An oligopeptide derived from goose plasma protein for improving liver lipid metabolism disorder and application thereof

Abstract

The application discloses a goose plasma protein-derived oligopeptide capable of improving liver lipid metabolism disorder and application thereof, and relates to the field of biotechnology.The goose plasma protein-derived oligopeptide capable of improving liver lipid metabolism disorder is screened by the application, and the amino acid sequence is GDWIVK.Animal experiment verification shows that the goose plasma protein-derived oligopeptide can effectively reduce the levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in serum and liver affected by high-fat diet, increase the content of high-density lipoprotein cholesterol, reduce liver lipid deposition and improve liver pathological damage.Meanwhile, the goose plasma protein-derived oligopeptide can regulate the structure and diversity of intestinal flora, increase the abundance of beneficial bacteria and the content of short-chain fatty acids in feces, reduce the inflammatory response of colon, and play a synergistic regulation role through the intestinal-liver axis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a goose plasma protein-derived oligopeptide that can improve hepatic lipid metabolism disorders and its applications. Background Technology

[0002] As the core organ of lipid metabolism in the human body, the liver undertakes the key functions of lipid synthesis, decomposition, transport and excretion. When the intake of high-fat diet continues to exceed the metabolic carrying capacity of the liver, it will cause a large amount of triglycerides (TG) to be deposited in liver cells, forming non-alcoholic fatty liver (NAFLD), which may further develop into non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and even liver cancer, seriously threatening human health.

[0003] Currently, clinical and daily interventions for hepatic lipid metabolism disorders induced by a high-fat diet mainly include lifestyle interventions (such as dietary control and weight loss through exercise) and drug therapy. However, lifestyle interventions require long-term adherence, leading to poor patient compliance and difficulty in achieving ideal intervention effects. Traditional chemical drugs (such as fibrates and statins) can regulate blood lipid levels to some extent, but long-term use can easily cause side effects such as gastrointestinal discomfort, liver and kidney damage, and rhabdomyolysis, limiting their clinical application due to safety and tolerability concerns. Therefore, screening safe and effective active ingredients from natural plant and animal resources to improve hepatic lipid metabolism disorders has become a research hotspot and cutting-edge direction in the fields of food science, nutrition, and medicine.

[0004] Protein-derived oligopeptides are small molecule compounds composed of 2-10 amino acid residues linked by peptide bonds. Compared to large protein molecules, they have significant advantages such as good water solubility, rapid digestion and absorption, high bioavailability, and diverse biological activities. Numerous studies in recent years have confirmed that oligopeptides derived from whey protein, soy protein, and fish protein have a clear function in regulating lipid metabolism. For example, they can reduce hepatic lipid deposition and alleviate lipid metabolism disorders by inhibiting the activity of fat synthesis-related enzymes (such as acetyl-CoA carboxylase ACC and fatty acid synthase FAS), promoting the expression of fat breakdown-related genes (such as peroxisome proliferator-activated receptor αPPARα and carnitine palmitoyltransferase 1CPT1), and improving insulin sensitivity. These research findings provide a solid theoretical basis for the development of functional foods or dietary supplements based on protein-derived oligopeptides.

[0005] The slaughtering and processing of geese generates a large number of byproducts, among which goose blood plasma accounts for approximately 3%-5% of the goose's body weight and is a valuable resource rich in high-quality protein. However, the utilization rate of goose blood plasma for high-value purposes is currently extremely low, with most of it being directly discarded or used as cheap feed. This not only results in a serious waste of protein resources but may also cause environmental pollution problems. Given the excellent properties of protein-derived oligopeptides and the resource potential of goose blood plasma protein, preparing oligopeptides with specific biological activities from goose blood plasma protein through enzymatic hydrolysis, separation, and purification techniques, and realizing its transformation from "waste" to "high-value active ingredients," has significant economic and ecological value.

[0006] To date, no research has been reported on the improvement of hepatic lipid metabolism disorders induced by a high-fat diet using oligopeptides derived from goose plasma protein. The active ingredients, mechanisms of action, and application value of these oligopeptides remain unexplored. Therefore, addressing the shortcomings of existing intervention methods, the application prospects of protein-derived oligopeptides, and the development potential of goose plasma protein resources, this invention aims to screen and identify active oligopeptides from goose plasma protein that can specifically improve hepatic lipid metabolism disorders, clarify their mechanisms of action, and provide new ideas and scientific basis for developing novel functional food ingredients, dietary supplements, or adjuvant therapeutic drugs. Simultaneously, it promotes the high-value utilization of by-products from the goose industry, contributing to the green and sustainable development of the livestock farming industry chain. Summary of the Invention

[0007] The purpose of this invention is to provide a goose plasma protein-derived oligopeptide that can improve hepatic lipid metabolism disorders and its application, thereby solving the problems existing in the prior art. This goose plasma protein-derived oligopeptide possesses the function of improving hepatic lipid metabolism disorders, providing a novel natural active ingredient for the intervention of hepatic lipid metabolism disorders.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides an oligopeptide derived from goose plasma protein that can improve hepatic lipid metabolism disorders, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The present invention also provides the use of the above-mentioned goose plasma protein-derived oligopeptide in the preparation of a medicament for treating liver lipid metabolism disorders.

[0011] Furthermore, the liver lipid metabolism disorder is at least one of hyperlipidemia and fatty liver.

[0012] The present invention also provides a medicament for treating liver lipid metabolism disorders, wherein the active ingredient includes the above-mentioned goose plasma protein-derived oligopeptide.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

[0015] Furthermore, the dosage form of the drug is powder, tablet, granule, capsule, pill, or oral liquid.

[0016] The present invention also provides the application of the above-mentioned goose plasma protein-derived oligopeptides in the preparation of health products that help maintain healthy blood lipid levels.

[0017] The present invention also provides a health product that helps maintain healthy blood lipid levels, the active ingredient of which includes the above-mentioned goose plasma protein-derived oligopeptide.

[0018] The present invention also provides a method for preparing the above-mentioned goose plasma protein-derived oligopeptide, comprising the step of preparing the goose plasma protein-derived oligopeptide by solid-phase synthesis.

[0019] The present invention discloses the following technical effects:

[0020] This invention screened and obtained a goose plasma protein-derived oligopeptide that can improve hepatic lipid metabolism disorders, with the amino acid sequence GDWIVK (SEQ ID NO.2). This oligopeptide was obtained through multi-step chromatographic separation and purification combined with mass spectrometry identification. Animal experiments verified that it can effectively reduce serum and liver levels of triglycerides, total cholesterol, and low-density lipoprotein cholesterol affected by a high-fat diet, increase high-density lipoprotein cholesterol levels, reduce hepatic lipid deposition, and improve liver pathological damage. Simultaneously, it can regulate the structure and diversity of intestinal flora, increase the abundance of beneficial bacteria and the content of short-chain fatty acids in feces, alleviate colonic inflammation, and exert a synergistic regulatory effect through the gut-liver axis.

[0021] This oligopeptide can be used to prepare drugs for treating diseases such as hyperlipidemia and fatty liver, as well as health products that help maintain healthy blood lipid levels, providing a novel natural active ingredient for intervention in liver lipid metabolism disorders. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1Statistical graphs showing the serum triglyceride (TG) (A), serum total cholesterol (TC) (B), serum high-density lipoprotein cholesterol (HDL-C) (C), and serum low-density lipoprotein cholesterol (LDL-C) (D) levels in mice under different doses of goose plasma protein crude peptide (GBP).

[0024] Figure 2 Statistical graphs showing the results of gel size exclusion chromatography (A) and the serum triglyceride (B), total cholesterol (C), high-density lipoprotein cholesterol (D), and low-density lipoprotein cholesterol (E) levels in mice under the influence of different gel size exclusion chromatography components.

[0025] Figure 3 Statistical graphs showing the cation exchange chromatography separation results (A) and the serum triglyceride (B), total cholesterol (C), high-density lipoprotein cholesterol (D), and low-density lipoprotein cholesterol (E) levels of mice in each group under the influence of different cation exchange chromatography separation components.

[0026] Figure 4 The results of reversed-phase chromatography (A) are presented in a statistical graph, showing the serum triglyceride (B), total cholesterol (C), high-density lipoprotein cholesterol (D), and low-density lipoprotein cholesterol (E) levels of mice in each group under the influence of different reversed-phase chromatography separation components.

[0027] Figure 5 For E-1- MS / MS spectra of the components;

[0028] Figure 6 For E-1- Primary mass spectra of the components;

[0029] Figure 7 This is the second-order mass spectrum of GDFIVK;

[0030] Figure 8 This is the secondary mass spectrum of GDWIVK;

[0031] Figure 9 The mass spectrum of GDWIVKK is shown below.

[0032] Figure 10 Statistical graphs showing serum triglyceride (A), serum total cholesterol (B), serum high-density lipoprotein cholesterol (C), and serum low-density lipoprotein cholesterol (D) levels in mice under different oligopeptide treatments; where I: GDWIVK, II: GDFIVK, III: GDWIVQK.

[0033] Figure 11Statistical graphs showing the changes in body weight (A), weight gain (B), food intake (C), Lee's index (D), and organ indices (liver and white fat) (E) of mice in each group under GDWIVK intervention.

[0034] Figure 12 This is a graph analyzing the effects of GDWIVK on HFD-induced intestinal barrier dysfunction and inflammation. A shows Alsin blue staining of the colonic mucus layer in different groups of mice; B and C show Western blotting (WB) results and expression levels of tight junction proteins in the colon of different groups of mice, respectively; D and G show statistical graphs of LPS, TNF-α, IL-6, and IL-1β concentrations in the colon of different groups of mice, respectively; H and I show WB results and expression levels of colitis-related proteins in different groups of mice, respectively; J shows a statistical graph of the ratio of phosphorylated NF-κB (p-NF-κB) to total NF-κA protein expression levels in different groups; the scale bar in A is 200 μm.

[0035] Figure 13 This is a statistical graph showing the biodiversity index of the gut microbiota in each group of mice; where A is the Ace index and B is the Shannon index.

[0036] Figure 14 PCA analysis diagram of gut microbiota in each group of mice;

[0037] Figure 15 A diagram showing the composition of the gut microbiota at the phylum level in each group of mice;

[0038] Figure 16 A graph showing the composition of the gut microbiota at the genus level in each group of mice;

[0039] Figure 17 A comparative diagram showing the differences in gut microbiota at the phylum (A) and genus (B) levels among different groups of mice;

[0040] Figure 18 This is a statistical chart showing the content of short-chain fatty acids in the feces of mice in each group under the intervention of goose blood oligopeptide (GDWIVK); where A, F are statistical charts showing the content of acetic acid, propionic acid, n-butyric acid, iso-butyric acid, n-valeric acid and iso-valeric acid, respectively.

[0041] Figure 19This is an analysis of GDWIVK-regulated HFD-induced lipid accumulation in mice; where A is HE and Oil Red O staining images of liver tissue from different groups of mice; B is a statistical graph of TG levels in the liver of different groups of mice; C is a statistical graph of TC levels in the liver of different groups of mice; the scale bar in A is 200 μm. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] Example 1

[0048] Preparation method of goose plasma protein-derived oligopeptides:

[0049] (1) Fresh goose blood was allowed to stand at 4°C to coagulate, and then centrifuged to obtain goose blood plasma. The goose blood plasma was packaged into a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed with flowing deionized water at 4°C for 24 h. The goose blood plasma protein was then freeze-dried to obtain goose blood plasma protein freeze-dried powder.

[0050] (2) The goose blood plasma protein freeze-dried powder was reconstituted in water to form a protein solution. Trypsin was added at a mass ratio of 1:50 to substrate. The solution was enzymatically hydrolyzed at 37°C and pH 8.0 for 3.6 h to obtain goose blood plasma protein hydrolysate.

[0051] (3) Add 2% activated clay to the goose blood plasma protein hydrolysate, stir in an ice bath for 20 min and centrifuge (3000 rpm) to collect the supernatant; pass the decolorized supernatant through a hollow fiber column with a molecular weight cutoff of 3000 Da, collect the filtrate to obtain components with a molecular weight cutoff of less than 3000 Da, freeze dry to obtain crude goose blood plasma protein peptide powder (GBP).

[0052] (4) The crude peptide powder was reconstituted with phosphate buffer to a crude peptide solution with a final concentration of 100 mg / mL. The crude peptide solution was purified by gel size exclusion chromatography to obtain 6 oligopeptide components (AF) with different molecular weights. The components were collected and their lipid-regulating activity was verified.

[0053] In the gel size exclusion chromatography separation, the injection volume was 2 mL each time, the flow rate was 1 mL / min, and the mobile phase was ultrapure water.

[0054] (5) The oligopeptide component E with the highest lipid regulation activity obtained in step (4) was further purified by cation exchange chromatography to obtain two oligopeptide components (E-1 and E-2) with different net negative charge numbers. They were collected and their lipid regulation activity was verified.

[0055] In the cation exchange chromatography separation, the injection volume was 5 mL per injection, the flow rate was 2 mL / min, the mobile phase A was 20 mM Tris-HCl buffer (pH 8.5) without NaCl, and the mobile phase B was 20 mM Tris-HCl buffer (pH 8.5) + 1 M NaCl. The gradient elution program was: 0-12 min, 100% A; 12-42 min, 100%-70% A, 0-30% B; 42-47 min, 100% B.

[0056] (6) The oligopeptide component E-1 with the highest lipid-regulating activity obtained in step (5) was further purified by reversed-phase chromatography to obtain five oligopeptide components with different hydrophobicities (E-1- E-1- E-1- E-1- E-1- ), and their lipid-regulating activity was verified separately.

[0057] In the reversed-phase chromatography separation, the injection volume was 2 mL per injection, the flow rate was 0.8 mL / min, the mobile phase A was 0.1% formic acid solution, the mobile phase B was 100% acetonitrile solution, and the gradient elution program was: 0-10 min, 100% A; 10-22 min, 30%-80% B; 22-23 min, 100% A.

[0058] (7) Take the oligopeptide component E-1- with the highest lipid-regulating activity obtained in step (6). The amino acid sequences were identified by LC-MS / MS, yielding three oligopeptides: GDFIVK (SEQ ID NO.1), GDWIVK (SEQ ID NO.2), and GDWIVKK (SEQ ID NO.3). Figures 7-9 (As shown).

[0059] The amino acid sequence was identified by LC-MS / MS using the following methods: Column type: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 mm ID × 100 mm); injection volume: 5 μL; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution; gradient elution program: 0–2 min, 95%–90% A, 5%–10% B; 2–10 min, 90%–60% A, 10%–40% B; 10–12 min, 60%–10% A, 40%–90% B; 12–15 min, 10%–95% A, 90%–5% B. Flow rate: 1 mL / min.

[0060] The target oligopeptide was artificially synthesized using solid-phase synthesis, and its lipid-regulating activity was subsequently verified.

[0061] Example 2

[0062] 1. Experimental Materials

[0063] Laboratory mice: Eight-week-old male C57BL / 6 mice [SPF], weighing 20±2 g, were purchased from Kevin's Laboratory Animal Co., Ltd., Changzhou City, Jiangsu Province. The light / dark cycle in the housing was repeated every 12 hours. The air conditioning temperature was controlled at 18~26℃, and the relative humidity at 40~70%.

[0064] The formulations of the basal feed and the high-fat feed are shown in Table 1.

[0065] Table 1. Formulation of basal feed and high-fat feed

[0066]

[0067] 2. Experimental Methods

[0068] 2.1 Experimental Grouping

[0069] Mice were first given an acclimatization period of one week, and then randomly divided into groups of eight. Experimental treatments were performed according to Table 1, and the experiment lasted for 10 weeks. Blood, liver tissue, and fecal samples were collected from each group after the experiment.

[0070] Table 1 Experimental Groups

[0071]

[0072] 2.2 Serum and Liver Biochemical Indicators Analysis

[0073] Collected blood was placed in 2 mL centrifuge tubes and centrifuged at 4°C and 1500 g for 10 min. The supernatant was collected as serum. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), total cholesterol (TC), triglycerides (TG), free fatty acids (NEFA), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) in serum were measured according to the instructions of the commercial kits. All commercial kits used were purchased from Nanjing Jiancheng Biotechnology Institute. 1 g of liver tissue was placed in a 10 mL centrifuge tube, 5 mL of pre-chilled physiological saline was added, and the mixture was homogenized in an ice-water bath to form a liver homogenate. The homogenate was then centrifuged at 4°C and 3000 g for 30 min, and the supernatant was collected. The levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver were measured according to the instructions of the commercial kits. The protein concentration in the supernatant was determined using a BCA kit (purchased from Nanjing Jiancheng Biotechnology Institute).

[0074] 2.3 Pathological tissue section analysis

[0075] Fresh tissue was fixed with fixative for at least 24 hours. The tissue was removed from the fixative and trimmed smooth in a fume hood using a scalpel. The trimmed tissue and corresponding labels were placed in a dehydration box. The dehydration box was placed in a basket and dehydrated sequentially using graded alcohols in a dehydrator. The tissue was then placed in an embedding machine for embedding. Melted wax was first placed in the embedding frame. Before the wax solidified, the tissue was removed from the dehydration box, placed in the embedding frame according to the required embedding surface, and labeled accordingly. The tissue was cooled on a -20°C freezing stage. After the wax solidified, the wax block was removed from the embedding frame and trimmed. The trimmed wax block was sectioned using a paraffin microtome to a thickness of 4 μm. The sections were floated on a 40°C warm water spreader to flatten the tissue. The tissue was then retrieved onto a glass slide and baked in a 60°C oven. After the wax was melted and dried by baking, the slides were removed and stored at room temperature for later use. Finally, the tissue was stained with hematoxylin and eosin (H&E) and alexandrite blue, respectively. Place the stained tissue flat on a glass slide, wipe away excess xylene around the tissue, cover with a coverslip, add an appropriate amount of neutral adhesive to secure the slide, and tilt the coverslip (to avoid bubbles). Finally, allow it to air dry. Observe and photograph the dried sections under an optical microscope.

[0076] 2.4 Immunofluorescence analysis

[0077] Wash paraffin sections according to the following procedure: 15 min - xylene I, 15 min - xylene II, 15 min - anhydrous ethanol I, 15 min - anhydrous ethanol II, 5 min - 85% ethanol, 5 min - 75% ethanol, 5 min - distilled water. Then place the tissue sections in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0) and microwave for antigen retrieval. Microwave on medium heat for 8 min, turn off for 8 min, then microwave on medium-low for 7 min. During this process, prevent excessive evaporation of the buffer and do not dry the sections. After natural cooling, place the slides in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 min each time. After slightly drying the sections, draw circles around the tissue with a histochemical pen (to prevent antibody migration), add autofluorescence quencher to the circle for 5 min, rinse with running water for 10 min. Add BSA to the circle and incubate for 30 min. Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a specific ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add the secondary antibody corresponding to the species of the primary antibody to the circle to cover the tissue, and incubate at room temperature in the dark for 50 min. Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add DAPI staining solution to the circle, and incubate at room temperature in the dark for 10 min. Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, mount it with anti-fluorescence quenching mounting medium. Observe the slide under a fluorescence microscope (FITC excitation wavelength 465-495 nm, emission wavelength 515-555 nm, emitting green light) and acquire images.

[0078] 2.5 Analysis of Liver Inflammatory Factors

[0079] One g of liver tissue was placed in a 10 mL centrifuge tube, and 5 mL of pre-chilled physiological saline was added. The mixture was homogenized in an ice-water bath to form a liver homogenate, and then centrifuged at 3000g for 30 min at 4℃. The supernatant was collected for later use. The levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the liver were measured according to the instructions of a commercial enzyme-linked immunosorbent assay (ELISA) kit. The level of lipopolysaccharide (LPS) in serum was also measured using the same method. All commercial ELISA kits used were purchased from Wuhan Xinqidi Biotechnology Co., Ltd. In addition, the protein concentration in the supernatant was determined using a BCA kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0080] 2.6 Gut microbiota analysis

[0081] According to Omega EZNA TM Metagenomic analysis of gut bacteria was performed on colon contents using the instructions of a fecal DNA extraction kit (Omega Bio-tek, Norcross, GA, US). The concentration of DNA in the extract was quantitatively analyzed using a NanoDrop 1000 nucleic acid analyzer (Thermo Scientific, Wilmington, USA), and the quality of the DNA was qualitatively analyzed by 1% agarose gel electrophoresis. Samples meeting sequencing requirements (DNA concentration greater than 50 ng / μL and clear bands) were stored at -80°C for later use. The structure of the gut microbiota was analyzed using thermal cycling PCR (GeneAmp 9700, ABI, USA) based on the V3-V4 variable region of the bacterial 16S rRNA gene.

[0082] 2.7 Analysis of short-chain fatty acid content in feces

[0083] Weigh 600 mg of fecal sample and reconstitute it with 1.2 mL of phosphate buffer (pH 7.3), then vortex at 1000 rpm for 5 minutes. Centrifuge the resulting mixture at 10000 rpm for 5 minutes at 4°C and collect the supernatant. Take 200 μL of the supernatant, add 0.1 mL of 50% (v / v) sulfuric acid, vortex for 1 minute, and let stand for 2 minutes. Add 0.4 mL of diethyl ether to the mixture to extract short-chain fatty acids, and filter the organic phase through a 0.22 μm filter membrane. Analyze the short-chain fatty acid composition of the filtrate using an Agilent 7980A gas chromatograph (Agilent Technologies, Santa Clara, California, USA) equipped with a flame ionization detector and an HP-INNOWAX capillary column (30 m × 0.32 mm, 0.5 μm), using nitrogen as the carrier gas and hydrogen as the auxiliary gas. The flame ionization detector and injection port temperature were both set to 220℃; the column temperature was initially set to 60℃ and held for 3 minutes, then increased to 190℃ at a rate of 3.5℃ / min, and then increased to 230℃ at a rate of 25℃ / min; the split ratio was set to 5:1, and the injection volume was 6.0 μL. Short-chain fatty acids were quantitatively analyzed using the external standard method, and the results are expressed as micromoles per gram of feces (μmol / g).

[0084] 3. Experimental Results

[0085] 3.1 Effects of different doses of goose plasma protein crude peptide (GBP) on blood lipids in mice on a high-fat diet

[0086] Triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) are core serum biomarkers of systemic lipid metabolism disorders. They not only reflect the progression of systemic dyslipidemia but are also directly related to the severity of hepatic steatosis in mice with high-fat diet (HFD) induced non-alcoholic fatty liver disease (NAFLD). Therefore, this invention detected these four serum lipid biomarkers to verify the successful establishment of a high-fat diet-induced mouse NAFLD model and to evaluate the preventive effect and dose-dependent improvement effect of GBP (protein-bound lipids) with a molecular weight less than 3 kDa on this model.

[0087] like Figure 1 As shown, the serum concentrations of TG, TC, HDL-C, and LDL-C in mice in the normal diet (ND) group were 0.89±0.08, 4.02±0.35, 5.24±0.42, and 0.41±0.02 mmol / L, respectively. After 10 weeks of high-fat diet intervention, compared with the normal diet group, the serum TG (1.81±0.15 mmol / L), TC (8.34±0.89 mmol / L), and LDL-C (1.46±0.12 mmol / L) levels in the high-fat diet group were significantly increased, while the serum HDL-C level was significantly decreased (2.61±0.34 mmol / L), and the differences between the groups were statistically significant (P<0.05). These characteristic changes in lipid metabolism disorder confirm the successful establishment of the high-fat diet-induced mouse NAFLD model in this invention.

[0088] Subsequently, this invention investigated the dose-dependent effect of GBP on high-fat diet-induced NAFLD in mice. The results showed that a dose of 200 mg / kg GBP had no significant regulatory effect on the abnormal lipid profile in mice, while doses of 300–500 mg / kg GBP could restore abnormal lipid markers to normal levels in a dose-dependent manner. Figure 1 It is worth noting that increasing the dosage from 400 mg / kg to 500 mg / kg did not significantly improve the lipid-regulating effect of GBP. Therefore, this invention selected 400 mg / kg as the optimal dosage for subsequent experiments.

[0089] 3.2 Effects of Gel Size Exclusion Chromatography Separates on Blood Lipids in Mice on a High-Fat Diet

[0090] GBP is a complex mixture of oligopeptides of varying molecular weights. To screen for active oligopeptide components in GBP that can improve lipid metabolism disorders in non-alcoholic fatty liver disease (NAFLD), this invention employs a dextran-filled gel. TMA 10 / 300 GL size exclusion column was used to separate the oligopeptide mixture into different components. Based on molecular weight differences, GBP was successfully separated into six components, which were named GBP-A, GBP-B, GBP-C, GBP-D, GBP-E, and GBP-F, respectively. Figure 2 (A). Of these, GBP-A is a large-molecule oligopeptide with a relatively high molecular weight, and therefore eluted first; GBP-F is a small-molecule oligopeptide, and its retention time in the column is significantly prolonged. All collected fractions were freeze-dried for subsequent activity assays.

[0091] This invention subsequently examined the serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in NAFLD mice induced by a high-fat diet (HFD) after treatment with different GBP components. The results showed that among all treatment groups, the GBP-E component treatment had the most significant improvement in lipid profile: serum TG (1.01±0.11 mmol / L), TC (4.52±0.41 mmol / L), and LDL-C (0.64±0.05 mmol / L) levels were significantly reduced, while HDL-C levels were significantly increased (4.26±0.34 mmol / L). All lipid indicators were closest to those of the normal diet (ND) group, and the differences between groups were statistically significant (P<0.05). Figure 2 (BE). The above results indicate that the GBP-E component has the strongest regulatory activity on lipid metabolism in NAFLD mice induced by a high-fat diet. To further identify the physicochemical characteristics of this active component, the GBP-E component was dried using a vacuum freeze dryer and stored for subsequent analytical experiments.

[0092] 3.3 Effects of cation exchange chromatography-separated components on blood lipids in mice on a high-fat diet

[0093] GBP-E is a mixture of oligopeptides with similar molecular weights, but its cation exchange capacity varies. Therefore, based on this difference, the present invention employs ion exchange chromatography (IEC) to further separate the GBP-E components to identify the specific active components. Figure 3As shown in Figure A, after ion-exchange chromatography separation, the GBP-E fraction was divided into two subfractions, E-1 and E-2. E-1 had a weaker cation exchange capacity, while E-2 had a stronger one. To verify the lipid-regulating effects of these two subfractions, this invention measured the serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mice with high-fat diet (HFD) induced non-alcoholic fatty liver disease (NAFLD) after treatment with E-1 and E-2, respectively. The results showed that, compared with the E-2 treatment group, the E-1 treatment group exhibited a more significant improvement in lipid metabolism profiles: serum TG (1.23±0.11 mmol / L), TC (4.43±0.42 mmol / L), and LDL-C (0.51±0.04 mmol / L) levels were significantly reduced, while HDL-C levels were significantly increased (4.28±0.41 mmol / L). All lipid indicators were closer to the baseline levels of the normal diet (ND) group, and the differences between groups were statistically significant (P<0.05). Figure 3 (BE). The above results indicate that in a high-fat diet-induced NAFLD mouse model, the E-1 subfraction exhibits superior regulatory activity on lipid metabolism compared to the E-2 subfraction. The E-1 subfraction was subsequently collected, freeze-dried, and stored for later analytical experiments.

[0094] 3.4 Effects of reversed-phase chromatographic separation components on blood lipids in mice fed a high-fat diet

[0095] The E-1 subfraction consists of a mixture of oligopeptides with similar molecular weights and weak cation exchange capacities, but the hydrophobicity of each component differs. Therefore, based on this difference in hydrophobicity, this invention employs reversed-phase liquid chromatography (RP-LC) to further separate the E-1 subfraction and obtain its core active ingredient. Figure 4 As shown in Figure A, after separation by reversed-phase liquid chromatography, the E-1 subfraction was separated into 5 secondary components, which were named E-1- To E-1- To verify the lipid-regulating effects of the secondary components obtained by reversed-phase liquid chromatography, this invention detected the concentrations of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum of mice with high-fat diet (HFD) induced non-alcoholic fatty liver disease (NAFLD) after treatment with each secondary component. Figure 4 As shown in BE, among all treatment groups, E-1- The secondary component treatment showed the best improvement in lipid metabolism profile in mice: the serum TG (1.24±0.14 mmol / L), TC (4.36±0.34 mmol / L), and LDL-C (0.62±0.05 mmol / L) levels decreased most significantly, while the HDL-C level (4.35±0.37 mmol / L) increased most significantly. Furthermore, all lipid indicators were essentially consistent with the baseline levels in the normal diet (ND) group, and the differences between groups were statistically significant (P<0.05). These results indicate that in a high-fat diet-induced NAFLD mouse model, compared to other secondary components, E-1- It exhibits the strongest regulatory activity on lipid metabolism. This invention subsequently collected E-1- The secondary components were then freeze-dried to prepare for subsequent structural identification experiments of the active oligopeptide components.

[0096] 3.5 Identification and Activity Verification of Target Oligopeptides

[0097] like Figures 5-9 As shown, the present invention is in E-1- Three dominant molecular ion peaks corresponding to oligopeptides were identified in the component, namely GDFIVK ( Figure 7 ), GDWIVK ( Figure 8 ) and GDWIVKK ( Figure 9 The monoisotopic masses of these oligopeptides are 677.35 Daltons, 716.36 Daltons, and 845.46 Daltons, respectively. These oligopeptides are all derived from goose plasma albumin and are characterized by low molecular weight, moderate hydrophobicity (containing hydrophobic residues such as tryptophan, isoleucine, and valine), and weak cation exchange capacity. Their physicochemical properties are consistent with the characteristics of the oligopeptides identified in the previous separation scheme.

[0098] To further verify the lipid-regulating activity of the above-mentioned oligopeptides, GDFIVK, GDWIVK and GDWIVKK were chemically synthesized by solid-phase synthesis, and the effects of each oligopeptide were evaluated in a mouse model of non-alcoholic fatty liver induced by a high-fat diet.

[0099] like Figure 10 As shown, compared with the GDFIVK and GDWIVKK groups, the GDWIVK pretreatment group exhibited the strongest regulatory effect on the disordered serum lipid profile. The serum triglyceride (1.12±0.15 mmol / L), total cholesterol (4.52±0.38 mmol / L), and low-density lipoprotein cholesterol (0.52±0.04 mmol / L) levels of mice in this group were significantly reduced, while the high-density lipoprotein cholesterol (4.42±0.45 mmol / L) level was significantly increased (P<0.05).

[0100] The above results fully demonstrate that among all oligopeptides isolated from goose plasma protein hydrolysate, GDWIVK exhibits the strongest lipid-regulating activity in a mouse model of non-alcoholic fatty liver disease induced by a high-fat diet.

[0101] 3.6 Effects of GDWIVK oligopeptide intervention on body weight, Lee's index, and organ indices (liver and white adipose tissue) in mice fed a high-fat diet

[0102] To investigate whether the peptide GDWIVK could improve obesity-related physiological disorders in mice fed a high-fat diet, this invention examined a series of core indicators, including body weight, body weight gain, food intake, Lee's index, and organ coefficients of liver and white adipose tissue (WAT). After 10 weeks of high-fat diet intervention, compared with the normal diet group, the body weight of mice in the high-fat diet group ( Figure 11 (A) Body weight gain ( Figure 11 Both the B index and the Lee index (D in the figure) were significantly increased (p<0.05); at the same time, the organ coefficients of the liver and white adipose tissue of this group of mice were also significantly increased (p<0.05). Figure 11 The result (E) indicates excessive fat accumulation in the body. It is noteworthy that there was no significant difference in food intake among the experimental groups (E). Figure 11 (C) This indicates that the obesity-related physiological changes in high-fat diet mice were not caused by changes in food intake. Supplementation with GDWIVK effectively restored the increased body weight, body weight gain, and Lee's index in high-fat diet mice to normal levels. Figure 11 (A, B, D); Correspondingly, after GDWIVK supplementation intervention, the elevated organ coefficients of liver and white adipose tissue in high-fat diet mice were also significantly reduced ( Figure 11 The results (E) suggest that the fat accumulation in the mice has been alleviated.

[0103] 3.7 Effects of oligopeptide GDWIVK intervention on colonic barrier function and inflammation in high-fat diet mice

[0104] The intestinal barrier is a crucial defense mechanism against the invasion of pathogens and harmful substances into the intestinal lumen. Extensive research has confirmed that a long-term high-fat diet damages the structure and function of the intestinal barrier, a process often accompanied by disordered expression of tight junction proteins. Impaired intestinal barrier function leads to increased intestinal mucosal permeability, promoting the translocation of toxins such as lipopolysaccharide (LPS) to the colonic tissue, exacerbating local inflammation, and creating a vicious cycle. This invention employs multiple complementary detection indicators to evaluate high-fat diet-induced intestinal dysfunction in mice and the protective effect of supplemental GDWIVK. The detection methods include: observing pathological changes in colonic tissue using Alsin blue staining; detecting the expression levels of core tight junction proteins Claudin-1, Occludin, and ZO-1, which maintain the integrity of the intestinal epithelial barrier structure; and quantitatively detecting the levels of LPS and pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in colonic tissue.

[0105] Compared with the normal diet group, mice in the high-fat diet group showed significant impairment of intestinal barrier function, specifically manifested as: Alsin blue staining results showed a significant reduction in the number of colonic goblet cells and a significant decrease in mucus secretion. Figure 12 In the middle A group, the expression levels of Claudin-1, Occludin, and ZO-1 were significantly downregulated. Figure 12 In the middle C, the levels of LPS, TNF-α, IL-6, and IL-1β in colon tissue were all significantly increased. Figure 12 The above changes all indicate a significantly aggravated local inflammatory response in the mouse colon. Notably, GDWIVK supplementation effectively alleviated the above-mentioned intestinal dysfunction induced by a high-fat diet: it significantly restored the number of goblet cells and mucus secretion levels in the mouse colon. Figure 12 (A), upregulates the expression levels of Claudin-1, Occludin, and ZO-1 ( Figure 12 (F), while reducing the content of LPS and various pro-inflammatory cytokines in colon tissue ( Figure 12 (BE).

[0106] In summary, this invention demonstrates that GDWIVK can alleviate intestinal barrier dysfunction induced by a high-fat diet by maintaining the homeostasis of goblet cells and mucus in the mouse colon and protecting the structural integrity of tight junction proteins; at the same time, it can reduce local inflammatory responses in the colon induced by a high-fat diet by inhibiting LPS translocation and the production of pro-inflammatory cytokines.

[0107] 3.8 Effects of GDWIVK oligopeptide intervention on gut microbiota in high-fat diet mice

[0108] Mounting evidence suggests that gut microbiota dysbiosis is a key driver in the pathogenesis of high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD), which can disrupt intestinal barrier integrity, promote inflammatory responses, and impair hepatic lipid metabolism by altering microbial metabolites. Previous studies have confirmed that bioactive peptides can exert prebiotic-like effects by remodeling gut microbiota composition. Therefore, this invention hypothesizes that GDWIVK may improve NAFLD by regulating gut microbiota dysbiosis. To verify this hypothesis, this invention used 16S rRNA gene sequencing technology to analyze the fecal microbial community structure of mice in each group.

[0109] This invention uses alpha diversity indicators such as the Ace index and Shannon index to quantify the richness and diversity of gut microbiota. For example... Figure 13 As shown, compared with the normal diet (ND) group, mice fed a high-fat diet exhibited significantly lower Ace and Shannon indices, indicating impaired gut microbial richness and diversity, respectively; however, these indices recovered after GDWIVK treatment. β-diversity analysis employed principal coordinate analysis (PCoA) based on Bray-Curtis distance, a method that can distinguish differences in microbial community structure between different groups. Figure 14 As shown, the normal diet group and the high-fat diet group were clearly separated along the first principal component, indicating that the high-fat diet can induce significant changes in the composition of the gut microbiota. In contrast, the high-fat diet + GDWIVK group and the normal diet group had tightly clustered microbial communities with significant overlap in community characteristics, suggesting that GDWIVK intervention can reverse the gut microbiota structure disorder induced by the high-fat diet.

[0110] To further clarify the taxonomic changes in the gut microbiota, this invention analyzed the relative abundance of dominant bacterial groups at the phylum and genus levels. The results are shown in [Figure number missing]. Figures 15-17 .

[0111] At the level of the door ( Figure 15 In the normal diet group, the gut microbiota of mice was dominated by Firmicutes and Bacteroidota, with small amounts of Desulfobacterota and Verrucomicrobiota. A high-fat diet significantly disrupted the balance of the gut microbiota in mice, showing an increased relative abundance of Firmicutes and a decreased relative abundance of Bacteroidota and Verrucomicrobiota. GDWIVK intervention could improve these abnormal microbial composition by reducing Firmicutes abundance and increasing Bacteroidota and Verrucomicrobiota abundance, bringing the phylum-level microbiota distribution closer to that of the normal diet group.

[0112] At the genus level ( Figure 16Several key bacterial genera showed significant responses to GDWIVK intervention. High-fat diets significantly reduced the abundance of various beneficial bacterial genera in feces, including *Faecalibaculum*, *Lachnospira* (UBA3282), *Oscillospiraceae* (88309), *Akkermansia*, *Unclassified* *Muribaculaceae*, and *Muribaculum*. Simultaneously, pro-inflammatory and lipopolysaccharide (LPS)-related genera were significantly enriched, such as *Desulfovibrio* (R446353), *Prevotella* (CAG95), *Romboutsia* (B), and *Clostridium* (CAG269). GDWIVK intervention reversed the opposite trend in a dose-dependent manner: compared with the high-fat diet group, GDWIVK treatment significantly increased the relative abundance of *Femtobacter*, *Trichophyton* UBA3282, *Oscillatoria* 88309, *Akkermansia*, *Trichophyton* unclassified, and *Muribaculum*, while decreasing the relative abundance of *Desulfovibrio* R_446353, *Prevotella* CAG95, *Rombutz* B, and *Clostridium* CAG269.

[0113] It is noteworthy that the aforementioned genus-level changes in gut microbiota exhibit clear functional correlations, supported by the unique biological functions of key genera: *Femobacterium*, *Trichophyton* UBA3282, *Oscillatoria* 88309, *Trichophyton* var. *unclassified*, and *Muribaculum* are all major producers of short-chain fatty acids (especially butyrate), which can enhance intestinal barrier function and inhibit hepatic lipid synthesis. Simultaneously, *Akkermansia* and *Muribaculum* var. *unclassified* can promote mucus layer integrity and reduce intestinal mucosal permeability. Furthermore, *Trichophyton* var. *unclassified* can regulate immune responses and suppress inflammation. Conversely, *Desulfovibrio* R_446353 can produce LPS, thereby triggering a TLR4 / NF-κB-mediated inflammatory response in the liver; similarly, *Prevotella* CAG95, *Rombutzella* B, and *Clostridium* CAG269 are all associated with intestinal barrier disruption and metabolic endotoxemia.

[0114] 3.9 Effects of Oligopeptide GDWIVK Intervention on Short-Chain Fatty Acid Content in Feces of Mice Dieted on a High-Fat Diet

[0115] Short-chain fatty acids (SCFAs) are core metabolites produced by gut microbiota fermentation of dietary substrates, playing an irreplaceable role in mediating the interactions between diet, gut microbiota, and host health. Acetic acid, propionic acid, and butyric acid are the three most bioactive subtypes. Functionally, propionic acid can alleviate inflammatory responses by downregulating the expression of pro-inflammatory cytokines and chemokine genes, while butyric acid can enhance intestinal barrier function and promote the synthesis of tight junction-related proteins. This invention quantitatively detected the content of major SCFA subtypes (acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid) in mouse fecal samples. The relevant results are shown in […]. Figure 18 Compared with the normal diet group, high-fat diet intervention alone significantly reduced the levels of all short-chain fatty acids detected in mouse feces (p<0.05), suggesting that the gut microbiota fermentation function of mice induced by a high-fat diet was impaired. Notably, supplementation with GDWIVK reversed the aforementioned decrease in short-chain fatty acid levels induced by a high-fat diet.

[0116] This regulatory effect of GDWIVK on short-chain fatty acid production is consistent with previous research findings. For example, studies on animal-derived protein peptides have shown that whey protein hydrolysate can promote the biosynthesis of short-chain fatty acids in the feces of mice with high-fat diet-induced fatty liver by enriching short-chain fatty acid-producing bacteria in the mouse gut microbiota. These findings are consistent with the present invention, both indicating that a high-fat diet inhibits the production of short-chain fatty acids in the gut microbiota, while supplementation with GDWIVK can effectively restore or even enhance the level of short-chain fatty acids in the feces of mice fed a high-fat diet.

[0117] 3.10 Effects of GDWIVK oligopeptide intervention on hepatic lipid metabolism in high-fat diet mice

[0118] To investigate the regulatory effect of GDWIVK on lipid accumulation in the liver of mice induced by a high-fat diet (HFD), this invention detected the levels of triglycerides (TG) and total cholesterol (TC) in mouse liver tissue. Hematoxylin-eosin (HE) staining was used to observe the morphological characteristics of liver tissue, and Oil Red O staining was used for visual analysis of lipid droplet distribution within the liver tissue. Compared with the normal diet (ND) group, the TG levels in the liver tissue of mice in the high-fat diet group were significantly higher. Figure 19 (B), TC ( Figure 19 The levels of C and C were significantly increased (p<0.05), confirming that a high-fat diet induces excessive lipid accumulation in the liver of mice. After GDWIVK intervention in mice on a high-fat diet, the aforementioned elevated liver lipid levels were significantly reduced (p<0.05), and the levels were close to the baseline levels of the normal diet group.

[0119] Subsequent histological supplementary analysis ( Figure 19(A) Further revealed the specific differences in liver morphology among the groups of mice: the normal diet group showed well-organized hepatocyte structure and minimal lipid droplet deposition; while the high-fat diet group showed a large accumulation of lipid droplets upon Oil Red O staining, and HE staining revealed disordered hepatocyte arrangement, all of which are typical pathological features of hepatic lipid accumulation. The high-fat diet + GDWIVK intervention group showed significant improvement in the above-mentioned abnormal liver structure, specifically a reduction in the distribution range of lipid droplets in the liver tissue and a near-normal restoration of hepatocyte morphology.

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A goose plasma protein-derived oligopeptide that can improve hepatic lipid metabolism disorders, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. The use of the goose plasma protein-derived oligopeptide as described in claim 1 in the preparation of a medicament for treating liver lipid metabolism disorders, characterized in that, The liver lipid metabolism disorder mentioned is non-alcoholic fatty liver disease.

3. A drug for treating liver lipid metabolism disorders, characterized in that, The active ingredient includes the goose plasma protein-derived oligopeptide as described in claim 1; The liver lipid metabolism disorder mentioned is non-alcoholic fatty liver disease.

4. The drug according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The drug according to claim 4, characterized in that, The excipients include fillers, binders, disintegrants, emulsifiers, flavoring agents, preservatives, or colorants.

6. The drug according to claim 3, characterized in that, The dosage form of the drug is powder, tablet, granule, capsule, pill, or oral liquid.

7. The use of the goose plasma protein-derived oligopeptide as described in claim 1 in the preparation of health products that help maintain healthy blood lipid levels.

8. A health supplement that helps maintain healthy blood lipid levels, characterized in that, The active ingredient includes the goose plasma protein-derived oligopeptide as described in claim 1.

9. A method for preparing the goose blood plasma protein-derived oligopeptide as described in claim 1, characterized in that, The method includes the step of preparing the goose plasma protein-derived oligopeptide using a solid-phase synthesis method.

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