSF = 100:1) was added. Cells were lysed on ice for 20 minutes. Cells were gently and thoroughly scraped from the 6-well plates to ensure all cells were collected. The cell-containing solution was then transferred to 1.5 mL centrifuge tubes and centrifuged at 12,000 rpm for 15 minutes. After centrifugation, the centrifuge tubes were carefully removed, and the supernatant was collected using a pipette for subsequent experiments. Triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels were measured according to the manufacturer's instructions. Six replicates were performed for each group. Results are shown below. Figure 4 As shown, the concentrations of TG and LDL-C in HepG2 cells were significantly reduced after treatment with different doses of VIE and the positive control (p<0.05), indicating that a certain dose of VIE has an inhibitory effect on the increase of TG and LDL-C concentrations in HepG2 cells. Compared with the high-fat model group, HDL-C in HepG2 cells was significantly increased after treatment with different doses of VIE and the positive control (p<0.05), with increases of 57.85%, 53.02%, and 24.33%, respectively. This indicates that a certain dose of VIE can effectively increase the HDL-C level in HepG2 cells. These results indicate that VIE can effectively improve lipid metabolism in high-fat model cells.

[0071] Regulation of key genes in lipid metabolism by vitamin E

[0072] The expression levels of SREBP-1c, FAS, ATGL, PPARα, CPT1A, MTTP, APOB, and CD36 mRNA were detected by qRT-PCR. Among them, SREBP-1c and FAS are lipid synthesis-related genes, ATGL is a lipid breakdown-related gene, PPARα and CPT1A are fatty acid oxidation-related genes, MTTP and APOB are lipid export-related genes, and CD36 is a lipid uptake-related gene.

[0073] like Figure 5As shown, after intervention with different concentrations of VIE, the expression levels of SREBP-1 cmRNA and FAS mRNA in cells were significantly decreased compared with the model group (p<0.05), indicating that VIE has a regulatory effect on lipid synthesis.

[0074] like Figure 6 As shown, co-treatment with different concentrations of VIE and FFAs significantly increased the expression level of ATGL mRNA in cells (p<0.05). This finding suggests that VIE may upregulate ATGL mRNA expression, thereby affecting intracellular lipid breakdown and metabolism. Furthermore, intervention with different concentrations of VIE significantly increased the expression levels of PPARα mRNA and CPT1A in cells (p<0.05), effectively accelerating the entry of intracellular fatty acids into subsequent oxidative breakdown processes and improving the efficiency of fatty acid metabolism.

[0075] like Figure 7 As shown, compared with the high-fat group, the VIE treatment group significantly increased the expression levels of lipid export-related genes MTTP and APOB mRNA, thereby accelerating the rate of new lipid export cells and reducing intracellular lipid content. It also significantly promoted the expression of CD36 mRNA, a key gene for fatty acid uptake.

[0076] VIE Regulation of FXR / PPARα Pathway Proteins

[0077] The expression differences of SREBP-1c, FAS, ATGL, PPARα, CPT1A and CD36 proteins were determined by Western blot.

[0078] like Figure 8 As shown, compared with the control group, the relative expression levels of SREBP-1c and CD36 proteins in the model group treated with 500 μM FFAs were significantly increased (p<0.05); the relative expression level of FAS protein was significantly decreased, which is opposite to its gene expression. This may be because the post-translational FAS protein level regulates its gene expression, or other pathways increase the extracellular secretion of FAS. The specific reasons need further investigation. The relative expression levels of ATGL and CPT1A proteins were significantly increased, consistent with their gene expression trends; the expression level of PPARα protein was significantly decreased. Compared with the model group, the relative expression levels of SREBP-1c and CD36 proteins were significantly reduced in different concentrations of VIE treatment groups (p<0.05) in a dose-dependent manner; the 100μM and 200μM VIE treatment groups significantly reduced the relative expression of Fas protein (p<0.05) in a dose-dependent manner, while the expression of Fas protein in the low-dose group (50μM) was reduced, but there was no statistical difference; different concentrations of VIE treatment significantly promoted the expression of ATGL, PPARα and CPT1A.

[0079] In conclusion, the Schizochytrium sp. polypeptide (VIEIPDDVAEK) can significantly reduce lipid accumulation caused by 500 μM free fatty acids (FFAs), increase HDL-C content, and reduce LDL-C and TG content. By inhibiting the expression of genes and proteins related to cell lipid synthesis (SREBP-1c and FAS), increasing the expression of genes and proteins related to lipid decomposition and fatty acid oxidation (ATGL, PPARa and CPT1A), promoting the expression of genes related to lipid uptake and output (APOB, MTTP and CD36), and reducing the expression of the protein CD36 related to lipid uptake, the Schizochytrium sp. polypeptide effectively improves the lipid accumulation of FFAs-induced high-fat HepG2 cells.

[0080] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Schizochytrium sp. polypeptide having a modulating lipid metabolism effect, characterized by: The amino acid composition of the Schizochytrium sp. polypeptide is SEQ ID NO.

1.

2. The Schizochytrium polypeptide with lipid metabolism regulating effect according to claim 1, characterized in that: The molecular weight of the Schizochytrium sp. polypeptide is less than 3 kDa.

3. The Schizochytrium polypeptide with lipid metabolism regulating effect according to claim 1, characterized in that: The concentration of the polypeptide is 50-200 μM.

4. A method for preparing a Schizochytrium sp. polypeptide having a modulating lipid metabolism effect according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1: preparing a Schizochytrium sp. protein suspension; S2: adjusting the Schizochytrium sp. protein suspension obtained in step S1 to neutral, adding neutral protease for hydrolysis, boiling and cooling, and obtaining a Schizochytrium sp. protein hydrolysate; S3: filtering the Schizochytrium sp. protein hydrolysate obtained in step S2 through a membrane to obtain a polypeptide with a molecular weight of less than 3 kDa and an amino acid sequence of SEQ ID NO. 1, thereby obtaining a Schizochytrium sp. polypeptide with the function of regulating lipid metabolism.

5. The method for preparing a Schizochytrium polypeptide with lipid metabolism regulating effect according to claim 4, characterized in that: Step S1 of preparing a Schizochytrium sp. protein suspension comprises preparing a Schizochytrium sp. protein suspension by mixing Schizochytrium sp. protein and phosphate buffer; The mass concentration of the Schizochytrium sp. protein suspension is 3.50%-4.30%.

6. The method for preparing a Schizochytrium polypeptide with lipid metabolism regulating effect according to claim 5, characterized in that: The method for preparing Schizochytrium sp. protein comprises the following steps: A1: weighing Schizochytrium sp. powder, mixing the Schizochytrium sp. powder with n-hexane to obtain a mixture, stirring the mixture for defatting, and air-drying to obtain defatted Schizochytrium sp. powder; A2: mixing the defatted Schizochytrium sp. powder with deionized water, adjusting to alkaline, stirring and centrifuging, collecting the supernatant, adjusting to acidic, standing and centrifuging, collecting the precipitate, dialyzing the precipitate, and freeze-drying to obtain Schizochytrium sp. protein.

7. The method for preparing a Schizochytrium polypeptide with lipid metabolism regulating effect according to claim 6, characterized in that: In step A1, the mass-to-volume ratio of the Schizochytrium sp. powder to n-hexane is 1:4-6; In step A1, the stirring of the mixture for defatting comprises stirring the mixture in a fume hood at room temperature for 11-13 h for defatting; In step A2, the mass-to-volume ratio of the defatted Schizochytrium sp. powder to deionized water is 1:8-12; In step A2, adjusting to alkaline comprises adjusting the pH to 9.5-10.5 using NaOH; In step A2, stirring and centrifuging comprises stirring at 35-45℃ for 3.5-4.5 h, and then centrifuging at 3-5℃ and 9000-11000 rpm for 18-22 min using a centrifuge; In step A2, adjusting to acidic comprises adjusting the pH of the collected supernatant to 4.0-4.4 using HCl; In step A2, standing and centrifuging comprises standing for 0.3-0.8 h, and then centrifuging at 3-5℃ and 9000-11000 rpm for 12-18 min using a centrifuge; In step A2, dialyzing the precipitate and freeze-drying to obtain Schizochytrium sp. protein comprises placing the precipitate in a dialysis bag, and then dialyzing against distilled water for 46-50 h, with the dialysis solution being replaced every 7.5-8.5 h. After pre-freezing the protein in the dialysis bag, the dialysis bag is placed in a vacuum freeze dryer for vacuum freeze-drying, thereby obtaining Schizochytrium sp. protein.

8. The method for preparing a Schizochytrium sp. polypeptide having a modulating lipid metabolism effect according to claim 4, characterized in that: In step S2, the mass fraction of the neutral protease is 9.60%; In step S2, the temperature of the neutral protease hydrolysis is 40-50℃, and the hydrolysis time is 3.5-4.5 h; In step S2, the boiling and cooling time is 8-12 min.

9. The method of claim 4, wherein the preparation of the Schizochytrium sp. polypeptide having an effect of regulating lipid metabolism is characterized by: The proteolysis solution of the obtained Schizochytrium sp. obtained in step S2 in step S3 is filtered by membranes, including separation by 0.45 μm microporous filter, two ultrafiltration tubes with 10 kDa and 3 kDa are selected for step-by-step separation, and polypeptides with a molecular weight less than 3 kDa are obtained, and polypeptides with the amino acid sequence of SEQ ID NO. 1 are further screened.

10. The preparation method of the Schizochytrium sp. polypeptide with the function of regulating lipid metabolism according to any one of claims 5-9, wherein the Schizochytrium sp. polypeptide prepared by the method is applied in a medicine with the function of regulating lipid metabolism, and the method is characterized in that: Application in anti-fatty liver drugs, blood lipid-lowering drugs or metabolic syndrome treatment drugs.

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