A goat milk whey protein peptide that can maintain healthy blood lipid levels, its preparation method and application

Abstract

This invention discloses a sheep milk whey protein peptide capable of maintaining healthy blood lipid levels, its preparation method, and its applications. The invention involves proteolytic hydrolysis of sheep milk whey protein, followed by ultrafiltration, purification, and identification of the hydrolysate to obtain two sheep milk whey protein peptides, ECAQKK and STEYGL. These two peptides possess the functions of inhibiting cholesterol esterase activity, regulating serum lipid levels (lowering triglyceride, total cholesterol, and low-density lipoprotein levels, and increasing high-density lipoprotein levels), reducing body weight and white fat mass, alleviating hepatic lipid accumulation and steatosis, and improving the body's inflammatory response. This invention provides a new product for maintaining healthy blood lipid levels and offers technical support for the in-depth development and high-value utilization of whey protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a goat milk whey protein peptide that can maintain healthy blood lipid levels, its preparation method, and its application. Background Technology

[0002] Dyslipidemia, especially elevated levels of low-density lipoprotein cholesterol (LDL-C), is a major risk factor for atherosclerotic cardiovascular disease. Data from the Global Burden of Disease (GBD) study shows that between 2019 and 2023, the prevalence of dyslipidemia among adults worldwide reached 33.8%, and dyslipidemia-related diseases cause more than 4 million deaths annually, seriously endangering human health. Currently, statins are the primary treatment for dyslipidemia in clinical practice; however, doubling the dosage only increases the LDL-C-lowering effect by 6%, and long-term use can lead to adverse reactions such as myopathy, new-onset diabetes, and abnormal liver function. Therefore, developing natural, safe, and effective dietary cholesterol-lowering products is of great significance.

[0003] Cholesterol esterase (CEase) is a bile salt-activated lipase primarily secreted by the pancreas into the intestine. It catalyzes the hydrolysis of dietary cholesterol esters into cholesterol and free fatty acids, playing a crucial role in dietary cholesterol absorption and transport to intestinal epithelial cells. Since cholesterol esters cannot be directly taken up by intestinal epithelial cells, inhibiting cholesterol esterase activity has become an important strategy for limiting dietary cholesterol absorption and regulating blood lipid levels.

[0004] Milk proteins are characterized by high water solubility, good biocompatibility, and high bioavailability, making them an important source of dietary bioactive peptides. Numerous studies have confirmed that milk protein peptides have the potential to lower cholesterol, inhibiting cholesterol esterase and pancreatic lipase, reducing serum LDL-C and triglyceride (TG) levels, improving hepatic lipid accumulation and steatosis, thereby maintaining cholesterol homeostasis in the body.

[0005] However, current research on specific whey protein peptide sequences with cholesterol esterase inhibitory activity is lacking. Therefore, this invention uses ultrafiltration, mass spectrometry sequencing, and molecular docking techniques to screen cholesterol-lowering peptides with cholesterol esterase inhibitory activity from sheep milk whey protein hydrolysates. In vitro activity evaluation and in vivo functional verification are then conducted, providing a theoretical basis and technical support for the development of novel functional ingredients for maintaining healthy blood lipids. Summary of the Invention

[0006] In order to fully explore the efficacy of whey protein peptides and expand products for maintaining healthy blood lipids, this invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a goat milk whey protein peptide, wherein the goat milk whey protein peptide is ECAQKK and / or STEYGL.

[0008] Preferably, ECAQKK exhibits an in vitro cholesterol esterase inhibition rate of 77.32%.

[0009] Preferably, STEYGL exhibits an in vitro cholesterol esterase inhibition rate of 67.43%.

[0010] In a second aspect, the present invention provides a composition comprising the sheep milk whey protein peptides described in the first aspect.

[0011] Thirdly, the present invention provides a cholesterol esterase inhibitor, the cholesterol esterase inhibitor comprising the sheep milk whey protein peptide described in the first aspect or the composition described in the second aspect.

[0012] Preferably, the cholesterol esterase inhibitor has an in vitro activity of ≥67%.

[0013] Thirdly, the present invention provides a method for preparing the goat milk whey protein peptide described in the first aspect, the method comprising the following steps: S1, dissolve goat milk whey protein powder in water, adjust pH to alkaline, then add protease to hydrolyze and inactivate the enzyme, and adjust the hydrolysate to neutral; S2, after ultrafiltration of the enzymatic hydrolysate obtained in step S1, the ultrafiltrate is freeze-dried to obtain whey protein hydrolysate freeze-dried powder. S3, the whey protein hydrolysate lyophilized powder obtained in step S2 is separated and purified by gel chromatography, high performance liquid chromatography, and the whey protein peptide is obtained. S4. The whey protein peptides obtained in step S3 are sequence identified.

[0014] Preferably, in step S1, the mass ratio of whey protein powder to water is 1:10~25, for example: 1:10, 1:15, 1:20, 1:25.

[0015] Preferably, the protease is an alkaline protease, papain, or a complex protease, and more preferably an alkaline protease.

[0016] Preferably, the amount of protease added is 2000~5000 U / g whey protein powder, for example: 2000 U / g whey protein powder, 2500 U / g whey protein powder, 3000 U / g whey protein powder, 3500 U / g whey protein powder, 4000 U / g whey protein powder, 4500 U / g whey protein powder, and 5000 U / g whey protein powder.

[0017] Preferably, the enzymatic hydrolysis temperature is 50~60℃, for example: 50℃, 53℃, 55℃, 57℃, 60℃.

[0018] Preferably, the enzymatic hydrolysis time is 1.5 to 3 hours, for example: 1.5 hours, 2 hours, 2.5 hours, or 3 hours.

[0019] Preferably, the enzyme inactivation temperature is 80~90℃, for example: 80℃, 83℃, 85℃, 87℃, 90℃.

[0020] Preferably, the time is 10 to 20 minutes, for example: 10 minutes, 12 minutes, 15 minutes, 18 minutes, or 20 minutes.

[0021] Preferably, in step S2, the molecular weight of the ultrafiltration membrane used for ultrafiltration is 3~8 kDa, for example: 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa.

[0022] Preferably, in step S3, the stationary phase for the gel chromatography is Sephadex G-10, Sephadex G-15, Bio-Gel P-2, or Toyopearl HW-40.

[0023] Preferably, in step S3, the eluent is Tris-HCl.

[0024] Preferably, in step S3, the elution flow rate is 0.2~0.6 mL / min, for example: 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, more preferably 0.4 mL / min.

[0025] Preferably, in step S3, the conditions for high performance liquid chromatography are: C18 column; flow rate: 0.1~0.5 mL / min; elution program: the proportion of mobile phase B increases linearly from 0% to 80% within 100 min; mobile phase A is water containing 0.1% trifluoroacetic acid, and mobile phase B is a 60% acetonitrile aqueous solution containing 0.05% trifluoroacetic acid.

[0026] Furthermore, the C18 chromatographic column has an inner diameter of 2.1 mm, a column length of 100 mm, and a particle size of 1.7 μm.

[0027] Furthermore, the flow rate is 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min, more preferably 0.3 mL / min.

[0028] Fourthly, the present invention provides the use of the goat milk whey protein peptides described in the first aspect or the composition described in the second aspect in the preparation of products that help maintain healthy blood lipid levels.

[0029] Preferably, the application includes at least one of the following: (1) Application in the preparation of cholesterol esterase inhibitors; (2) Application in the preparation of products that can improve dyslipidemia; (3) Application in the preparation of products that can prevent or treat obesity; (4) Application in the preparation of products that can improve inflammatory responses caused by hyperlipidemia; (5) Application in the preparation of products that can improve liver damage and lipid accumulation in the liver caused by hyperlipidemia.

[0030] Preferably, the product is a pharmaceutical, health food, or feed.

[0031] Furthermore, the product also includes excipients permitted to be added to pharmaceuticals, health foods, or animal feed.

[0032] The beneficial effects of this invention are: This invention involves the enzymatic hydrolysis of sheep whey protein powder, followed by ultrafiltration, purification, and identification of the hydrolysate to obtain two sheep whey protein peptides, ECAQKK and STEYGL. These two peptides possess functions such as inhibiting cholesterol esterase activity, reducing body weight and white fat mass, regulating serum lipid levels, alleviating hepatic lipid accumulation and steatosis, and improving the body's inflammatory response. This invention provides a new product for maintaining healthy blood lipid levels and offers technical support for the in-depth development and high-value utilization of whey protein. Attached Figure Description

[0033] Figure 1 The diagram shows the molecular docking of ECAQKK with cholesterol esterase; A represents the molecular docking conformation, and B represents the interaction mode. Figure 2 The diagram shows the docking of STEYGL with cholesterol esterase; A represents the molecular docking conformation, and B represents the interaction mode. Figure 3 The figure shows the in vitro cholesterol esterase inhibition rates of ECAQKK and STEYGL. Figure 4 The image shows the effect of whey protein peptides on the weight gain and white adipose tissue weight in mice; A represents the weight gain result, and B represents the white adipose tissue weight result. Figure 5 The figure shows the effect of whey protein peptides on serum lipid levels in mice; A represents serum TC content, B represents serum TG content, C represents serum LDL-C content, and D represents serum HDL-C content. Figure 6 The image shows the effects of whey protein peptides on serum inflammatory factors and endotoxin levels in mice; A represents serum TNF-α levels, B represents serum IL-6 levels, C represents serum IL-1β levels, and D represents serum LPS levels. Figure 7 The image shows the effects of whey protein peptides on serum liver function indicators (enzymes) and liver lipid levels in mice; A represents serum ALT content, B represents serum AST content, C represents liver TC content, and D represents liver TG content.

[0034] Figure 8 The image shows the effect of whey protein peptides on the histopathological results of mouse liver tissue; A is the result of liver H&E staining, and B is the result of liver Oil Red O staining. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1: Preparation of whey protein peptides capable of maintaining healthy blood lipid levels In this embodiment, whey protein peptides capable of maintaining healthy blood lipid levels are prepared by the following method: (1) Enzymatic hydrolysis: Sheep whey protein was dissolved in distilled water at a ratio of 5%, and after heat treatment at 85℃ for 15 min, the pH was adjusted to 9. Alkaline protease was added at an enzyme / substrate ratio of 3000 U / g. After enzymatic hydrolysis at 55℃ for 2 h, the enzyme was inactivated by heating in a water bath at 85℃ for 15 min. The pH of the hydrolysate was then adjusted to 7.0.

[0037] (2) Ultrafiltration: The enzymatic hydrolysate obtained in step (1) was ultrafiltered using a 5 kDa ultrafiltration membrane. The ultrafiltrate was collected and freeze-dried under vacuum to obtain whey protein hydrolysate lyophilized powder.

[0038] (3) Purification and identification: The whey protein peptides were obtained by separating and purifying the lyophilized whey protein hydrolysate obtained in step (2) by gel column chromatography and high performance liquid chromatography.

[0039] The specific separation and purification steps are as follows: Gel column chromatography: Lyophilized whey protein hydrolysate powder was dissolved in Tris-HCl buffer (20 mmol / L, pH 8.0) and loaded onto a Sephadex G-10 gel permeation chromatography column equilibrated with Tris-HCl buffer. Elution was performed with Tris-HCl buffer at a flow rate of 0.4 mL / min. The eluent was collected periodically using an automated collector, one tube every 5 min. OD values ​​were measured at 220 nm to obtain the distribution curves of the enzymatic ultrafiltration products in each chromatographic fraction, thus collecting peptide fractions of different molecular weights.

[0040] High-performance liquid chromatography (HPLC) separation and purification: The active components separated by a Sephadex G-10 column were isolated using reversed-phase HPLC. Chromatographic conditions were as follows: C18 column (2.1 × 100 mm, 1.7 μm); flow rate: 0.3 mL / min; detection wavelength: 220 nm; mobile phase: Solution A: water containing 0.1% trifluoroacetic acid; Solution B: 60% acetonitrile aqueous solution containing 0.05% trifluoroacetic acid; elution program: the proportion of mobile phase B increased linearly from 0% to 80% within 100 min.

[0041] Example 2: Screening of whey protein peptides capable of maintaining healthy blood lipid levels (1) Peptide sequence identification: The lyophilized whey protein peptide powder was dissolved in an aqueous solution containing 0.1% (v / v) formic acid, and the peptide sequence was identified by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS / MS). Chromatographic conditions: Mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid, and mobile phase B was an aqueous solution of 80% (v / v) acetonitrile containing 0.1% (v / v) formic acid. During elution, the solvent B was increased from 8% to 50% (v / v) at a flow rate of 200 nL / min. Mass spectrometry conditions: The first-order spectral detection range was 50-1500 m / z with a scan resolution of 60000, and the second-order spectral detection range was 50-1400 m / z with a scan resolution of 15000. The LC-ESI MS / MS data were collected and compared with the UniProt (https: / / www.uniprot.org / ) and NCBI databases to identify the amino acid sequence of the whey protein peptide.

[0042] (2) Molecular docking screening of cholesterol esterase inhibitory peptides: The three-dimensional structure of cholesterol esterase (PDB ID: 1CLE) was obtained from the protein database (PDB, https: / / www.rcsb.org / ). Before molecular docking, water molecules and endogenous ligands were removed, incomplete amino acid residues were added, and hydrogen atoms were added. The 3D structure of the peptide was constructed using HyPerChem software, and CHARMM was used as the force field for geometry optimization and energy minimization. A semi-flexible docking method was adopted, and the binding site was defined by the endogenous linoleic cholesterol ester ligand. Screening was performed based on the -CDOCKER Energy score, with a higher score indicating stronger binding strength.

[0043] (3) Peptide property prediction: AllerCatPro (https: / / allercatpro.bii.a-star.edu.sg / ) was used to predict the potential sensitization of peptides; ToxinPred (https: / / webs.iiit d.edu.in / raghava / toxinpred / index.html) was used to predict the potential toxicity of peptides; admetSAR (http: / / lmmd.ecust.edu.cn / admetsar1 / predict / ) was used to predict the absorption of peptides in the human intestine; and Expasy (https: / / web.expasy.org / protparam / ) was used to predict the hydrophilicity and hydrophobicity of peptides, with negative values ​​indicating hydrophilicity.

[0044] Cholesterol esterase catalyzes the hydrolysis of dietary cholesterol esters into cholesterol and free fatty acids, playing a crucial role in cholesterol absorption and transport. Inhibiting its activity can effectively reduce blood total cholesterol and LDL cholesterol levels. Based on the docking energy between whey protein peptides and cholesterol esterase, two cholesterol-lowering peptides were screened from the identified peptides: ECAQKK (SEQ ID NO.1) and STEYGL (SEQ ID NO.2). Their docking binding energies and predicted properties with cholesterol esterase are shown in Table 1. Both peptides ECAQKK and STEYGL are non-toxic, non-allergenic, and exhibit good intestinal absorption in humans, and demonstrate a strong binding affinity to cholesterol esterase.

[0045] Table 1. Docking binding energy and property prediction of peptides with cholesterol esterase Figures 1-2Molecular docking results and interaction modes of the peptides ECAQKK and STEYGL with cholesterol esterase showed that peptide ECAQKK can interact with 7 amino acid residues of cholesterol esterase. Peptide ECAQKK can form hydrogen bonds with residues Glu123, Gly124, Glu208, Ala210, and His449, and electrostatic interactions with residues Phe345 and Phe415. Peptide STEYGL can interact with 6 amino acid residues of cholesterol esterase. Peptide STEYGL can form hydrogen bonds with residues Gly124, Ala210, and His449, and electrostatic interactions with residues Met213, and hydrophobic interactions with residues Leu297 and Phe448. The active sites of cholesterol esterase include the catalytic triplet (Ser209-His449-Glu341) and the oxygen anion pore (Gly123, Gly124, Ala210). Whey protein peptides inhibit enzyme activity by binding to these active sites, inducing conformational changes in cholesterol esterases, reducing the enzyme's affinity for the substrate, and preventing the substrate from entering the active site.

[0046] The active peptides provided by this invention are derived from whey protein and can also be obtained through solid-phase synthesis. The peptides ECAQKK (SEQ ID NO.1) and STEYGL (SEQ ID NO.2) used in the following examples were obtained through solid-phase synthesis. The above peptides were synthesized in a solid-phase manner by Shanghai Nuoyou Biotechnology Co., Ltd. for subsequent experiments.

[0047] Example 3: Determination of the in vitro cholesterol esterase inhibitory activity of whey protein peptides The cholesterol-lowering activity of whey protein peptides ECAQKK and STEYGL obtained by molecular docking screening was evaluated by measuring their in vitro cholesterol esterase inhibition rate.

[0048] In a 96-well microplate, 25 μL of whey protein peptide solution (4 mg / mL, final concentration 0.5 mg / mL), 25 μL of PNPB (5 mM, prepared in acetonitrile), 25 μL of taurocholate sodium phosphate buffer (5.16 mmol / L taurocholate sodium, 0.1 mol / L pH 7.04 sodium phosphate buffer), and 100 μL of sodium chloride buffer (pH 7.3) were added sequentially. Finally, 25 μL of cholesterol esterase solution (2.5 µg / mL, 60 U / mg) was added to initiate the reaction. After reacting at 37℃ for 30 min, the absorbance was measured at 405 nm. Pure water was used to replace the sample solution as a sample control, pure water was used to replace both the enzyme solution and the sample solution as a blank control, and pure water was used to replace the enzyme solution as a blank sample. The in vitro cholesterol esterase inhibition rate was calculated using the following formula: In the formula, A is the absorbance value of the sample control, B is the absorbance value of the blank control, C is the absorbance value of the peptide-containing solution, and D is the absorbance value of the blank sample.

[0049] like Figure 3 As shown, the in vitro cholesterol esterase inhibition rates of peptides ECAQKK and STEYGL were 77.32%±2.41% and 67.43%±3.96%, respectively, indicating that both peptides have good cholesterol esterase inhibitory activity (>65%) and can maintain healthy blood lipids by inhibiting the absorption and transport of dietary cholesterol. Compared with STEYGL, ECAQKK showed a significantly higher in vitro cholesterol esterase inhibition rate (p<0.05), demonstrating superior cholesterol-lowering potential.

[0050] Half maximal inhibitory concentration (IC50) 50 IC50 refers to the sample concentration required to inhibit enzyme activity to 50%. 50 The lower the value, the stronger the inhibitory effect of the peptide on the enzyme. Table 2 shows the IC50 value of the whey protein peptide ECAQKK. 50 The value was significantly lower than that of STEYGL (p<0.05), indicating that it had a better inhibitory effect on cholesterol esterase.

[0051] Table 2. Half-inhibitory concentrations of cholesterol esterase inhibitory peptides Example 4: The effect of whey protein peptides on improving hyperlipidemia induced by a high-fat diet in mice. Fifty male C57BL / 6J mice were selected and housed at 23±3℃, 65±5% humidity, and a 12-hour light-dark cycle with good ventilation. The mice were housed for one week prior to the experiment to acclimatize to the laboratory environment.

[0052] Mice were randomly divided into a blank control group, a model group, an ECAQKK group, a STEYGL group, and a simvastatin group (n=10 per group). The model group, ECAQKK group, STEYGL group, and simvastatin group were fed a high-fat diet (60% fat to energy) for 9 weeks to establish a hyperlipidemia model, while the blank control group was fed a maintenance diet (10% fat to energy). After the modeling period, the blank control group and the model group were administered physiological saline by gavage, while the ECAQKK group, STEYGL group, and simvastatin group were administered ECAQKK (10 mg / kg bw), STEYGL (10 mg / kg bw), and simvastatin solution (5 mg / kg bw), respectively, by gavage. The dietary intake remained unchanged.

[0053] During the modeling and intervention period, the weight of mice was measured weekly. Nine weeks after drug administration, the weight of mice was measured again, and the weight gain of mice before and after whey protein peptide intervention was calculated. After the mice were sacrificed, blood, liver, and white adipose tissue were collected, and the weight of inguinal fat, epididymal fat, and perirenal fat was measured.

[0054] like Figure 4 As shown in Figure A, compared with the blank control group, the weight gain of mice in the model group was significantly increased (p<0.05). Compared with the model group, the weight gain of mice in the ECAQKK group, STEYGL group and simvastatin group was significantly decreased (p<0.05).

[0055] like Figure 4 As shown in Figure B, compared with the blank control group, the weight of inguinal fat, epididymal fat, and perirenal fat in the model group mice were significantly increased (p<0.05). After intervention with whey protein peptides ECAQKK and STEYGL, the weight of inguinal fat, epididymal fat, and perirenal fat in mice were significantly reduced (p<0.05), and the improvement effect was not significantly different from that of the positive drug simvastatin (p>0.05).

[0056] Furthermore, there were no significant differences in body weight gain and white adipose tissue weight between the ECAQKK group and the STEYGL group (p>0.05).

[0057] The above results indicate that a high-fat diet can induce obesity in mice, manifested as increased body weight and increased white adipose tissue mass; whey protein peptides ECAQKK and STEYGL can effectively improve lipid accumulation and obesity induced by a high-fat diet, and the two have comparable effects.

[0058] Dyslipidemia, characterized by elevated levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), is closely associated with atherosclerotic cardiovascular diseases. To investigate the effect of whey protein-derived cholesterol esterase inhibitory peptide on blood lipid levels in mice on a high-fat diet, serum TC and TG levels were measured using ELISA kits (MM-0632M, MM-0631M1, Jiangsu Enzyme Immunoassay Co., Ltd.); serum LDL-C and high-density lipoprotein cholesterol (HDL-C) levels were measured using a fully automated biochemical analyzer. The accompanying kits were purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd.

[0059] like Figure 5As shown, compared with the blank control group, the serum TC, TG, LDL-C and HDL-C levels in the model group mice were significantly increased (p<0.05), increasing from 0.36±0.01 mmol / L, 0.12±0.00 mmol / L, 0.26±0.02 mmol / L and 2.16±0.06 mmol / L to 0.54±0.00 mmol / L, 0.19±0.01 mmol / L, 1.11±0.08 mmol / L and 3.07±0.10 mmol / L, respectively, indicating that the hyperlipidemia model was successfully established. Compared with the model group, the serum TC, TG, and LDL-C levels in mice in the ECAQKK and STEYGL groups were significantly decreased, while the HDL-C level was significantly increased (p<0.05). After intervention with ECAQKK and STEYGL, TC levels decreased by 19.3% and 14.3%, respectively; TG levels decreased by 23.2% and 21.7%, respectively; LDL-C levels decreased by 44.3% and 38.9%, respectively; and HDL-C levels increased by 22.8% and 18.6%, respectively. Furthermore, the two whey protein peptides showed comparable efficacy in improving dyslipidemia to the positive control drug simvastatin. This indicates that whey protein peptides ECAQKK and STEYGL can effectively improve high-fat diet-induced dyslipidemia, significantly improve lipid clearance efficiency, and have good lipid-lowering effects.

[0060] Excessive dietary cholesterol intake can promote the secretion of pro-inflammatory cytokines, induce systemic inflammatory responses, and consequently damage vascular endothelial function, increasing the risk of atherosclerotic cardiovascular disease, hypertension, diabetes, and other diseases. To investigate the effect of whey protein-derived cholesterol esterase inhibitory peptides on the inflammatory response in mice on a high-fat diet, the levels of several pro-inflammatory cytokines in serum were measured.

[0061] like Figure 6 As shown, compared with the blank control group, the serum levels of TNF-α, IL-6, IL-1β, and lipopolysaccharide (LPS) in the model group mice were significantly increased (p<0.05), indicating that a high-fat diet induces LPS entry into the blood, thereby activating the expression of pro-inflammatory factors. After intervention with whey protein peptides ECAQKK and STEYGL, the serum levels of inflammatory factors TNF-α, IL-6, IL-1β, and LPS in mice were significantly decreased (p<0.05). Furthermore, the effect of ECAQKK in reducing IL-6 and IL-1β, and the effect of STEYGL in reducing IL-6, were not significantly different from those of the positive control drug simvastatin (p>0.05). This indicates that whey protein peptides ECAQKK and STEYGL can improve the inflammatory state induced by hyperlipidemia by downregulating the levels of pro-inflammatory cytokines.

[0062] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important indicators for evaluating liver function, and their serum levels can directly reflect the degree of hepatocyte damage. When the liver is damaged, ALT and AST are released from hepatocytes into the blood, leading to elevated serum levels. To investigate the ameliorative effect of whey protein-derived cholesterol esterase inhibitory peptide on liver damage in mice fed a high-fat diet, the serum ALT and AST levels of mice were measured using a fully automated biochemical analyzer.

[0063] like Figure 7 As shown in Figures A and B, compared to the blank control group, the serum ALT and AST levels in the model group mice significantly increased from 42.00±3.51 U / L and 151.2±1.26 U / L to 179.53±21.24 U / L and 279.43±6.55 U / L, respectively (p<0.05), indicating that a high-fat diet causes hepatocellular damage, leading to impaired liver function. Compared with the model group, the serum ALT levels in the ECAQKK group and STEYGL group mice decreased by 56.5% and 54.0%, respectively, and the serum AST levels decreased by 29.6% and 27.2%, respectively. There was no significant difference in the effects of the two peptides (p>0.05).

[0064] As the central organ for lipid metabolism, abnormal accumulation of total cholesterol (TC) and triglycerides (TG) in the liver can induce hepatocyte steatosis, inflammatory damage, and even lead to liver fibrosis and cirrhosis. To investigate the effect of whey protein-derived cholesterol esterase inhibitory peptide on lipid metabolism in the liver of mice on a high-fat diet, the levels of TC and TG in the liver of mice were measured.

[0065] like Figure 7 As shown in C-D, compared with the blank control group, the liver TC and TG levels of mice in the model group were significantly increased (p<0.05). Compared with the model group, the liver TC and TG levels of mice in the ECAQKK and STEYGL groups were significantly decreased (p<0.05), with improvement effects comparable to those of the positive control drug simvastatin. Furthermore, the peptide ECAQKK was significantly more effective than the peptides STEYGL and simvastatin in reducing liver TC levels (p<0.05). These results indicate that whey protein peptides ECAQKK and STEYGL can significantly improve high-fat diet-induced liver injury and hepatic lipid accumulation, thereby contributing to the regulation of lipid metabolism.

[0066] The effects of whey protein-derived cholesterol esterase inhibitory peptide on the pathological structure of mouse liver were analyzed using H&E staining.

[0067] like Figure 8As shown in Figure A, compared with the blank control group, the hepatocytes of the model group mice were loosely and disordered, with a large number of fat vacuoles and obvious inflammatory cell infiltration, presenting typical pathological features of steatosis. After intervention with whey protein peptides ECAQKK and STEYGL, the morphology of mouse hepatocytes tended to be normal, with neat arrangement and obvious intercellular spaces; at the same time, the number of hepatic fat vacuoles was significantly reduced, and the inflammatory cell infiltration was significantly improved. The overall improvement effect was similar to that of the positive drug simvastatin.

[0068] Oil Red O is a lipid-soluble dye that specifically binds to neutral fats such as triglycerides in tissues and is commonly used to visualize lipid distribution within tissues. To evaluate the effect of whey protein-derived cholesterol esterase inhibitory peptide on lipid accumulation in the liver of mice with high-fat diet-induced hyperlipidemia, Oil Red O staining was used to analyze mouse liver sections.

[0069] like Figure 8 As shown in Figure B, compared with the blank control group, a large number of clustered red lipid droplets were observed in the liver slices of the model group mice. After intervention with whey protein peptides ECAQKK and STEYGL, the number of red lipid droplets in hepatocytes was significantly reduced. Among them, the peptide ECAQKK showed better effect in improving lipid accumulation, similar to the positive control drug simvastatin.

[0070] The above results indicate that whey protein peptides ECAQKK and STEYGL can alleviate high-fat diet-induced hepatic steatosis, inflammatory cell infiltration, and hepatic lipid accumulation.

[0071] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A sheep milk whey protein peptide, characterized in that, The goat milk whey protein peptides are ECAQKK and / or STEYGL.

2. A composition, characterized in that, The composition comprises the sheep milk whey protein peptide of claim 1.

3. A cholesterol esterase inhibitor, characterized in that, The cholesterol esterase inhibitor comprises the goat milk whey protein peptide of claim 1 or the composition of claim 2.

4. The method for preparing the goat milk whey protein peptide according to claim 1, characterized in that, The preparation method includes the following steps: S1, dissolve goat milk whey protein powder in water, adjust pH to alkaline, then add protease to hydrolyze and inactivate the enzyme, and adjust the hydrolysate to neutral; S2, after ultrafiltration of the enzymatic hydrolysate obtained in step S1, the ultrafiltrate is freeze-dried to obtain whey protein hydrolysate freeze-dried powder. S3, the whey protein hydrolysate lyophilized powder obtained in step S2 is separated and purified by gel chromatography, high performance liquid chromatography, and the whey protein peptide is obtained. S4. The whey protein peptides obtained in step S3 are sequenced.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass ratio of the goat milk whey protein powder to water is 1:10~25; and / or The amount of protease added is 2000~5000 U / g whey protein powder; and / or The enzymatic hydrolysis is performed at a temperature of 50-60°C for 1.5-3 hours; and / or The enzyme inactivation temperature is 80~90℃, and the time is 10~20 min.

6. The preparation method according to claim 4, characterized in that, In step S2, the molecular weight of the ultrafiltration membrane used in the ultrafiltration process is 3~8 kDa.

7. The preparation method according to claim 4, characterized in that, In step S3, the stationary phase of the gel chromatography is Sephadex G-10, Sephadex G-15, Bio-Gel P-2, or Toyopearl HW-40, the eluent is Tris-HCl, and the elution flow rate is 0.2~0.6 mL / min; and / or In step S3, the conditions for high performance liquid chromatography are as follows: C18 column; flow rate: 0.1~0.5 mL / min; elution program: the proportion of mobile phase B increases linearly from 0% to 80% within 100 min; mobile phase A is water containing 0.1% trifluoroacetic acid, and mobile phase B is a 60% acetonitrile aqueous solution containing 0.05% trifluoroacetic acid.

8. The use of the goat milk whey protein peptide of claim 1 or the composition of claim 2 in the preparation of products that help maintain healthy blood lipid levels.

9. The application according to claim 8, characterized in that, The application includes at least one of the following: (1) Application in the preparation of cholesterol esterase inhibitors; (2) Application in the preparation of products that can improve dyslipidemia; (3) Application in the preparation of products that can prevent or treat obesity; (4) Application in the preparation of products that can improve inflammatory responses caused by hyperlipidemia; (5) Application in the preparation of products that can improve liver damage and lipid accumulation in the liver caused by hyperlipidemia.

10. The application according to claim 8, characterized in that, The product is a pharmaceutical, health food, or animal feed.

Images