Dry-cured ham source oligopeptide and application thereof

Abstract

The invention discloses a dry-cured ham source oligopeptide and application thereof. The dry-cured ham source oligopeptide comprises at least one of the following amino acid sequences: ELISDQDARDLY; a YHEHRSDLN, a YHEHRSDL YKATEPVIAF is used as a template; the method comprises the following steps: carrying out INKVEELKKKY; and GEKLKRQKY is used as a reference. The dry-cured ham source oligopeptide is applied to preparation of medicines and health-care foods for treating glycolipid metabolism disorder diseases. The oligopeptide extracted and verified by the invention can be specifically combined with an AdipoR2 receptor, shows stronger glycolipid metabolism regulation activity compared with the existing KRQKYD polypeptide, can obviously improve various glycolipid metabolism disorder diseases, and has a better medical and edible application prospect.

Description

Technical Field

[0001] The present invention relates to a medicinal polypeptide, in particular to a dry-cured ham-derived oligopeptide and application thereof in the preparation of medicines and health-care foods. Background Art

[0002] Glucose and lipid metabolism disorder is a chronic disease caused by multiple factors, and its initial characteristic is lipid accumulation in hepatocytes. In addition, glucose and lipid metabolism disorder is also a key factor in inducing metabolic syndromes such as diabetes, obesity, fatty liver, and atherosclerosis. The pathogenesis of glucose and lipid metabolism disorder is closely related to insulin resistance, liver fat synthesis, liver lipid peroxidation, oxidative stress, obesity-related cytokine expression, and intestinal microbial disorders. Fat accumulation and degeneration are the main characteristics of glucose and lipid metabolism disorder, so maintaining glucose and lipid metabolism homeostasis may be one of the effective strategies for preventing and treating glucose and lipid metabolism disorder. There is currently a lack of therapeutic drugs for glucose and lipid metabolism disorder, so the development of effective prevention strategies is urgent. Since glucose and lipid metabolism disorder is closely related to diet, dietary intervention is considered to be an effective strategy for preventing glucose and lipid metabolism disorder.

[0003] Food-derived bioactive peptides refer to functional peptides that are taken up by organisms from outside the body. They are directly or indirectly derived from food proteins and are generally found in dietary proteins with specific amino acid sequences. Food-derived peptides are very safe and easy to industrially produce, and therefore have received special attention in the fields of nutrition and food science. Currently, the development of peptides containing functional ingredients is an innovative strategy for functional foods, health products, and pharmaceuticals. The large amount of food-derived proteins and waste generated during the processing are usually used as fertilizers or directly discharged, which not only wastes resources but also causes environmental pollution.

[0004] During the maturation cycle of fermented ham, a large amount of protein is hydrolyzed into amino acids and small peptides, which are enriched in the final product. These small peptides have the ability to scavenge free radicals in the human body and prevent various chronic diseases. Currently, there are no reports on the extraction of dry-cured ham-derived oligopeptides from fermented ham that target the AdipoR2 receptor to improve glucose and lipid metabolism disorders. This approach addresses the problems of low functional factor content, difficult extraction, and low efficiency in dietary interventions for glucose and lipid metabolism disorders. Summary of the Invention

[0005] Objectives of the Invention: This invention aims to provide a dry-cured ham-derived oligopeptide to address the problem of how to effectively improve glucose and lipid metabolism disorders by targeting the AdipoR2 receptor. Another objective of the invention is to propose the use of a dry-cured ham-derived oligopeptide in the preparation of a drug and health food for treating glucose and lipid metabolism disorders, thereby addressing the problem of how to prepare such a drug and health food.

[0006] Technical solution: The dry-cured ham-derived oligopeptide of the present invention comprises at least one of the following amino acid sequences: ELIDQDARDLY; YHEHRSDLN; YKATEPVIAF; INKVEELKKKY; GEKLKRQKY.

[0007] Preferably, the dry-cured ham-derived oligopeptide contains the following amino acid sequence:

[0008] ELIDQDARDLY and / or YHEHRSDLN.

[0009] The second aspect of the present invention discloses the use of the dry-cured ham-derived oligopeptide in the preparation of a drug for treating glucose and lipid metabolism disorders.

[0010] Preferably, the glucose and lipid metabolism disorder disease includes at least one of insulin resistance, hyperglycemia, diabetes, hyperlipidemia, and fatty liver.

[0011] Preferably, the content of the dry-cured ham-derived oligopeptide in the drug is 1-200 mg / mL. Pharmaceutically acceptable excipients can be selectively added to the drug according to different formulation requirements.

[0012] The third aspect of the present invention discloses the use of the dry-cured ham-derived oligopeptides in the preparation of health foods.

[0013] The method for preparing health food using the dry-cured ham-derived oligopeptides comprises the following steps:

[0014] (1) dissolving dry-cured ham-derived oligopeptides in water to obtain a polypeptide aqueous solution;

[0015] (2) The polypeptide aqueous solution and the oil phase are mixed and emulsified to obtain an emulsion.

[0016] Preferably, in step (1), the final concentration of the dry-cured ham-derived oligopeptide in the aqueous polypeptide solution is 100-200 mg / mL;

[0017] In step (2), the volume ratio of the polypeptide aqueous solution to the oil phase is 5-7:3-5.

[0018] Preferably, in step (2), the oil phase comprises ham fat and castor oil glycerol; the ham fat is prepared as follows:

[0019] The fat in the ham is mixed with petroleum ether, subjected to heating and ultrasonic extraction, and the supernatant is taken out. The petroleum ether is removed by rotary evaporation to obtain the ham fat.

[0020] In some embodiments, the solid-liquid ratio of fat in ham to petroleum ether is 0.5-1.5 g: 3-7 mL, and the conditions for heated ultrasonic extraction are ultrasonic extraction at 45-55° C. for 30-240 min.

[0021] In some embodiments, the volume ratio of ham fat to ricinolein is 90-110:8.

[0022] The above method for preparing health food further comprises the following steps:

[0023] Using the emulsion, nutritional excipients and formulation excipients as raw materials to prepare capsules or soft candies;

[0024] The nutritional auxiliary materials include at least one of vitamins and fruit juices; the preparation auxiliary materials include at least one of gelatin, water, glycerin, maltodextrin, xylitol, carrageenan, and xanthan gum.

[0025] In some embodiments, the preparation method of the soft candy is as follows:

[0026] By weight, 50-70 parts of the above-mentioned emulsion, 0.5-1.5 parts of vitamin C, 1-5 parts of fruit juice, 0.2-0.8 parts of maltodextrin, 0.1-1 parts of xylitol, 0.5-1.5 parts of carrageenan, and 0.5-1.5 parts of xanthan gum are mixed and heated at 60-80°C for 20-30 minutes. The mixture is then poured into a mold, cooled and formed, and then demolded and sterilized to obtain the soft candy. The fruit juice is concentrated juice of at least one of apple, banana, strawberry, orange, and watermelon.

[0027] In some embodiments, the capsule is prepared as follows:

[0028] By weight, 60-80 parts of the above emulsion and 1-2 parts of vitamins are mixed to obtain capsule contents; a soft capsule shell is prepared using gelatin: water: glycerol = 80-100: 80-100: 20-40 as raw materials, and the capsule contents are encapsulated in the soft capsule shell to obtain a soft capsule.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] The oligopeptide extracted and verified by the present invention can specifically bind to the AdipoR2 receptor, exhibits stronger glycolipid metabolism regulating activity than the existing KRQKYD polypeptide, can significantly improve a variety of glycolipid metabolism disorders, and has good application prospects for both medicinal and edible purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the HPLC chromatogram of the target oligopeptide powder;

[0032] Figure 2 is the secondary mass spectrum of the target oligopeptide AE;

[0033] Figure 3 The effects of five oligopeptides on hepatocyte lipid deposition in a non-alcoholic fatty liver disease (NAFLD) cell model;

[0034] Figure 4 The effects of peptide D and peptide E on the body weight and organ indexes of NAFLD mice; (A) shows the body weight change; (B) shows the body weight change; (C) shows the liver index; and (D) shows the white fat index.

[0035] Figure 5 Effects of peptide D and peptide E on lipid accumulation in NAFDL mice; (A) shows a representative image of H&E staining of adipose tissue, scale bar, 50 μm; (B) shows serum total cholesterol level; (C) shows serum triglyceride level; (D) shows serum low-density lipoprotein cholesterol level; (E) shows serum high-density lipoprotein cholesterol level; and (F) shows free fatty acid level.

[0036] Figure 6 Figure 3: Effects of peptide D and peptide E on lipid deposition in the liver of NAFLD mice; (A) shows representative images of liver H&E and Oil Red O staining, scalebar, 50 μm; (B) shows liver TC content; (C) shows liver TG content; (D) shows liver HDL-C content; (E) shows liver LDL-C content; (F) shows serum ALT activity; (G) shows serum AST activity.

[0037] Figure 7 Figure 3 shows the effects of peptide D and peptide E on oxidative stress in liver tissue of NAFLD mice; (A) shows liver SOD activity; (B) shows liver GSH-PX activity; (C) shows liver CAT activity; (D) shows liver MDA level.

[0038] Figure 8 Figure 3: Effects of peptide D and peptide E on insulin resistance in NAFLD mice. (A) shows the fasting blood glucose levels of each group after 14 weeks of oligopeptide intervention; (B) shows the serum insulin level; (C) shows the blood glucose level in an oral glucose tolerance test (OGTT); (D) shows the area under the curve; (E) shows the QUICK index; and (F) shows the HOMA-IR index.

[0039] Figure 9 The regulatory effects of peptide D and peptide E on the blood glucose metabolism signaling pathway in NAFLD mice.

[0040] Figure 10 Figure 3: Effects of peptide D and peptide E intervention on the AMPK / PPAR-α signaling pathway in the liver of NAFLD mice; (A) shows the expression levels of each protein under Western blot detection; (B) shows the quantitative analysis of p-AMPK / AMPK; (C) shows the quantitative analysis of PPAR-α; (D) shows the quantitative analysis of CPT-1; (D) shows the quantitative analysis of SREBP-1. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0042] Example 1: A dry-cured ham-derived oligopeptide, the sequence of which is as follows:

[0043] Peptide A: YKATEPVIAF;

[0044] B-peptide: INKVEELKKKY;

[0045] C-peptide: GEKLKRQKY;

[0046] D-peptide: ELIDQDARDLY;

[0047] Peptide E: YHEHRSDLN.

[0048] The above five peptides are all extracted from dry-cured ham. The extraction method is as follows:

[0049] (1) Degreasing: Select mature dry-cured ham, remove visible fat and connective tissue, and cut the lean meat into 0.5cm pieces. 3 Small pieces were mixed with meat sample and n-hexane at a solid-liquid ratio of 1:3 (g / mL), extracted twice with shaking at 30°C for 30 min each time, centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the residue was vacuum dried to remove the residual solvent;

[0050] (2) Desalting: The defatted meat residue obtained in step 1 was dialyzed with flowing deionized water at 4°C for 24 h to remove small molecule salts, and then freeze-dried and ground for later use;

[0051] (3) Oligopeptide extraction: The lyophilized powder obtained in step (2) was mixed with 0.1 mol / L hydrochloric acid solution at a ratio of 1:4 (g / mL), and homogenized in an ice-water bath (speed: 12000 rpm, average number of times: 4 times, each time 1 min). The homogenized solution was then ultrasonically treated for 1 h (ultrasonic power: 300 W, temperature 30°C, pulse time: 30 s / 30 s), filtered through 4 layers of gauze, and the filtrate was centrifuged at 8000 rpm for 20 min. The supernatant was taken for later use;

[0052] (4) Decolorization: 0.1%-0.3% activated clay was added to the supernatant obtained in step (3), and the mixture was stirred in an ice-water bath for 15 min (stirring rate: 80 rpm), and then centrifuged at 4000 rpm for 5 min, and the supernatant was collected for later use;

[0053] (5) Fractional separation: The supernatant obtained in step (4) was subjected to gradient separation through hollow fiber columns with different molecular weight cut-offs (3000 Da, 500 Da), and the fractions between 500 and 3000 Da were collected and lyophilized to obtain crude peptide powder;

[0054] (6) Structure-guided purification:

[0055] Assembly and polymerization of molecular imprinting polymer (MIP): 4-vinylpyridine (4-VP), 3-aminophenylboronic acid (APBA), methacrylic acid (MAA), acrylamide (AAm), and ethylene glycol dimethacrylate (EGDMA) were mixed in a solvent system (acetonitrile: water = 7:3 v / v) at a ratio of n(APBA):n(4-VP):n(MAA):n(AAm):n(EGDMA) = 2:3:1.5:1:20. The assembly temperature was 4°C, the assembly time was 12 h, and the molar ratio of the template peptide (sequence KRQKY) to the MIP monomer was 1:7.5. Subsequently, a free radical polymerization reaction was initiated by the addition of 2,2'-azobisisobutyronitrile (0.5% w / v) at 60°C under nitrogen protection. The polymerization reaction was maintained at 60°C for 24 h and then annealed at 4°C for 12 h to form a rigid backbone and fine recognition cavities. A porogen (toluene:dodecanol = 3:1 v / v) was then added to form a porous structure. The template peptide was eluted from the MIP pores using a Soxhlet extractor. A 0.1% TFA / acetonitrile solution was added to disrupt hydrogen bonds and π-π stacking interactions between the template peptide and the MIP. The strong solubility of acetonitrile facilitated the dissolution of the template peptide. Ultrasonic elution was then performed (300W ultrasound at 40°C) to minimize MIP structural damage. The temperature was kept within the polymer's thermal resistance range. A pH gradient elution (pH 2.0→10.0) was then performed to modulate the dissociation of reversible covalent bonds, such as boronate bonds, through pH variation, resulting in complete elution of the template peptide. Finally, the molecularly imprinted polymer with triple recognition mechanism based on tyrosine residues was obtained by freeze-drying.

[0056] The crude peptide powder obtained in step (5) was prepared into a 100 mg / mL crude peptide solution with pH 7.4 PBS buffer (containing 0.1 M NaCl) and purified by passing through a medium-pressure glass chromatography column (inner diameter 10 mm, length 100 mm, wet-packed with 1 g molecularly imprinted polymer, column efficiency: theoretical plate number ≥8000 / m) at a flow rate of 0.5 mL / min. The filtrate was collected and then eluted with 10CV deionized water to remove salt, and then eluted with 5CV 10% acetonitrile to remove nonspecific adsorbed impurities. The filtrates were combined and discarded, and then eluted with pH 9.5 borax buffer (containing 10% acetonitrile) at 1.0 mL / min. The eluate was collected and dialyzed with flowing deionized water for 24 h at 4°C through a dialysis bag (molecular weight cutoff: 500 Da) to remove borax, and then lyophilized to obtain the target oligopeptide powder.

[0057] (7) Mass spectrometry detection method: The oligopeptides were analyzed by chromatography in negative ion mode using an UltiMate 3000 ultra-high performance liquid chromatography (UPLC) system coupled with a Q-Exactive electrospray ionization mass spectrometer (Thermo Fisher Scientific). The specific steps are as follows: The target oligopeptide powder was dissolved in HPLC-grade methanol to prepare a standard solution with a concentration of 100 μg / mL. Chromatographic column: Agilent Zorbax SB-C18 column (1.9 μm, 2.1×100 mm); column temperature: 35°C; flow rate: 0.2 mL / min; mobile phase: phase A is acidified water containing 0.1% formic acid, phase B is acetonitrile; injection volume: 2.0 μL; gradient elution program: 0–2 min, phase B is maintained at 10%; 2–20 min, phase B is linearly increased from 10% to 30%. The temperature was set to 550°C and the spray voltage was set to 5500 V (positive ion mode). After data acquisition, MSDIAL ver 4.6 software was used to perform data processing such as peak search and peak alignment on the converted data.

[0058] The results are as follows Figure 1 and Figure 2 As shown, Figure 1 A\B\C\D\E are the peaks of AE peptide respectively. Figure 2 From top to bottom are the mass spectrometry data of AE peptides. Figure 1 and Figure 2 It can be seen that the target oligopeptide powder contains at least five polypeptides including the above-mentioned AE peptides, and all of these five polypeptides have a tyrosine residue at their N-termini or C-termini.

[0059] MIPs were incubated with varying concentrations of target peptides (including the control peptide KRQKY and the test peptides YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN) in a buffered solution and shaken on a thermostatic shaker until adsorption equilibrium. Subsequently, the MIPs (solid phase) and the supernatant (liquid phase) were separated by filtration. Peptide concentrations were determined using high-performance liquid chromatography (HPLC).

[0060] The saturation binding capacity is calculated by plotting the binding isotherm (bound amount B versus free peptide concentration L). When L is sufficiently high, B reaches a plateau, and this plateau value is the saturation binding capacity (mg / g MIP). The high saturation binding capacity of KRQKY (shown in the table) directly explains its advantage as a template peptide—the MIP provides a richer and more compatible tailored binding site. However, the saturation binding capacity of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN) also exceeds 70% of that of the template peptide, indicating that this MIP is feasible for separating and screening target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN).

[0061] The dissociation constant Kd was calculated as follows: The Langmuir adsorption model was used to fit the binding isotherm, and the formula was: The dissociation constant, Kd, ​​(unit: μM) was obtained by fitting the curve using nonlinear regression analysis (Origin software). A smaller Kd indicates a stronger binding affinity between the MIP and the target peptide. The dissociation constants of the test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN) shown in the table are slightly larger than those of the template peptide, but the difference is not significant, indicating that the MIP still has strong binding ability to the target peptide.

[0062] The calculation formula of the selectivity coefficient α is as follows: Selectivity coefficient: The binding affinity of the test peptides to MIP is weaker than that of the control peptide KRQKY; α in the table is all >1, indicating that the binding ability of the test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) to MIP is slightly weaker than that of the template peptide KRQKY, but the difference is not large, indicating that MIP still has strong selectivity for the target peptide. The results are shown in Table 1:

[0063] Table 1 Analysis of saturated binding capacity, dissociation constant and selectivity coefficient of target oligopeptides and IMP

[0064]

[0065]

[0066] Example 2: The effect of the AE peptide identified in Example 1 on lipid deposition in hepatocytes in a non-alcoholic fatty liver disease (NAFLD) cell model was investigated. The experimental method was as follows:

[0067] HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin and plated at 5×10 5 cells / well were seeded into 6-well plates (or 2×10 4 Cells were seeded into 24-well plates (10 cells / well, selected according to the test requirements), 2 mL of DMEM medium containing 10% FBS was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere and become 70%-80% confluent. The original culture medium was discarded and the cells were gently washed twice with PBS (to avoid damaging the adherent cells). Model group (HFD group): Serum-free DMEM medium containing 1 mM mixed fatty acids (PA:OA=1:2) + 1% BSA was added and the cells were returned to the incubator for a further 24 hours. Control group (ND group): Serum-free DMEM medium containing 1% BSA was added and the cells were returned to the incubator for a further 24 hours. Experimental group (AE group): Serum-free DMEM medium containing 4 μM oligopeptides (A, B, C, D, E peptides) + 1 mM mixed fatty acids (PA:OA = 1:2) + 1% BSA was added, and the cells were returned to the incubator and cultured for 24 hours. The cells were collected, washed with PBS, and then added with cell lysis buffer (containing PMSF to inhibit protease). Ultrasonic lysis was performed on ice (power 200 W, 3 seconds / time, 5 seconds interval, a total of 5 times). Centrifugation was performed at 12000 rpm for 10 minutes, and the supernatant was collected. The content of each indicator in hepatocytes was measured according to the instructions of the TG, TC, HDL-C, and LDL-C detection kits.

[0068] The experimental results are as follows Figure 3 As shown, among the five peptides, only peptide D and peptide E had the effect of significantly inhibiting lipid deposition in hepatocytes, while peptide AC had no such effect.

[0069] Example 3: The efficacy of peptide D and peptide E in improving glucose and lipid metabolism disorders in Example 1 was tested using the following experimental methods:

[0070] (1) Seventy male C57BL / 6 mice (4 weeks old, weighing 18–20 g) were purchased from Beijing Sibeifu Biotechnology Co., Ltd., with animal qualification certificate number SCXK (Tianjin) 2022-006. All animals were housed in a temperature-controlled environment (21 ± 2°C) and relative humidity (60 ± 10%), with a 12-h light-dark cycle and free access to food and water. All experimental procedures strictly adhered to the Regulations on the Administration of Laboratory Animals in China and were conducted under the guidance of the Guide for the Care and Use of Laboratory Animals. They were approved by the Animal Research Ethics Committee of Chuzhou University.

[0071] (2) All mice were adapted to feeding in an SPF laboratory for 7 days and then randomly divided into 6 groups (n = 8 per group). The specific groups and treatments were as follows: control group (ND group): given basal feed and gavage with an equal volume of normal saline; D-peptide and E-peptide combined control group (OP group): given basal feed + a mixture of D-peptide and E-peptide in a mass ratio of 1:1 by gavage (400 mg / kg / d); model group (HFD group): fed a high-fat feed (formula: 61.8% basal feed, 20% lard, 3% cholesterol, 0.2% bile salt, 15% egg yolk powder) and gavage with an equal volume of normal saline; oligopeptide D group (D group): high-fat feed + oligopeptide D gavage (200 mg / kg / d); oligopeptide E group (E group): high-fat feed + oligopeptide E gavage (200 mg / kg / d). Both basal feed and high-fat feed were provided by Nanjing Shengmin Co., Ltd. Gavage was continued for 14 weeks, once a day. Food intake, water intake, and body weight were monitored weekly in each group. During the final week, all mice were fasted overnight and euthanized by CO2. Eyeballs were removed and blood was collected for serum isolation. Liver, colon, and pancreas tissues were collected for biochemical and histopathological analysis. Heart, kidney, spleen, and white adipose tissue (WAT, including perirenal, epididymal, and mesenteric fat) were collected and weighed, and organ index was calculated (organ weight / body weight). Cecal tissue was stored at −80°C for subsequent cecal microbiome analysis.

[0072] like Figure 4 As shown in Figures A and B, 14 weeks of high-fat diet feeding caused a significant increase in the body weight of mice in the NAFLD model group (HFD group), which increased by 25.49% compared with the normal diet-fed group (ND group). The intervention of peptide D and peptide E significantly inhibited the abnormal weight gain induced by a high-fat diet. The body weight of the oligopeptide D group and oligopeptide E group decreased by 8.40% and 12.29% respectively compared with the HFD group. It is worth noting that compared with the ND group, there was no significant decrease in the body weight of mice in the normal diet-fed group (OP group) with OP intervention, which indicates that peptide D and peptide E can inhibit the abnormal weight gain caused by a high-fat diet, but do not affect the normal weight gain during the development of normal mice and have no physiological toxicity. In order to preliminarily evaluate the liver pathological changes in NAFLD mice and the protective effect of oligopeptides on the liver, the organ coefficients (liver coefficient and fat coefficient) were evaluated, and the results are shown as follows. Figure 4 As shown in Figure C, the liver coefficient of mice in the HFD group increased significantly by 11.53% compared with the ND group, and decreased by 6.51% and 7.42% after intervention with oligopeptide D and oligopeptide E, respectively. At the same time, high-fat diet induced an increase in the fat coefficient of NAFLD mice, and oligopeptide intervention could effectively antagonize this abnormal change. Figure 4 As shown in Figure D, compared to the HFD group, the fat index of mice in the oligopeptide intervention groups (oligopeptide D and oligopeptide E) was significantly reduced, decreasing by 15.37% and 23.41%, respectively. This shows that oligopeptides can effectively improve the obesity characteristics of NAFLD mice.

[0073] White adipose tissue was cut into small blocks of 0.5 cm × 0.5 cm × 0.2 cm. The blocks were fixed in 10% neutral formalin (pH 7.2) at 4°C for 24 hours (the volume of fixative should be five times the volume of the tissue block to prevent lipid dissolution). Dehydration was then performed using a gradient of alcohol: 70% alcohol: 1 hour; 80% alcohol: 1 hour; 95% alcohol I: 1 hour; 95% alcohol II: 1 hour; anhydrous alcohol I: 30 minutes; anhydrous alcohol II: 30 minutes. Next, the blocks were immersed in xylene I: 20 minutes; and xylene II: 20 minutes. Embedding: The blocks were placed in melted paraffin (56°C) and immersed in wax three times (30 minutes each time). The blocks were then fixed in an embedding frame and cooled to solidify into wax blocks. Sectioning: The blocks were cut into 5 μm thick sections using a paraffin microtome and mounted on polylysine-treated slides. Spreading: Place the slide in a 37°C warm water bath to spread the sections (the water temperature should not be too high to prevent the fat droplets from melting). Then bake in a 60°C oven for 2 hours to ensure the sections adhere firmly. Dewaxing to water: Xylene I: Soak for 10 minutes; Xylene II: Soak for 10 minutes; Absolute Alcohol I: Soak for 5 minutes; Absolute Alcohol II: Soak for 5 minutes; 95% Alcohol: Soak for 5 minutes; 80% Alcohol: Soak for 5 minutes; 70% Alcohol: Soak for 5 minutes; Distilled Water: Soak for 5 minutes.

[0074] Hematoxylin staining: Place the sections in Harris hematoxylin solution and stain for 5 minutes. Differentiation: Differentiate with 1% hydrochloric acid alcohol (prepared with 70% alcohol) for 10 seconds, rinse with tap water for 10 minutes to return to blue. Eosin staining: Place in 0.5% eosin solution and stain for 1 minute (cytoplasm and matrix appear pink), then quickly rinse with tap water. Dehydration: 70% alcohol: soak for 30 seconds; 80% alcohol: soak for 30 seconds; 95% alcohol I: soak for 1 minute; 95% alcohol II: soak for 1 minute; anhydrous alcohol I: soak for 2 minutes; anhydrous alcohol II: soak for 2 minutes. Transparentization: Xylene I: soak for 3 minutes; xylene II: soak for 3 minutes. Mounting: Drop neutral gum (or Canada balsam) on the sections, cover with a coverslip (avoid bubbles), and let dry at room temperature. Finally, observe and photograph under an inverted microscope.

[0075] The collected blood was centrifuged at 1600 g for 10 minutes at 4°C to obtain serum. Serum levels of total cholesterol (TC), triglycerides (TG), non-esterified fatty acids (NEFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using biochemical kits (Nanjing Jiancheng Bioengineering Institute, China). These tests were performed according to the instructions provided by the biochemical kit manufacturer.

[0076] Test results such as Figure 5 As shown, Figure 5 Figure A in the middle is the H&E staining result of white adipose tissue. It can be seen that compared with the ND group, the morphology of the fat cells in the HFD group mice was chaotic and the volume was significantly increased. After 14 weeks of intervention with oligopeptide D and oligopeptide E, the morphology of the fat cells was significantly improved and the cell diameter was significantly reduced. These results show that the intervention of oligopeptides can effectively alleviate the abnormal enlargement of fat cells induced by a high-fat diet. The abnormal increase in the levels of TC, TG, and HDL-C in the serum is one of the main characteristics of lipid metabolism disorders in NAFLD mice induced by a high-fat diet. The blood lipid profile was further measured, and the results are as follows Figure 5 As shown in Figures B, C, D, E, and F, the results show that under the intervention of long-term high-fat diet, the TC, TG, and LDL-C levels in the serum of mice in the HFD group were significantly increased compared with those in the ND group, increasing by 85.16%, 133.00%, and 179.75%, respectively, and the HDL-C level was significantly reduced (reduced by 45.70%). These abnormal changes in blood lipid levels were significantly reversed by the administration of oligopeptide D and oligopeptide E, and the blood lipid levels of NAFLD mice induced by high-fat diet (HFD group) were restored to those of the ND group. It is worth noting that the administration of oligopeptide D could not significantly reduce the LDL-C level in the serum of NAFLD mice.

[0077] Liver tissue from each group of mice was cut into 0.3 cm × 0.3 cm × 0.2 cm sections from the target region (e.g., the left lobe) and stained with H&E and Oil Red O. For Oil Red O staining, liver tissue blocks were fixed with 4% paraformaldehyde for 1.5 hours (at 4°C to avoid over-fixation that could damage lipid droplets) and then immersed in 30% sucrose solution at 4°C overnight. Tissue blocks were embedded in OCT embedding medium and frozen at -20°C for 30 minutes. Sections were cut into 6 μm-thick sections using a cryostat, mounted on slides, and air-dried at room temperature for 30 minutes. Sections were then immersed in 60% isopropanol for 5 minutes. For Oil Red O staining, sections were stained in freshly prepared Oil Red O solution (0.5 g Oil Red O dissolved in 100 ml isopropanol, 60 ml diluted with 40 ml distilled water, filtered, and used) at 37°C for 15 minutes. Differentiation: Excess stain was removed with 60% isopropanol for 1 minute, followed by rinsing with tap water. Nuclei were counterstained with hematoxylin for 30 seconds, then rinsed with tap water to restore the blue color. Mounting was done with glycerol-gelatin. Finally, the slides were observed and photographed under an inverted microscope.

[0078] Liver tissue from each group of mice was completely homogenized in a homogenizer and centrifuged at 8000 g for 10 minutes at 4°C to obtain the supernatant. The levels of total cholesterol (TC), triglycerides (TG), non-esterified fatty acids (NEFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the supernatant were measured using biochemical kits (Nanjing Jiancheng Bioengineering Institute, China). These tests were performed according to the instructions provided by the biochemical kit manufacturer.

[0079] Blood was collected from each group and centrifuged at 1600 g for 10 minutes at 4°C to obtain serum. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were measured using biochemical kits (Nanjing Jiancheng Bioengineering Institute, China). These tests were performed according to the manufacturer's instructions.

[0080] Test results such as Figure 6 The results of liver tissue staining are shown in Figure 6As shown in Figure A, a large number of lipid vacuoles were present in the livers of NAFLD mice induced by a long-term high-fat diet, accompanied by focal necrosis and a small amount of inflammatory infiltration, showing severe liver damage. After intervention with two different doses of oligopeptides, the number and size of lipid vacuoles in the liver showed a dose-dependent reduction, while the intercellular spaces decreased and there was no obvious cell infiltration, and its pathological characteristics were significantly improved. Oil red O staining further showed that the liver cells in the ND group had regular morphology, clear boundaries between cells, and no lipid accumulation or degeneration; while the cells in the HFD group were disorderly arranged, the cell structure and edges were blurred, and there was a large amount of fat droplet distribution and lipid accumulation, showing severe liver fat infiltration and fatty degeneration. After 14 weeks of oligopeptide administration, the fat deposition induced by high-fat diet was improved, and liver damage was significantly restored. As shown in Figure A Figure 6 As shown in Figures B, C, D, and E, the levels of TC, TG, and LDL-C in the liver tissues of mice in the HFD group were abnormally elevated, increasing by 53.99%, 82.71%, and 81.84%, respectively, compared to the ND group. It is noteworthy that although the TG level in the oligopeptide D group (Group D) decreased compared to the HFD group, there was no statistical difference, while the oligopeptide E group (Group E) significantly reduced the TG level. At the same time, compared to the ND group, the HDL-C level in the liver tissues of mice in the HFD group decreased significantly. The HDL-C levels in the oligopeptide-intervention groups D and E increased by 33.80% and 37.97%, respectively, compared to the HFD group. Figure 6 The results in Figures F and G show that compared with the ND group, the ALT and AST activities in the HFD group increased significantly. Among them, ALT activity increased from 30.37U / L to 60.62U / L, and ALT activity increased from 48.70U / L to 117.14U / L. However, the intervention of low-dose and high-dose oligopeptides significantly reversed this trend. Compared with the HFD group, the ALT activity in groups D and E decreased by 21.88% and 36.85%, respectively, and the AST activity decreased by 35.36% and 41.79%, respectively. The above results fully demonstrate that the intervention of oligopeptides can significantly alleviate the liver damage of NAFLD mice induced by high-fat diet.

[0081] The collected mouse liver tissues were thoroughly homogenized in a homogenizer and centrifuged at 8000 g for 10 minutes at 4°C to obtain the supernatant. The levels of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) in the supernatant were measured using biochemical kits (Nanjing Jiancheng Bioengineering Institute, China). These assays were performed according to the kit manufacturer's instructions.

[0082] The results are as follows Figure 7 As shown, Figure 7 Figures A, B, and C show that compared with the ND group, the activities of antioxidant enzymes SOD, GSH-PX, and CAT in the liver tissue of the HFD group were significantly decreased. After intervention with two oligopeptides, D and E, they all increased significantly. MDA reflects the severity of free radical damage to liver cells. The results showed that the MDA level in the liver of mice in the HFD group was significantly higher than that in the ND group, while the rising trend of MDA levels in mice in the D and E groups that were gavage-fed with oligopeptides was significantly inhibited, accounting for only 62.46% and 57.76% of the HFD group (as shown in Figure 1). Figure 7 The above results show that oligopeptides can effectively reduce the oxidative stress level in the liver of NAFLD mice.

[0083] Example 4: The improvement effect of peptide D and peptide E on insulin resistance was investigated using the following method:

[0084] (1) After 14 weeks of oligopeptide intervention, C57BL / 6 mice were fasted for 12 hours overnight (with free access to water). Blood was then collected from the tail vein (1 mm from the tail tip was cut and 5 μL of blood was drawn using a capillary tube). Whole blood glucose was then directly measured using a portable blood glucose meter (Roche Active).

[0085] (2) The serum collected and separated was used to detect and calculate the mouse serum insulin concentration using an ELISA kit (Mercodia mouse insulin kit);

[0086] (3) After 14 weeks of oligopeptide intervention, C57BL / 6 mice were fasted for 12 hours and weighed. They were then gavaged with 20% glucose solution at 2 g / kg body weight. Blood was collected before gavage (0 min, i.e., fasting blood glucose), and at 30, 60, 120, and 180 minutes after gavage according to the "fasting blood glucose" method to measure blood glucose concentration.

[0087] (4) Area under the curve (AUC): It is usually used to quantify the overall blood glucose level of the OGTT curve, reflecting the total blood glucose exposure after the glucose load, and can better reflect impaired glucose tolerance than a single time point. The calculation method of AUC is based on the blood glucose values ​​at each time point of the OGTT (C0, C30, C60, C120, C180, unit: mmol / L), and the time interval is in minutes (Δt): Unit: mmol·L-1·min.

[0088] The QUICK index is an insulin sensitivity assessment indicator based on fasting blood glucose and insulin. The higher the index, the better the insulin sensitivity. It is calculated as follows: Fasting blood glucose (FBG) and fasting insulin (FINS) are required, and the formula is:

[0089]

[0090] The HOMA-IR index is the most commonly used indicator for evaluating insulin resistance. It is based on fasting blood glucose and insulin. The higher the index, the more severe the insulin resistance. It is calculated as follows: fasting blood glucose (FBG, mmol / L) and fasting insulin (FINS, mU / L) are required. The formula is:

[0091] like Figure 8 As shown in Figures A and B, serum insulin levels in mice on the HFD were significantly elevated compared to those in the ND group. In contrast, serum insulin levels in mice on the high-fat diet (groups D and E) that were given different oligopeptide interventions were significantly decreased compared to those in the HFD group. HOMA-IR (homeostasis model assessment of insulin resistance) was further calculated from FBG and serum insulin levels.

[0092] like Figure 8 As shown in Figures C, D, and E, in the OGTT experiment, blood glucose levels in each group rose sharply after oral administration of glucose solution, and the maximum value reached was positively correlated with the relative FBG levels in each group. The maximum blood glucose level in the HFD group was 23.16 mmol / L, which was 156.06% of the ND group (14.84 mmol / L). Meanwhile, after 14 weeks of oligopeptide intervention, the maximum blood glucose levels in Groups D and E were only 86.83% and 78.84% of those in the HFD group. The blood glucose levels of mice in the HFD group peaked at 30 minutes and then declined. In contrast, the blood glucose levels of mice in the ND, OP, D, and E groups showed an earlier downward trend. In all four groups, blood glucose peaked at 15 minutes and began to decline by 30 minutes. At the end of the 120-min OGTT experiment, the blood glucose levels of the ND, OP, D, and E groups were significantly lower than those of the HFD group, reaching only 83.03%, 74.46%, 54.96%, and 50.45% of those in the HFD group. Further quantification of the area under the curve of the OGTT experiment revealed that compared with the HFD group, the areas under the curve of the 120-min OGTT in groups D and E decreased by 18.84% and 32.88%, respectively, showing a dose-dependent decrease in the oligopeptide. This result indicates that oligopeptides can significantly improve glucose tolerance in NAFLD mice.

[0093] like Figure 8 As shown in Figure F, the HOMA-IR index of mice in the HFD group increased significantly, by 70.52% compared to the ND group. This increase was significantly suppressed by oligopeptide intervention in groups D and E. Compared to the HFD group, the HOMA-IR index in groups D and E decreased by 30.20% and 49.12%, respectively. These results clearly demonstrate that oligopeptides can significantly improve insulin resistance in NAFLD mice induced by a high-fat diet.

[0094] Example 5: To further explore the regulatory mechanism of peptide D and peptide E on abnormal lipid metabolism in NAFLD mice, Western blotting was used to determine the expression levels of related proteins in the lipid metabolism pathway as follows:

[0095] Liver tissue samples were rinsed three times with ice-cold 1× PBS to remove residual blood, minced, and transferred to a homogenization tube. RIPA buffer containing 1% protease inhibitors was added to the sample at 10 times the tissue volume, followed by two 2-mm magnetic beads, and homogenization for 60 seconds. The homogenized sample tube was removed from the ice bath for half an hour, vortexed every 5 minutes for 30 seconds to promote complete tissue lysis. The mixture was centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was collected as the total protein fraction. Protein content in the supernatant was quantified using a commercial BCA protein assay kit. The extracted protein sample was then mixed with 5× protein loading buffer at a 4:1 (v / v) ratio and denatured by heating at 100°C for 15 minutes. The voltage of the stacking gel was set at 75 V, and the voltage of the separating gel was set at 120 V. The membrane was transferred at 300 mA for 30 minutes. The membrane was then blocked by incubation in 5% nonfat dry milk containing 0.5% TBST on a shaking platform for 1 hour at room temperature. Primary antibodies targeting phosphorylated proteins were diluted in 5% nonfat dry milk and 5% BSA dissolved in TBST and incubated overnight at 4°C. Secondary antibodies were diluted 1:3000 in TBST and incubated for 5 minutes each on a shaker at room temperature. After a 30-minute incubation at room temperature, the cells were washed three times with TBST on a shaker at room temperature for 5 minutes each. Western blotting was performed using an ECL chemiluminescence kit (Thermo Fisher Scientific, CA, USA), and band optical density was analyzed using Image J software.

[0096] The results are as follows Figure 9 and Figure 10 As shown, Figure 9The results showed that in the normal diet (ND) and control treatment (OP) groups, the AdipoR2-AMPK-AKT pathway was continuously activated (high ratios of p-AdipoR2, p-AMPK, and p-AKT), the glucose transporter GLUT4 was adequately expressed, the glycogen synthase kinase GSK3β remained active (low ratio of p-GSK3β), and the transcription factor FoxO1 was dephosphorylated (low ratio of p-FoxO1); a high-fat diet (HFD) significantly inhibited this pathway, leading to decreased GLUT4 expression (obstructed glucose uptake), increased GSK3β phosphorylation (inhibited glycogen synthesis), and abnormal upregulation of FoxO1 phosphorylation (disordered gluconeogenesis regulation); intervention treatments (D-peptide and E-peptide) effectively restored pathway activity, promoted GLUT4 expression, GSK3β activation, and reset FoxO1 phosphorylation status, and the differences were statistically significant. In summary, HFD disrupts glucose metabolism by blocking the AdipoR2-AMPK-AKT signaling axis, while intervention with peptide D and peptide E can synergistically improve glucose uptake, glycogen synthesis, and gluconeogenesis balance by rebuilding this pathway. Figure 10 Results showed that long-term high-fat diet-induced AMPK phosphorylation significantly decreased, while oligopeptide intervention significantly activated AMPK phosphorylation, with expression levels in groups D and E increased by 43.71% and 57.47%, respectively, compared to the HFD group. Furthermore, oligopeptide intervention significantly upregulated the expression of PPAR-α and CPT-1. Compared to the HFD group, low- and high-dose oligopeptide intervention increased PPAR-α expression by 40.23% and 58.35%, respectively, and CPT-1 expression by 54.75% and 71.36%, respectively. SREBP-1, a major regulator of hepatic lipid synthesis genes, was significantly elevated in the livers of high-fat diet-induced NAFLD mice, with expression levels in the HFD group increasing by 48.91% compared to the ND group. High-dose oligopeptide intervention reduced SREBP-1 expression by 38.46%, while low-dose oligopeptide administration had no significant inhibitory effect on the high-fat diet-induced abnormal increase in SREBP-1 expression. The above results indicate that oligopeptides D and E can activate the AMPK / PPAR-α pathway by upregulating AMPK phosphorylation expression, thereby upregulating CPT1 expression and further enhancing the level of fatty acid oxidation; at the same time, they can reduce lipid deposition caused by a high-fat diet by downregulating SREBP-1 expression.

[0097] Example 6: Molecular docking simulation method was used for spatial structure matching, chemical group recognition and hydrogen bonding, as follows:

[0098] The structures of the control peptide KRQKYD and target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN) were predicted using AlphaFold. The structure of the AdipoR2 receptor protein was directly retrieved from the PDB database. A docking region (surrounding the target binding site) was defined, and a genetic algorithm was used to search for the optimal binding conformation between the peptide and target. A custom score (0-10, with higher scores indicating higher conformational fit) was assigned based on the spatial complementarity of the conformations (contact area, conformational fit, and RMSD value). Scores ranged from 8-10, with higher scores indicating near-perfect conformational fit and no steric clashes, and from 0-5, with distorted conformations and severe steric clashes. A custom score (0-10, with higher scores indicating higher functional group fit and interaction strength) was assigned based on the chemical group identity (binding enthalpy change, binding constant, and stoichiometry) of the single binding site model. Scores ranged from 8-10, with higher scores indicating perfect functional group complementarity and strong interactions (π-π stacking and electrostatic synergy), and from 0-5, with weak functional group clashes and weak interactions. The results are shown in Table 2:

[0099] Table 2 Analysis results of target oligopeptides' ability to bind to AdipoR2 receptor

[0100]

[0101]

[0102] The interaction scores between the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) in Table 2 were all >5 points, indicating that the intermolecular interaction force between the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) and AdipoR2 receptor protein was stronger than that of KRQKYD. At the same time, the number of hydrogen bonds formed between the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) and the AdipoR2 receptor protein was significantly greater than that of the control peptide KRQKYD, indicating that the hydrogen bond binding ability of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) and the AdipoR2 receptor protein was stronger than that of KRQKYD.

[0103] Comprehensive binding ability (total score): The spatial structure matching score (0-10), chemical group recognition score (0-10), and number of hydrogen bonds are added together to obtain a total score (e.g., KRQKYD: 4.3+4.7+1=10.0). The higher the score, the stronger the binding ability of the oligopeptide to the AdipoR2 receptor protein. The comprehensive binding ability (total score) of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) in the table are significantly higher than that of KRQKYD, indicating that the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) have significantly stronger specific binding ability to the AdipoR2 receptor protein than KRQKYD.

[0104] Example 7: Peptide D and peptide E in Example 1 were prepared into soft candies as follows:

[0105] (1) Peptide D and peptide E (molar ratio 1:1) were dissolved in water at a final concentration of 200 mg / mL to obtain a peptide aqueous solution;

[0106] (2) The fat portion of three-year-old Jinhua ham was mixed with petroleum ether at a material-liquid ratio of 1 g:5 mL, and then ultrasonically extracted at 50°C for 2 h. The supernatant was removed by rotary evaporation to obtain ham fat;

[0107] (3) The polypeptide aqueous solution was mixed with ham fat and castor oil glycerol oleate in a volume ratio of 60:40:3.2, and high-speed shearing (40s / time, 4 times) was performed at 25000rpm to obtain an emulsion with the function of improving glycolipid metabolism disorder.

[0108] (4) By weight, 60 parts of the above emulsion, 1 part of vitamin C, 3 parts of apple juice concentrate, 0.5 parts of maltodextrin, 0.5 parts of xylitol, 1 part of carrageenan, and 1 part of xanthan gum were mixed and heated at 70°C for 25 minutes. The mixture was then poured into a mold, cooled and formed, and then demolded and sterilized to obtain a soft candy. The apple juice concentrate can be replaced with concentrated juice of banana, strawberry, orange, watermelon, etc.

[0109] Example 7: Peptide D or peptide E in Example 1 was prepared into soft capsules as follows:

[0110] (1) Dissolve peptide D or peptide E in water to a final concentration of 100 mg / mL to obtain a polypeptide aqueous solution;

[0111] (2) The fat portion of three-year-old Jinhua ham was mixed with petroleum ether at a material-liquid ratio of 1.5 g:7 mL, and then ultrasonically extracted at 45 °C for 4 h. The supernatant was removed by rotary evaporation to obtain ham fat;

[0112] (3) The polypeptide aqueous solution was mixed with ham fat and castor oil glycerol oleate in a volume ratio of 70:30:1.8, and high-speed shearing (40s / time, 4 times) was performed at 22000 rpm to obtain an emulsion with the function of improving glycolipid metabolism disorder.

[0113] (4) By weight, 70 parts of the above emulsion and 2 parts of vitamin C were mixed to obtain capsule contents; a soft capsule shell was prepared using gelatin: water: glycerol = 80-100: 80-100: 20-40 as raw materials, and the capsule contents were encapsulated in the soft capsule shell to obtain a soft capsule.

Claims

1. A dry-cured ham-derived oligopeptide, characterized in that: Contains at least one of the following amino acid sequences: ELIDQDARDLY; YHEHRSDLN; YKATEPVIAF; INKVEELKKKY; GEKLKRQKY.

2. The dry-cured ham-derived oligopeptide according to claim 1, characterized in that: Contains the following amino acid sequence: ELIDQDARDLY and / or YHEHRSDLN.

3. Use of the dry-cured ham-derived oligopeptide according to claim 1 or 2 in the preparation of a drug for treating glycolipid metabolism disorders.

4. The use according to claim 3, characterized in that The glucose and lipid metabolism disorder disease includes at least one of insulin resistance, hyperglycemia, diabetes, hyperlipidemia, and fatty liver.

5. The use according to claim 3, characterized in that The content of the dry-cured ham-derived oligopeptide in the medicine is 1-200 mg / mL.

6. Use of the dry-cured ham-derived oligopeptide according to claim 1 or 2 in the preparation of health food.

7. The use according to claim 6, characterized in that The steps include: (1) dissolving dry-cured ham-derived oligopeptides in water to obtain a polypeptide aqueous solution; (2) The polypeptide aqueous solution and the oil phase are mixed and emulsified to obtain an emulsion.

8. The use according to claim 7, characterized in that In step (1), the final concentration of the dry-cured ham-derived oligopeptide in the aqueous polypeptide solution is 100-200 mg / mL; In step (2), the volume ratio of the polypeptide aqueous solution to the oil phase is 5-7:3-5.

9. The use according to claim 7, characterized in that In step (2), the oil phase comprises ham fat and castor oil glycerol oleate; the ham fat is prepared as follows: The fat in the ham is mixed with petroleum ether, subjected to heating and ultrasonic extraction, and the supernatant is taken out. The petroleum ether is removed by rotary evaporation to obtain the ham fat.

10. The use according to claim 7, characterized in that The following steps are also included: Using the emulsion, nutritional excipients and formulation excipients as raw materials to prepare capsules or soft candies; The nutritional auxiliary materials include at least one of vitamins and fruit juices; the preparation auxiliary materials include at least one of gelatin, water, glycerin, maltodextrin, xylitol, carrageenan, and xanthan gum.

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