A polypeptide composition for assisting in reducing blood lipid and improving fatty liver and a preparation method and application thereof

Abstract

The present application provides a kind of polypeptide composition for assisting reducing blood fat and improving fatty liver and its preparation method and application, it is related to marine active peptide field.The polypeptide composition for assisting reducing blood fat and improving fatty liver of the present application includes the mass ratio 65-75:15-25:8-12 of phosphorus shrimp protein peptide, kudzu root oligopeptide, corn oligopeptide.The preparation method of the phosphorus shrimp protein peptide includes the following steps: taking phosphorus shrimp, grinding and pulping, to obtain phosphorus shrimp slurry;Add water to prepare protein suspension;Heat treatment;Adjust pH value, add complex protease constant temperature enzymolysis, the complex protease is composed of alkaline protease, neutral protease, flavour protease compound;Enzyme inactivation, centrifugation, collect supernatant;Membrane separation is carried out to supernatant;After vacuum concentration, spray drying is carried out, to obtain powder phosphorus shrimp protein peptide.The polypeptide composition of the present application has the effect of assisting reducing blood fat, can also improve fatty liver, and has good safety.

Description

Technical Field

[0001] This invention relates to the field of marine bioactive peptides, and in particular to a polypeptide composition for assisting in lowering blood lipids and improving fatty liver, as well as its preparation method and application. Background Technology

[0002] With the improvement of people's living standards and changes in dietary structure, the incidence of hyperlipidemia and fatty liver is increasing year by year, becoming a common chronic metabolic disease that seriously threatens human health. Hyperlipidemia is mainly characterized by elevated levels of serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and decreased levels of high-density lipoprotein cholesterol. It is an important risk factor for cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, and stroke. Fatty liver is a pathological change in the liver characterized by excessive accumulation of fat in hepatocytes. In recent years, the prevalence of non-alcoholic fatty liver disease has increased significantly, and some patients may further develop into steatohepatitis, liver fibrosis, or even cirrhosis.

[0003] Hyperlipidemia and fatty liver often coexist, influencing and promoting each other. Disorders of lipid metabolism can lead to increased fat uptake and synthesis in the liver, exacerbating fat accumulation; conversely, abnormal liver fat metabolism can further aggravate dyslipidemia, creating a vicious cycle. Therefore, regulating liver fat metabolism while intervening in hyperlipidemia is of great significance for the health management of relevant populations.

[0004] Currently, interventions for hyperlipidemia and fatty liver mainly include lifestyle interventions and drug therapy. Commonly used lipid-lowering drugs, such as statins and fibrates, can lower blood lipid levels to some extent, but long-term use may cause adverse reactions such as abnormal liver function and muscle damage, and are not suitable for individuals with simple fatty liver or mild dyslipidemia. There is currently no specific treatment for fatty liver; clinical treatment often employs comprehensive measures such as liver protection, lipid-lowering, and improvement of insulin resistance, but the efficacy is limited and certain side effects exist. Therefore, developing safe and effective natural functional ingredients or combinations to assist in lowering blood lipids and improving fatty liver has significant practical importance and broad market demand. Summary of the Invention

[0005] In view of this, the present invention proposes a polypeptide composition for assisting in lowering blood lipids and improving fatty liver, as well as its preparation method and application, to solve the above problems.

[0006] The technical solution of the present invention is as follows: a polypeptide composition for assisting in lowering blood lipids and improving fatty liver, comprising krill protein peptide, kudzu root oligopeptide and corn oligopeptide in a mass ratio of 65-75:15-25:8-12.

[0007] Furthermore, it includes krill protein peptides, kudzu root oligopeptides, and corn oligopeptides in a mass ratio of 70:20:10.

[0008] Furthermore, the preparation method of the krill protein peptide includes the following steps: (1) Take krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add water to the krill slurry, stir well, and make a protein suspension; (3) Heat treatment of protein suspension; (4) Adjust the pH value of the heated protein suspension and add a complex protease for isothermal enzymatic hydrolysis. The complex protease is composed of alkaline protease, neutral protease and flavor protease. (5) Inactivate enzymes, centrifuge, and collect the supernatant; (6) Perform membrane separation on the supernatant; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

[0009] Furthermore, the preparation method of the krill protein peptide includes the following steps: (1) Take krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add water to the krill slurry at a ratio of 1:8 to 1:20 by mass, stir well, and prepare a protein suspension; (3) Heat the protein suspension at 85-95℃ for 10-20 minutes; (4) Adjust the pH of the heated protein suspension to 6.5-8.5, add a complex protease at 45-58°C and hydrolyze for 2-6 hours. The complex protease is composed of alkaline protease, neutral protease and flavor protease in a mass ratio of (0.8-1.2):(0.8-1.2):(0.4-0.6). (5) Inactivate enzymes at 90-95℃ for 10-15 minutes, centrifuge, and collect the supernatant; (6) Perform membrane separation on the supernatant, with a molecular weight cutoff of 300-1000 Da; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

[0010] Furthermore, the amount of enzyme added is 1.0% to 3.0% of the weight of the protein suspension.

[0011] Furthermore, the molecular weight of the kudzu oligopeptide is ≤500 Da.

[0012] Furthermore, the molecular weight of the corn oligopeptide is 300~500 Da.

[0013] The application of the polypeptide composition of the present invention in the preparation of health food products that help lower blood lipids.

[0014] The application of the polypeptide composition of the present invention in the preparation of drugs that help lower blood lipids or improve fatty liver.

[0015] A product comprising the polypeptide composition of the present invention.

[0016] Furthermore, the product is a health food or a medicine.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention uses a compound polypeptide composition of krill protein peptides, kudzu root oligopeptides, and corn oligopeptides, which has the effect of assisting in lowering blood lipids, improving liver function, and has good safety.

[0018] (1) The polypeptide composition of the present invention is a combination of krill protein peptide, kudzu root oligopeptide and corn oligopeptide. The three work synergistically to regulate lipid metabolism in the body through multiple pathways and targets, and have a significant auxiliary effect in lowering blood lipids. Among them, the krill protein peptide of the present invention can reduce serum total cholesterol, triglycerides and low-density lipoprotein cholesterol levels, while helping to increase high-density lipoprotein cholesterol levels; kudzu root oligopeptide and corn oligopeptide further synergistically regulate liver fat metabolism, reduce liver lipid accumulation, thereby improving fatty liver and having a positive regulatory effect on liver function-related indicators.

[0019] (2) The polypeptide composition of the present invention has the dual function of assisting in lowering blood lipids and improving fatty liver. Compared with single active peptides or conventional compositions, its efficacy is more comprehensive and its effects are more synergistic. It can play a role in both blood lipid regulation and liver protection at the same time, and is suitable for daily nutritional intervention for people with hyperlipidemia and fatty liver.

[0020] (3) The krill protein peptides, kudzu root oligopeptides and corn oligopeptides used in this invention are all derived from natural food ingredients or food and medicine homologous raw materials. They have good safety, no obvious toxic side effects, are suitable for long-term use, and have good application prospects.

[0021] (4) In the preparation method of the krill protein peptide of the present invention, the pretreatment process such as defatting and shell removal can effectively remove impurities such as lipids and shells from the krill, reduce the fishy smell of the product, and improve the purity and quality of the protein peptide. The enzymatic hydrolysis is carried out by combining alkaline protease, neutral protease and flavor protease, which can take advantage of the differences in the cleavage sites of different proteases to improve the hydrolysis efficiency of krill protein and the yield of active peptides, so as to obtain krill protein peptides with suitable molecular weight and high biological activity.

[0022] (5) In the preparation method of the present invention, the supernatant is separated and purified by membrane separation technology after enzymatic hydrolysis, which can effectively enrich the polypeptide fragments with lipid-lowering activity, remove large molecular proteins and small molecular impurities, and improve the purity and bioavailability of the product; the subsequent vacuum concentration and spray drying process is beneficial to maintain the biological activity of the polypeptides, and the resulting krill protein peptide powder has good solubility and high stability, which is convenient for subsequent formulation processing and application. Detailed Implementation

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0024] The kudzu oligopeptide used in the embodiments of the present invention was purchased from Xi'an Yahe Pharmaceutical Co., Ltd., with a molecular weight ≤500 Da; the corn oligopeptide was purchased from Fufeng Sinote Biotechnology Co., Ltd., with a molecular weight distribution of 300-500 Da.

[0025] Example 1 A polypeptide composition for assisting in lowering blood lipids and improving fatty liver, comprising krill protein peptides, kudzu root oligopeptides, and corn oligopeptides in a mass ratio of 70:20:10.

[0026] The preparation method of the above-mentioned krill protein peptides includes the following steps: (1) Take 1 kg of krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add 12 kg of purified water to the krill slurry, stir well, and make a protein suspension; (3) Heat the protein suspension at 90°C for 15 minutes; (4) Adjust the pH of the heated protein suspension to 7.5, add a complex protease at 50°C and hydrolyze for 4 hours. The complex protease is composed of alkaline protease, neutral protease and flavor protease in a mass ratio of 1:1:0.5. The amount of enzyme added is 2.0% of the weight of the protein suspension. (5) Inactivate enzymes at 92℃ for 13 minutes, centrifuge, and collect the supernatant; (6) The supernatant was subjected to membrane separation with a molecular weight cutoff of 500 Da; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

[0027] Preparation of polypeptide composition: Krill protein peptide, kudzu root oligopeptide and corn oligopeptide are mixed evenly to obtain polypeptide composition.

[0028] Example 2 A polypeptide composition for assisting in lowering blood lipids and improving fatty liver, comprising krill protein peptides, kudzu root oligopeptides, and corn oligopeptides in a mass ratio of 65:25:10.

[0029] The preparation method of the above-mentioned krill protein peptides includes the following steps: (1) Take 1 kg of krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add 15 kg of purified water to the krill slurry, stir well, and make a protein suspension; (3) Heat the protein suspension at 85°C for 10 minutes; (4) Adjust the pH of the heated protein suspension to 7.8, add a complex protease at 58°C and hydrolyze for 6 hours. The complex protease is composed of alkaline protease, neutral protease and flavor protease in a mass ratio of 1.2:0.8:0.6. The amount of enzyme added is 2.5% of the weight of the protein suspension. (5) Inactivate enzymes at 90℃ for 15 minutes, centrifuge, and collect the supernatant; (6) The supernatant was subjected to membrane separation with a molecular weight cutoff of 1000 Da; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

[0030] Preparation of polypeptide composition: Krill protein peptide, kudzu root oligopeptide and corn oligopeptide are mixed evenly to obtain polypeptide composition.

[0031] Example 3 A polypeptide composition for assisting in lowering blood lipids and improving fatty liver, comprising krill protein peptides, kudzu root oligopeptides, and corn oligopeptides in a mass ratio of 75:15:10.

[0032] The preparation method of the above-mentioned krill protein peptides includes the following steps: (1) Take 1 kg of krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add 10 kg of purified water to the krill slurry, stir well, and make a protein suspension; (3) Heat the protein suspension at 95°C for 20 minutes; (4) Adjust the pH of the heated protein suspension to 8.5, add a complex protease at 45°C and hydrolyze for 2 hours. The complex protease is composed of alkaline protease, neutral protease and flavor protease in a mass ratio of 0.8:1.2:0.4, and the amount of enzyme added is 3.0% of the weight of the protein suspension. (5) Inactivate enzymes at 95℃ for 10 minutes, centrifuge, and collect the supernatant; (6) The supernatant was subjected to membrane separation with a molecular weight cutoff of 300 Da; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

[0033] Preparation of polypeptide composition: Krill protein peptide, kudzu root oligopeptide and corn oligopeptide are mixed evenly to obtain polypeptide composition.

[0034] Comparative Example 1 A polypeptide composition, mainly differing from Example 1 in that it does not contain kudzu oligopeptides, and instead consists of krill protein peptides and corn oligopeptides in a mass ratio of 70:10. Other treatments are the same as in Example 1.

[0035] Comparative Example 2 A polypeptide composition, mainly differing from Example 1 in that it does not contain corn oligopeptides, and instead consists of krill protein peptides and kudzu root oligopeptides in a mass ratio of 70:20. Other treatments are the same as in Example 1.

[0036] Comparative Example 3 A polypeptide composition, mainly differing from Example 1 in that the polypeptide composition consists of krill protein peptides, kudzu root oligopeptides, and corn oligopeptides in a mass ratio of 50:30:20. Other treatments are the same as in Example 1.

[0037] Comparative Example 4 A polypeptide composition, the main difference from Example 1, is that a single alkaline protease is used instead of a complex protease. Other treatments are the same as in Example 1.

[0038] Comparative Example 5 A polypeptide composition differs from Example 1 primarily in that the complex protease is composed of alkaline protease, neutral protease, and flavor protease in a mass ratio of 1:1:1. Other treatments are the same as in Example 1.

[0039] Experimental Example 1 1. Materials and Methods 1.1 Test samples: polypeptide compositions prepared in Examples 1-3 and Comparative Examples 1-5.

[0040] 1.2 Experimental animals: 100 healthy male SD rats, weighing between 180 and 220 g.

[0041] 1.3 High-fat feed formulation: Based on the basal maintenance feed, add 20.0% sucrose, 15.0% lard, 1.2% cholesterol, and 0.2% sodium cholate by mass, and supplement with appropriate amounts of casein, dicalcium phosphate, limestone powder, etc. Except for the crude fat content, the moisture, crude protein, crude fiber, crude ash, calcium, and phosphorus content of the model feed all meet the national standards for maintenance feed.

[0042] 1.4 Reagents and Instruments: Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) test kits were all purchased from Solarbio Biotechnology Co., Ltd.

[0043] 1.5 Dosage: The dosage for rats administered via gavage was 10 mg / (d·kg) body weight.

[0044] 1.6 Experimental methods: After 3 days of acclimatization feeding with a basic maintenance diet, rats were randomly divided into two groups according to their body weight: 10 rats were continuously fed the basic maintenance diet and served as the blank control group; the remaining 90 rats were fed a high-fat diet and served as the model preparation group.

[0045] After the model preparation group was fed a high-fat diet for 1-2 weeks, blood was collected from rats in the blank control group and the model preparation group without fasting. Serum was separated immediately after blood collection, and the serum levels of TC, TG, LDL-C and HDL-C were detected.

[0046] Based on serum TC levels, successfully modeled rats were randomly divided into a model control group and a model drug administration group. There were no statistically significant differences in TC, TG, LDL-C, and HDL-C levels between the two groups. The model control group consisted of 10 rats, and the model drug administration group consisted of 80 rats. Compared with the blank control group, the TC, TG, and LDL-C levels in both the model control group and the model drug administration group were significantly increased, while the HDL-C level was significantly decreased. The differences were statistically significant, indicating that the high-fat model was successfully established.

[0047] After grouping, the model drug administration group was further divided into 8 groups, with 10 rats in each group. Each group was administered the polypeptide compositions prepared in Examples 1-3 and Comparative Examples 1-5 by gavage daily. The blank control group and the model control group were administered an equal volume of purified water by gavage daily. During the experiment, the blank control group was continuously fed a maintenance diet, while the model control group and the model drug administration group were continuously fed a model diet. The rats' body weight was measured periodically. After 5 weeks of continuous administration, the experiment ended. Blood samples were collected from all rats without fasting. Serum was immediately separated after blood collection, and serum levels of TC (total cholesterol), TG (triglycerides), LDL-C (low-density lipoprotein), and HDL-C (high-density lipoprotein) were measured.

[0048] 2. Experimental Results 2.1 The effect of the polypeptide composition on total cholesterol (TC) in rats is shown in Table 1.

[0049] Table 1. Effects of the polypeptide composition on total cholesterol (TC) in rats.

[0050] Note: Compared with the blank control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0051] 2.2 The effect of the polypeptide composition on total cholesterol (TC) in rats is shown in Table 2.

[0052] Table 2. Effects of peptide composition on rat triglyceride (TG) levels.

[0053] Note: Compared with the blank control, ## P<0.01; compared with the model control, * P<0.05, ** P<0.01.

[0054] 2.3 The effects of the polypeptide composition on rat low-density lipoprotein LDL-C are shown in Table 3.

[0055] Table 3. Effects of peptide composition on rat low-density lipoprotein (LDL-C)

[0056] Note: Compared with the blank control, ## P<0.01; compared with the model control, * P<0.05, ** P<0.01.

[0057] 2.4 The effects of the polypeptide composition on rat high-density lipoprotein HDL-C are shown in Table 4.

[0058] Table 4. Effects of peptide composition on rat high-density lipoprotein (HDL-C)

[0059] Note: Compared with the blank control, ## P<0.01; compared with the model control, * P<0.05, ** P<0.01.

[0060] Compared to the blank control group, the model control group showed significantly increased serum TC, TG, and LDL-C, and significantly decreased HDL-C, indicating that the high-fat diet successfully induced hyperlipidemia. Compared to the model control group, the groups in Examples 1-3 showed significantly decreased TC, TG, and LDL-C, and increased HDL-C. These results demonstrate that the polypeptide composition of the present invention has an auxiliary lipid-lowering effect.

[0061] Experimental Example 2 A randomized, double-blind, controlled design was employed. One hundred and twenty patients with non-alcoholic fatty liver disease (NAFLD) meeting the criteria were enrolled, with a male-to-female ratio of 1:1 and an age range of 30–60 years. They were randomly assigned to either the experimental group or the control group, with 60 patients in each group. The experimental group received 20 g of the polypeptide composition described in Example 1 orally daily, divided into two doses, morning and evening. The control group received an equal amount of krill protein peptides (small molecule krill protein peptides, with a relative molecular mass mainly below 3000 Da, manufactured by Qingdao Antarctic Vicon Biotechnology Co., Ltd.). The intervention lasted for 12 weeks. Before and after the intervention, MRI-PDFF, ALT, AST, GGT, TG, HOMA-IR, and CAP were measured.

[0062] Among them, MRI-PDFF (Magnetic Resonance Proton Density Fat Fraction): Liver fat content is quantitatively assessed by calculating the ratio of fat proton signal to total water proton signal using the Dixon technique of liver magnetic resonance scanning, with units of %.

[0063] ALT (alanine aminotransferase): Fasting venous blood was collected, serum was separated, and measured using a fully automated biochemical analyzer by rate method / enzyme-coupled method. The unit is U / L.

[0064] AST (aspartate aminotransferase): Fasting venous blood was collected, serum was separated, and measured using a rate method with a fully automated biochemical analyzer. The unit is U / L.

[0065] TG (triglycerides): Fasting venous blood was collected, serum was separated, and the GPO-PAP enzymatic method was used for determination. The unit is mmol / L.

[0066] TC (Total Cholesterol): Fasting venous blood was collected, serum was separated, and the concentration was determined using the CHOD-PAP enzymatic method. The unit is mmol / L.

[0067] The results are shown in Table 5.

[0068] Table 5 Changes before and after intervention in the two groups

[0069] Note: Compared with before intervention, *P<0.05, **P<0.01; compared with the control group after intervention, #P<0.05, ##P<0.01.

[0070] No adverse events related to the test substance occurred during the entire experiment.

[0071] The results showed that after 12 weeks of intervention, the MRI-PDFF rate in the experimental group decreased from 18.6% to 11.2%, and in the control group from 18.3% to 15.9%, with a statistically significant difference between the groups (P<0.01). The proportion of patients in the experimental group with a ≥30% decrease in MRI-PDFF was 65.0%, significantly higher than that in the control group (P<0.01). The polypeptide composition of this invention can significantly reduce liver fat content and improve liver function in patients with non-alcoholic fatty liver disease, and is well-tolerated.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A polypeptide composition for assisting in lowering blood lipids and improving fatty liver, characterized in that, Including krill protein peptides, kudzu root oligopeptides, and corn oligopeptides with a mass ratio of 65-75:15-25:8-12.

2. The polypeptide composition for assisting in lowering blood lipids and improving fatty liver according to claim 1, characterized in that, It includes krill protein peptides, kudzu root oligopeptides, and corn oligopeptides in a mass ratio of 70:20:

10.

3. The polypeptide composition for assisting in lowering blood lipids and improving fatty liver according to claim 1, characterized in that, The preparation method of the krill protein peptide includes the following steps: (1) Take krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add water to the krill slurry, stir well, and make a protein suspension; (3) Heat treatment of protein suspension; (4) Adjust the pH value of the heated protein suspension and add a complex protease for isothermal enzymatic hydrolysis. The complex protease is composed of alkaline protease, neutral protease and flavor protease. (5) Inactivate enzymes, centrifuge, and collect the supernatant; (6) Perform membrane separation on the supernatant; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

4. The polypeptide composition for assisting in lowering blood lipids and improving fatty liver according to claim 1, characterized in that, The preparation method of the krill protein peptide includes the following steps: (1) Take krill, clean, remove impurities, defatted, shelled, crush and pulp to obtain krill pulp; (2) Add water to the krill slurry at a ratio of 1:8 to 1:20 by mass, stir well, and prepare a protein suspension; (3) Heat the protein suspension at 85-95℃ for 10-20 minutes; (4) Adjust the pH of the heated protein suspension to 6.5-8.5, and add a complex protease at 45-58°C for 2-6 hours of constant-temperature enzymatic hydrolysis. The complex protease is composed of alkaline protease, neutral protease, and flavor protease in a mass ratio of (0.8-1.2):(0.8-1.2):(0.4-0.6). The amount of enzyme added is 1.0%-3.0% of the weight of the protein suspension. (5) Inactivate enzymes at 90-95℃ for 10-15 minutes, centrifuge, and collect the supernatant; (6) Perform membrane separation on the supernatant, with a molecular weight cutoff of 300-1000 Da; (7) After vacuum concentration, spray drying is performed to obtain powdered krill protein peptides.

5. The polypeptide composition for assisting in lowering blood lipids and improving fatty liver according to claim 1, characterized in that, The molecular weight of the kudzu oligopeptide is ≤500 Da.

6. The polypeptide composition for assisting in lowering blood lipids and improving fatty liver according to claim 1, characterized in that, The molecular weight of the corn oligopeptide is 300~500 Da.

7. The use of the polypeptide composition according to any one of claims 1-6 in the preparation of health foods that help lower blood lipids.

8. The use of the polypeptide composition according to any one of claims 1-6 in the preparation of a drug for assisting in lowering blood lipids or improving fatty liver.

9. A product characterized in that, The product comprises the polypeptide composition according to any one of claims 1-6.

10. The product according to claim 9, characterized in that, The product is either a health food or a medicine.

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