Euphausia superba peptide with effects of reducing blood fat and protecting liver as well as preparation method and application of euphausia superba peptide

Abstract

The invention provides euphausia superba peptide or an euphausia superba peptide mixture as well as a preparation method and application thereof. Specifically, the euphausia superba peptide has an amino acid sequence as shown in any one of SEQ ID NO: 1-13 or a polypeptide of which the homology (or identity) with the amino acid sequence as shown in any one of SEQ ID NO: 1-13 is greater than or equal to 80%. The euphausia superba peptide or the euphausia superba peptide mixture disclosed by the invention has an excellent effect on treating and / or preventing related diseases of lipid metabolism disorder, and can be used for developing products such as medicines, health foods and special medical foods for the related diseases of the lipid metabolism disorder.

Description

Technical Field

[0001] The present invention relates to the field of functional polypeptides, and particularly to Antarctic krill peptides with lipid-lowering and liver-protecting effects, their preparation methods and applications. Background Art

[0002] Hyperlipidemia is a type of chronic disease mainly characterized by abnormal lipid metabolism. Currently, the commonly used lipid-lowering drugs in clinical practice are mainly statin drugs. Although statin drugs are generally safe, there are still quite a number of patients who will experience varying degrees of intolerance. Moreover, most patients are concerned about the adverse reactions of statins. Therefore, it is particularly important to clarify the key regulatory mechanisms of lipid metabolism in the body and discover safe and effective means and methods for regulating blood lipids.

[0003] Food and nutritional interventions have played an outstanding role in the prevention, management, treatment, and reversal of chronic diseases. The components of food-derived polypeptides are safe and have no side effects, and they have become a research and development hotspot in industries such as functional foods and health foods.

[0004] Therefore, there is an urgent need in this field to develop a food-derived polypeptide with lipid-lowering effects. Summary of the Invention

[0005] The purpose of the present invention is to provide a food-derived polypeptide for preventing and / or treating diseases related to lipid metabolism disorders and its preparation method.

[0006] In the first aspect of the present invention, a lipid-lowering Antarctic krill peptide, the Antarctic krill peptide is selected from the following groups:

[0007] (a) a polypeptide having the amino acid sequence shown in any one of SEQ ID NO: 1 to 13;

[0008] (b) a polypeptide having a homology (or identity) of ≥ 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% with the amino acid sequence shown in any one of SEQ ID NO: 1 to 13, and the polypeptide has the biological function shown in any one of SEQ ID NO: 1 - 13; or

[0009] (c) a derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 - 3, more preferably 1 - 2) amino acid residues to the amino acid sequence shown in any one of SEQ ID NO: 1 to 13, and retaining the biological function shown in any one of SEQ ID NO: 1 to 13.

[0010] In another preferred example, a polypeptide having the amino acid sequence shown in any one of SEQ ID NO: 1 to 5.

[0011] In another preferred example, the Antarctic krill peptide is composed of the amino acid sequence shown in any one of SEQ ID NO: 1-5.

[0012] In another preferred example, the Antarctic krill peptide binds to hydroxymethylglutaryl coenzyme A reductase.

[0013] In another preferred example, the binding energy of the Antarctic krill peptide to hydroxymethylglutaryl coenzyme A reductase is ≤ -7 kcal / mol, preferably ≤ -7.5 kcal / mol, more preferably ≤ -8 kcal / mol.

[0014] In another preferred example, the Antarctic krill peptide is prepared by the following method:

[0015] (s1) Provide an Antarctic krill solution; and

[0016] (s2) Add protease to the Antarctic krill solution and react to obtain the Antarctic krill peptide;

[0017] Wherein, the protease is selected from the following group: alkaline protease, neutral protease, flavor protease, or a combination thereof.

[0018] In the second aspect of the present invention, a method for preparing the Antarctic krill peptide as described in the first aspect of the present invention is provided, including the steps:

[0019] (s1) Provide an Antarctic krill solution; and

[0020] (s2) Add protease to the Antarctic krill solution and react to obtain the Antarctic krill peptide;

[0021] Wherein, the protease is selected from the following group: alkaline protease, neutral protease, flavor protease, or a combination thereof.

[0022] In another preferred example, the protease is derived from Bacillus or Aspergillus oryzae; preferably Bacillus licheniformis, Bacillus amyloliquefaciens, Aspergillus oryzae.

[0023] In another preferred example, the protease is selected from the following group: Novozymes neutral protease, Novozymes flavor protease, Novozymes alkaline protease, IFF alkaline protease, IFF neutral protease, Angel alkaline protease, or a combination thereof; preferably IFF alkaline protease and IFF neutral protease.

[0024] In another preferred example, the mass ratio of the protease to Antarctic krill is 0.001-10:100, preferably 0.005-5:100, more preferably 0.1-3:100.

[0025] In another preferred example, in step (s2), the reaction is carried out at 40 to 75 °C, preferably 45 to 70 °C, more preferably 50 to 65 °C, even more preferably 50 to 60 °C, for example, about 55 °C.

[0026] In another preferred example, the protease is added at 0.1% to 10% (w / w, based on the mass of Euphausia superba), preferably 0.5% to 5%, more preferably 1 to 3%, for example, about 2.2%, 1.4%.

[0027] In another preferred example, the mass ratio of the alkaline protease to the neutral protease is 1 to 5:1 to 5, preferably 1 to 3:1 to 3, more preferably 1 to 2:1 to 2, for example, about 1.2:1.

[0028] In another preferred example, 0.5% to 5% (w / w, based on the mass of Euphausia superba) of alkaline protease and 0.5% to 5% (w / w, based on the mass of Euphausia superba) of neutral protease are added; preferably 1% to 3% of alkaline protease and 0.5% to 3% of neutral protease; more preferably 1% to 3% of IFF alkaline protease and 0.5% to 3% of IFF neutral protease, for example, about 1.2% of alkaline protease and 1% of neutral protease.

[0029] In another preferred example, the pH value of the Euphausia superba solution is 7 to 10, preferably 7.5 to 9, more preferably 8 to 9, for example, about 8.5.

[0030] In another preferred example, in the Euphausia superba solution, the content of Euphausia superba is 50 to 500 mg / L, preferably 80 to 300 mg / L, more preferably 100 to 200 mg / L.

[0031] In another preferred example, the Euphausia superba solution is prepared from Euphausia superba powder.

[0032] In another preferred example, the Euphausia superba solution is a mixture of Euphausia superba powder and water, and the mass ratio of the material to the liquid is 1:5 to 10, preferably 1:6 to 9, more preferably 1:7 to 9.

[0033] In another preferred example, in step (s2), the reaction time is 3 to 8 h, preferably 4 to 7 h, more preferably 4 to 6 h.

[0034] In another preferred example, the method further includes step (s3) of inactivating the enzyme of the Euphausia superba peptide obtained in step (s2).

[0035] In another preferred example, in step (s3), the enzyme inactivation is carried out at 80 to 100 °C, preferably 80 to 95 °C, more preferably 80 to 90 °C, for example, about 85 °C.

[0036] In another preferred embodiment, in step (s3), the enzyme inactivation treatment is carried out for 1 to 10 minutes, preferably 3 to 8 minutes, more preferably 4 to 6 minutes, for example, about 5 minutes.

[0037] In another preferred embodiment, the method further includes step (s4) of defluorinating the enzyme-inactivated Antarctic krill peptides.

[0038] In another preferred embodiment, in step (s4), calcium hydroxide is used to defluorinate the Antarctic krill peptides, preferably 0.1 to 5% calcium hydroxide.

[0039] In the third aspect of the present invention, there is provided a mixture of Antarctic krill peptides, which contains the Antarctic krill peptides as described in the first aspect of the present invention.

[0040] In another preferred embodiment, the fluorine content in the mixture of Antarctic krill peptides is ≤ 5 mg / L, preferably ≤ 3 mg / L, more preferably ≤ 2.8 mg / L.

[0041] In another preferred embodiment, the total nitrogen content in the mixture of Antarctic krill peptides is 10 to 20 g / 100 g, preferably 12 to 18 g / 100 g, more preferably 13 to 16 g / 100 g.

[0042] In another preferred embodiment, the ash content of the mixture of Antarctic krill peptides is 1 to 10 g / 100 g, preferably 3 to 8 g / 100 g, more preferably 5 to 8 g / 100 g.

[0043] In another preferred embodiment, the mixture of Antarctic krill peptides does not contain inorganic arsenic.

[0044] In another preferred embodiment, the content of peptide segments with a molecular weight less than 180 Da in the mixture of Antarctic krill peptides is ≤ 20%, preferably ≤ 15%, more preferably ≤ 10%.

[0045] In another preferred embodiment, the content of peptide segments with a molecular weight of 180 to 1000 Da in the mixture of Antarctic krill peptides is ≥ 70%, preferably ≥ 75%, more preferably ≥ 80%.

[0046] In another preferred embodiment, the mixture of Antarctic krill peptides is in a liquid, solid or semi-solid state, and the solid is preferably in powder form.

[0047] In another preferred embodiment, the mixture of Antarctic krill peptides is prepared by the method described in claim 2.

[0048] In the fourth aspect of the present invention, there is provided the use of the Antarctic krill peptides as described in the first aspect of the present invention or the mixture of Antarctic krill peptides as described in the third aspect of the present invention for preparing a drug, a pharmaceutical composition, a special medical food, or a health food for preventing and / or treating diseases selected from the following group;

[0049] (a) Diseases related to lipid metabolism disorders;

[0050] (b) Liver injury.

[0051] In another preferred embodiment, the diseases related to lipid metabolism disorders are selected from the group consisting of: hyperlipidemia, obesity, fatty liver, hepatomegaly, hypertension, coronary heart disease, pancreatitis, hyperglycemia, or a combination thereof.

[0052] In another preferred embodiment, the diseases related to lipid metabolism disorders are caused by a high-fat diet.

[0053] In another preferred embodiment, the liver injury includes acute hepatitis, chronic hepatitis, or fatty liver.

[0054] In another preferred embodiment, the liver injury is caused by a drug, preferably CCl 4 .

[0055] In another preferred embodiment, the drug or pharmaceutical composition is also used for the following:

[0056] (y1) Slowing down the increase in body weight and / or body fat;

[0057] (y2) Reducing the increase in liver and / or fat weight;

[0058] (y3) Improving abnormal serum lipid levels.

[0059] In another preferred embodiment, the abnormal serum lipid levels are selected from the group consisting of: elevated serum triglyceride content, elevated total cholesterol content, reduced high-density lipoprotein content, elevated low-density lipoprotein content, or a combination thereof.

[0060] In another preferred embodiment, the drug or pharmaceutical composition is administered to a subject having the following characteristics:

[0061] (x1) Suffering from hyperlipidemia or at risk of suffering from hyperlipidemia;

[0062] (x2) High-fat diet;

[0063] (x3) Abnormal lipid levels in the serum.

[0064] In another preferred embodiment, the elevated triglyceride content means that the content X1 of triglyceride in the serum of the subject compared with the content X0 of triglyceride in the serum of healthy people ((X1 - X0) / X0) ≥ 20%, preferably ≥ 30%, more preferably ≥ 50%.

[0065] In another preferred embodiment, the increase in total cholesterol content means that the content of total cholesterol Y1 in the serum of the subject compared to the content of total cholesterol Y0 in the serum of a healthy population ((Y1 - Y0) / Y0) ≥ 50%, preferably ≥ 80%, more preferably ≥ 100%.

[0066] In another preferred embodiment, the decrease in high-density lipoprotein content means that the content of high-density lipoprotein N1 in the serum of the subject compared to the content of high-density lipoprotein N0 in the serum of a healthy population ((N0 - N1) / N0) ≥ 30%, preferably ≥ 40%, more preferably ≥ 50%.

[0067] In another preferred embodiment, the increase in low-density lipoprotein content means that the content of low-density lipoprotein M1 in the serum of the subject compared to the content of low-density lipoprotein M0 in the serum of a healthy population ((M1 - M0) / M0) ≥ 50%, preferably ≥ 80%, more preferably ≥ 100%.

[0068] In another preferred embodiment, the dosage of the drug or drug composition is 100 - 2000 mg / kg in terms of Antarctic krill peptide, preferably 100 - 1500 mg / kg, more preferably 200 - 1000 mg / kg. For example, about 300 mg / kg or 800 mg / kg.

[0069] In the fifth aspect of the present invention, there is provided a drug or drug composition for preventing and / or treating lipid metabolism disorder-related diseases and / or liver injury, comprising

[0070] (a) Antarctic krill peptide as described in the first aspect of the present invention or Antarctic krill peptide mixture as described in the third aspect of the present invention; and

[0071] (b) a pharmaceutically acceptable carrier.

[0072] In another preferred embodiment, the lipid metabolism disorder-related diseases are selected from the group consisting of: hyperlipidemia, obesity, fatty liver, hepatomegaly, hypertension, coronary heart disease, pancreatitis, hyperglycemia, or a combination thereof.

[0073] In another preferred embodiment, the lipid metabolism disorder-related diseases are caused by a high-fat diet.

[0074] In the sixth aspect of the present invention, there is provided a method for reducing cellular triglyceride, comprising the steps of:

[0075] Contacting Antarctic krill peptide as described in the first aspect of the present invention or Antarctic krill peptide mixture as described in the third aspect of the present invention with cells, thereby reducing the triglyceride in the cells.

[0076] In another preferred embodiment, the cells include hepatocytes, preferably AML12, Huh7, HepG2, or a combination thereof.

[0077] In another preferred embodiment, the concentration of the Antarctic krill peptide is 1 to 500 μM, preferably 10 to 300 μM, more preferably 20 to 200 μM.

[0078] In another preferred embodiment, the triglyceride expression level of the cell is high.

[0079] In another preferred embodiment, the high triglyceride expression level of the cell is induced by oleic acid.

[0080] In another preferred embodiment, the high triglyceride expression level means that the content Z1 of triglyceride in the cell compared with the cell Z0 not induced by oleic acid ((Z1 - Z0) / Z0) ≥ 50%, preferably ≥ 80%, more preferably ≥ 100%.

[0081] In another preferred embodiment, the method is in vitro.

[0082] In another preferred embodiment, the method is obtained non-diagnostically and non-therapeutically.

[0083] In the seventh aspect of the present invention, there is provided a polynucleotide encoding the Antarctic krill peptide as described in the first aspect of the present invention.

[0084] In the eighth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the seventh aspect of the present invention.

[0085] In the ninth aspect of the present invention, there is provided a host cell containing the vector as described in the eighth aspect of the present invention or having the polynucleotide as described in the seventh aspect of the present invention integrated into its genome.

[0086] In the tenth aspect of the present invention, there is provided a method for preventing and / or treating diseases related to lipid metabolism disorders, comprising the steps of:

[0087] Administering to a subject in need a prophylactically or therapeutically effective amount of the Antarctic krill peptide as described in the first aspect of the present invention or the Antarctic krill peptide mixture as described in the third aspect of the present invention, thereby preventing and / or treating diseases related to lipid metabolism disorders.

[0088] In another preferred embodiment, the diseases related to lipid metabolism disorders are selected from the group consisting of: hyperlipidemia, obesity, fatty liver, hepatomegaly, hypertension, coronary heart disease, pancreatitis, hyperglycemia, or a combination thereof.

[0089] In another preferred embodiment, the diseases related to lipid metabolism disorders are caused by a high-fat diet.

[0090] In another preferred embodiment, the dosage of the Antarctic krill peptide is 100-2000 mg / kg, preferably 100-1500 mg / kg, more preferably 200-1000 mg / kg. For example, it is about 300 mg / kg or 800 mg / kg.

[0091] In another preferred embodiment, the subject has the following characteristics selected from the group:

[0092] (x1) suffering from hyperlipidemia or at risk of suffering from hyperlipidemia;

[0093] (x2) having a high-fat diet;

[0094] (x3) having abnormal lipid levels in the serum.

[0095] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 Shows the comparison of the molecular weights of Antarctic krill peptides under different enzymatic hydrolysis schemes.

[0097] Figure 2 Shows that Antarctic krill peptides can control the increase in triglyceride levels in oleic acid-induced cells. A: The content of triglycerides after Antarctic krill peptide intervention in oleic acid-induced AML12 cells; B: The content of triglycerides after Antarctic krill peptide intervention in oleic acid-induced Huh7 cells; C: The content of triglycerides after Antarctic krill peptide intervention in oleic acid-induced HepG2 cells. Data are expressed as mean ± SEM, n = 5, #P<0.05, ##P<0.01, P<0.001 represent significant differences compared with the normal diet group, *P<0.05, **P<0.01, ***P<0.001, represent significant differences compared with the high-fat model group.

[0098] Figure 3 Shows that high-dose T12 Antarctic krill peptides slow down the increase in body weight and body fat induced by a high-fat diet. The changes in body weight (A) and the percentage of body weight change (B) of rats after 6 weeks of different interventions; liver weight (C) and its ratio to body weight (D); epididymal fat weight (E); white fat weight (F). Data are expressed as mean ± SEM, n = 5. #P<0.05, ##P<0.01, P<0.001 represent significant differences compared with the normal diet group, *P<0.05, **P<0.01, ***P<0.001, represent significant differences compared with the high-fat model group.

[0099] Figure 4It shows the lipid accumulation in hyperlipidemic rats induced by high-fat diet controlled by Antarctic krill peptides.

[0100] Figure 5 It shows that high-dose T12 Antarctic krill peptides can control the abnormal lipid levels in the serum of obese rats induced by high-fat diet. A: Triglyceride in rat serum; B: Total cholesterol in rat serum; C: High-density lipoprotein cholesterol in rat serum; D: Low-density lipoprotein cholesterol in rat serum; E: Ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol in rat serum. Data are expressed as mean ± SEM, n = 5, #P < 0.05, ##P < 0.01, P < 0.001 represent significant differences compared with the normal diet group, *P < 0.05, **P < 0.01, ***P < 0.001 represent significant differences compared with the high-fat model group.

[0101] Figure 6 It shows that T12 Antarctic krill peptides alleviate CCl 4 -induced liver injury in mice. A: ALT levels in each group of mice after 4 weeks of different interventions; B: AST levels in each group of mice after 4 weeks of different interventions; C: H&E staining and Sirius red staining pictures of mouse liver sections. Data are expressed as mean ± SEM, n = 3 - 4. #P < 0.05 represents significant differences compared with the normal group, *P < 0.05 represents significant differences compared with the model group. Detailed implementation mode

[0102] Through extensive and in-depth research, a large number of experiments and screenings, the present inventors unexpectedly discovered for the first time a lipid-lowering polypeptide and polypeptide mixture derived from Antarctic krill, and the polypeptide has an amino acid sequence shown in any one of SEQ ID NO: 1 - 13. Cell experiments show that Antarctic krill peptides significantly improve triglyceride accumulation induced by oleic acid. Animal experiments show that Antarctic krill peptides improve in vivo fat accumulation, serum lipid abnormalities, liver enlargement and control diet-induced weight gain, etc. The Antarctic krill peptides of the present invention have excellent effects in the treatment and / or prevention of related diseases of lipid metabolism disorders. On this basis, the present invention is completed.

[0103] Terms

[0104] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0105] As used herein, the term "comprising" or variations thereof such as "including" or "having" etc. are understood to include the stated element or component without excluding other elements or other components.

[0106] The term "about" may refer to a value or a component within an acceptable error range of a specific value or component determined by those of ordinary skill in the art, which will depend in part on how the value or component is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0107] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the stated range and, where appropriate, fractional values thereof (e.g., one-tenth and one-hundredth of an integer).

[0108] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0109] As used herein, the terms "oleic acid" and "OA" may be used interchangeably.

[0110] As used herein, the terms "atorvastatin" and "Ator" may be used interchangeably.

[0111] "Identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. "Identity" represents the percentage of the number of identical residues between the polypeptide or nucleic acid sequences out of the total number of residues, and the calculation of the total number of residues is determined based on the type of mutation. The types of mutations include insertions (extensions) at either or both ends of the sequence, deletions (truncations) at either or both ends of the sequence, substitutions / replacements of one or more amino acids / nucleotides, insertions within the sequence, and deletions within the sequence.

[0112] Taking a polypeptide sequence as an example, if the mutation type is one or more of the following: substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence, the total number of residues is calculated based on the larger of the two molecules being compared. If the mutation type also includes insertion (extension) at either or both ends of the sequence or deletion (truncation) at either or both ends of the sequence, the number of amino acids inserted or deleted at either or both ends (e.g., the number inserted or deleted at both ends is less than 20) is not included in the total number of residues. When calculating the percentage identity, the sequences being compared are aligned in a way that produces the maximum match between the sequences, and gaps in the alignment (if any) are resolved by a specific algorithm.

[0113] Protease

[0114] The protease of the present invention is used to enzymatically hydrolyze Antarctic krill solution, thereby producing the Antarctic krill peptides of the present invention. The protease of the present invention is selected from the following group: alkaline protease, neutral protease, flavor protease, or a combination thereof.

[0115] In a preferred embodiment, the protease is derived from Bacillus or Aspergillus oryzae; preferably Bacillus licheniformis, Bacillus amyloliquefaciens, Aspergillus oryzae.

[0116] In a preferred embodiment, the protease is selected from the following group: Novozymes neutral protease, Novozymes flavor protease, Novozymes alkaline protease, IFF alkaline protease, IFF neutral protease, Angel alkaline protease, or a combination thereof; preferably IFF alkaline protease and IFF neutral protease.

[0117] Angel alkaline protein is an enzyme preparation refined from Bacillus licheniformis through deep liquid fermentation, extraction, multiple filtrations, concentration, and purification. IFF alkaline protease is a protease fermented from Bacillus licheniformis. Novozymes alkaline protease is produced by fermentation of Bacillus licheniformis. Novozymes flavor protease is produced by fermentation of Aspergillus oryzae. Novozymes neutral protease is produced by fermentation of Bacillus amyloliquefaciens.

[0118] The Antarctic krill peptides of the present invention

[0119] As used herein, the terms "polypeptides of the present invention" and "Antarctic krill peptides of the present invention" are used interchangeably.

[0120] The Antarctic krill peptides of the present invention are selected from the following group:

[0121] A polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13;

[0122] (b) A polypeptide having a homology (or identity) of ≥ 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13, and the polypeptide having the biological function shown in any one of SEQ ID NOs: 1 - 13;

[0123] (c) A derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 - 3, more preferably 1 - 2) amino acid residues to the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13, and retaining the biological function shown in any one of SEQ ID NOs: 1 to 13.

[0124] The yield of Antarctic krill is about 1 billion tons, and it has the largest animal genome so far. Antarctic krill protein contains sufficient essential amino acids, and has characteristics such as high digestibility and high biological value, and can fully support the growth and development of the human body. Antarctic krill peptides have various biological activities such as significant antioxidant activity, blood pressure - lowering activity, blood sugar - lowering activity, DPP - IV inhibitory activity, alleviating joint inflammation, and protecting the liver. These activities are related to the diversity of the peptide chain structure and can play regulatory roles in different biological processes.

[0125] Those skilled in the art are aware of examples and embodiments of conservative amino acid substitutions. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non - polar amino acid residue is replaced with another non - polar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. As long as the substitution does not result in the inactivation of the biological activity of the polypeptide, conservative substitutions in which one amino acid is replaced with another amino acid belonging to the same group fall within the scope of the present invention. Therefore, the polypeptides of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably made according to Table 1. In addition, the present invention also encompasses polypeptides that further contain one or more other non - conservative substitutions, as long as the non - conservative substitutions do not significantly affect the required functions and biological activities of the polypeptides of the present invention.

[0126] Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. A "non-essential" amino acid residue is an amino acid residue that can be altered (deleted, substituted, or replaced) without changing biological activity, while an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions can be made in non-conserved regions of the Cas enzyme. Generally, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs, where such residues are required for protein activity.

[0127] In certain embodiments, a selected group of amino acids that are considered to be conservative substitutions for each other:

[0128] Initial residue Representative substitution Preferred substitution Ala(A) Val(V); Leu(L); Ile(I) Val(V) Arg(R) Lys(K); Gln(Q); Asn(N) Lys(K) Asn(N) Gln(Q); His(H); Lys(K); Arg(R) Gln(Q) Asp(D) Glu(E) Glu(E) Cys(C) Ser(S) Ser(S) Gln(Q) Asn(N) Asn(N) Glu(E) Asp(D) Asp(D) Gly(G) Pro(P); Ala(A) Ala(A) His(H) Asn(N); Gln(Q); Lys(K); Arg(R) Arg(R) Ile(I) Leu(L); Val(V); Met(M); Ala(A); Phe(F) Leu(L) Leu(L) Ile(I); Val(V); Met(M); Ala(A); Phe(F) Ile(I) Lys(K) Arg(R); Gln(Q); Asn(N) Arg(R) Met(M) Leu(L); Phe(F); Ile(I) Leu(L) Phe(F) Leu(L); Val; Ile(I); Ala(A); Tyr(Y) Leu(L) Pro(P) Ala(A) Ala(A) Ser(S) Thr(T) Thr(T) Thr(T) Ser(S) Ser(S) Trp(W) Tyr(Y); Phe(F) Tyr(Y) Tyr(Y) Trp(W); Phe(F); Thr(T); Ser(S) Phe(F) Val(V) Ile(I); Leu(L); Met(M); Phe(F); Ala(A) Leu(L)

[0129] The Euphausia superba peptides of the present invention can be artificially synthesized by chemical synthesis methods, biosynthesis methods, etc., preferably solid-phase synthesis method, liquid-phase synthesis method, stepwise synthesis method, enzymatic synthesis method, gene expression method, artificial grafting method, enzymatic hydrolysis method, etc.

[0130] Preparation method of the Euphausia superba peptide of the present invention

[0131] The preparation method of the Euphausia superba peptide of the present invention includes the steps:

[0132] (s1) Providing a Euphausia superba solution; and

[0133] (s2) Adding a protease to the Euphausia superba solution and reacting to obtain the Euphausia superba peptide;

[0134] Wherein, the protease is selected from the group consisting of: alkaline protease, neutral protease, flavor protease, or a combination thereof.

[0135] In a preferred embodiment, the mass ratio of the protease to Euphausia superba is 0.01-10:100, preferably 0.05-5:100, more preferably 0.1-3:100. In a preferred embodiment, the protease is added at 0.01%-10% (by the mass of Euphausia superba), preferably 0.1%-5%, more preferably 0.2-3%.

[0136] In a preferred embodiment, in step (s2), the reaction is carried out at 40-75°C, preferably 45-70°C, more preferably 50-65°C, even more preferably 50-60°C, for example, about 55°C. In a preferred embodiment, in step (s2), the reaction time is 3-8 h, preferably 4-7 h, more preferably 4-6 h.

[0137] In a preferred embodiment, in the Antarctic krill solution, the content of Antarctic krill is 50 - 500 mg / L, preferably 80 - 300 mg / L, more preferably 100 - 200 mg / L. In a preferred embodiment, the Antarctic krill solution is prepared from Antarctic krill powder. In a preferred embodiment, the Antarctic krill solution is a mixture of Antarctic krill powder and water, and the mass ratio of the material to the liquid is 1:5 - 10, preferably 1:6 - 9, more preferably 1:7 - 9.

[0138] In a preferred embodiment, the method further includes step (s3) of inactivating the enzyme of the Antarctic krill peptide obtained in step (s2). In a preferred embodiment, the method further includes step (s4) of defluorinating the inactivated Antarctic krill peptide.

[0139] The Antarctic krill peptide mixture containing the Antarctic krill peptide of the present invention can be prepared by using the method of the present invention. In the Antarctic krill peptide mixture, the fluorine content ≤ 5 mg / L, preferably ≤ 3 mg / L, more preferably ≤ 2.8 mg / L; the content of peptide segments less than 180 Da ≤ 20%, preferably ≤ 15%, more preferably ≤ 10%; the content of peptide segments of 180 - 1000 Da ≥ 70%, preferably ≥ 75%, more preferably ≥ 80%.

[0140] The main advantages of the present invention include:

[0141] 1. The Antarctic krill peptide or Antarctic krill peptide mixture of the present invention has excellent effects in treating diseases related to lipid metabolism disorders, especially in slowing down weight and body fat gain, improving abnormal serum lipid levels, and controlling hepatomegaly.

[0142] 2. The Antarctic krill peptide or Antarctic krill peptide mixture of the present invention has excellent effects in treating liver injury, can increase the contents of ALT and AST in the serum, and reduce liver tissue injury.

[0143] 3. The Antarctic krill peptide of the present invention has a strong interaction with hydroxymethylglutaryl coenzyme A reductase (HMGCR), has the same mechanism of action as atorvastatin, and affects the synthesis of cholesterol in the body by inhibiting the activity of HMGCR.

[0144] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0145] Materials and Instruments

[0146] Male Sprague-Dawley rats at 6-8 weeks of age were purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0147] Defatted krill powder was purchased from Shandong Luhua Marine Biotechnology Co., Ltd. Alkaline protease 2.4L, neutral protease 0.8L, and flavor protease 1000L were purchased from Novozymes (China) Biotechnology Co., Ltd.; alkaline protease AP-200A was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.; alkaline protease FoodPro AP and neutral protease FoodPro PNL were purchased from International Flavors & Fragrances (China) Co., Ltd. - IFF. 60% kcal high-fat diet was purchased from Research Diets. Triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol detection kits (Nanjing Jiancheng Bioengineering Institute).

[0148] High-speed multi-functional pulverizer (2500Y) was purchased from Yongkang Boou Hardware Products Co., Ltd.; multi-functional microplate reader (Various Flash) was purchased from Thermo Fisher Scientific Co., Ltd.; ultrasonic instrument (SB25-12DT) was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; TSKgel G2000SWXL gel filtration chromatography column was purchased from Tosoh Bioscience (Shanghai) Co., Ltd.; high-performance liquid chromatography (Ultimate 3000) was purchased from Thermo Fisher Scientific Co., Ltd.; high-speed refrigerated centrifuge (Centrifuge 5424) was purchased from Eppendorf (China) Co., Ltd.

[0149] Example 1 Preparation of Antarctic Krill Peptide

[0150] The defatted Antarctic krill powder was pulverized into fine powder by a pulverizer, and the Antarctic krill powder was dissolved in deionized water at a material-liquid mass ratio of 1:8. The pH of the Antarctic krill solution was adjusted to 8.5 using 0.1 mol / L hydrochloric acid. Protease was added according to Table 1, the enzymatic hydrolysis temperature was controlled at 55 °C, the enzymatic hydrolysis time was 5 h, and the solution was stirred evenly during enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzymatic hydrolysis product was inactivated at 85 °C for 5 min. After the enzymatic hydrolysis product was cooled to room temperature, it was centrifuged at 4000 rpm for 20 min. After centrifugation, the supernatant was taken, and the pH of the supernatant was adjusted to 7 with phosphoric acid. 0.5% calcium hydroxide (based on the mass of the supernatant) was added to the supernatant for defluorination treatment at 55 °C for 1 h, and the solution was stirred evenly during defluorination. The defluorinated product was centrifuged at 4000 rpm for 15 min. The defluorinated supernatant (i.e., the hydrolyzed oligopeptide solution) was spray-dried to obtain powdered krill peptide.

[0151] Table 1 Design of the type and addition amount of enzymes in each group:

[0152]

[0153]

[0154] Example 2 Determination of molecular weight distribution and fluorine content of krill oil peptide solution

[0155] 2.1 Method

[0156] According to Appendix A of GB / T 22729-2008, a method for determining the molecular weight distribution of protein peptides (high performance gel filtration chromatography) was established. 1 ml of the Antarctic krill peptide solution prepared in Example 1 was filtered through a 0.22 μm filter membrane and then injected. The injection volume each time was 10 μl, and the molecular weight distribution of krill peptide was obtained after calculation. According to the third method (fluoride ion selective electrode method) of GB 5009.18 / -2003, a fluoride ion electrode was used to determine the fluorine content in Antarctic krill peptide. According to the first method of GB 5009.5-2016, the total nitrogen content in Antarctic krill peptide was detected; according to the first method of GB 5009.4-2016, the ash content in Antarctic krill peptide was detected; according to the first method in the second part of GB 5009.11-2014, the inorganic arsenic content in Antarctic krill peptide was detected.

[0157] 2.2 Results

[0158] Table 2 Molecular weight distribution of Antarctic krill peptide under different enzymatic hydrolysis conditions

[0159] Enzymolysis conditions > 1000 Da 400 - 1000 Da 180 - 400 Da < 180 Da T10 9.81 33.74 42.54 13.91 T12 8.89 38.67 44.75 7.69 T18 11.59 39.19 40.82 8.4 T19 8.72 33.05 49.26 8.97

[0160] Figure 1Table 2 shows the molecular weight distribution of Antarctic krill peptides prepared under the enzymolysis conditions of T10, T12, T18, and T19. Bioactive peptides with a molecular weight of 180 - 1000 Da may have higher bioavailability and biological activity. The proportion of peptides with a molecular weight of 180 - 1000 Da in the T12, T18, and T19 Antarctic krill peptide samples is higher, while the proportion of peptides with a molecular weight less than 180 Da in the T10 Antarctic krill peptide sample is higher, indicating that the T10 sample contains more free amino acids and a lower polypeptide proportion than the other three samples. Therefore, the enzymolysis conditions of T12, T18, and T19 were selected for subsequent research.

[0161] Table 3 Determination results of the physicochemical properties of T12 Antarctic krill peptides

[0162]

[0163] As shown in Table 3, the fluorine content in Antarctic krill peptides before defluorination was 117.9 mg / L, and after defluorination, it was 2.62 mg / L. The defluorination rate reached over 97%, and the fluorine content had reached the safety standard. The total nitrogen content in T12 krill peptides was 14.4 g / 100 g. After calculation, the protein content was approximately 90%. The ash content was 6.7 g / 100 g, and inorganic arsenic was not detected.

[0164] Example 3 Antarctic krill peptides can control the increase in triglyceride levels in oleic acid-induced cells

[0165] 3.1 Method

[0166] AML12, Huh7, and HepG2 cells were divided into 11 groups (Table 4) and cultured in high-glucose DMEM medium. The cells in Group 1 were not intervened, and the cells in Groups 2 - 12 were modeled with oleic acid (OA). Among them, on the second day after modeling, different intervention treatments were carried out on Groups 3 - 12: the cells in Groups 3 - 11 were respectively given high, medium, and low doses of T12, T18, and T19 krill peptide solutions, and the cells in Group 12 were given atorvastatin as a positive control. A triglyceride kit was used to detect the triglyceride content in each group of cells.

[0167] Table 4 Cell grouping and intervention plan

[0168] Group number Grouping Intervention plan 1 Normal group None 2 High-fat model group 500 μM OA 3 Low-dose T12 krill peptide group 500 μM OA + 50 μM T12 krill peptide 4 Medium-dose T12 krill peptide group 500 μM OA + 100 μM T12 krill peptide 5 High-dose T12 krill peptide group 500 μM OA + 200 μM T12 krill peptide 6 Low-dose T18 krill peptide group 500 μM OA + 50 μM T18 krill peptide 7 Medium-dose T18 krill peptide group 500 μM OA + 100 μM T18 krill peptide 8 High-dose T18 krill peptide group 500 μM OA + 200 μM T18 krill peptide 9 Low-dose T19 krill peptide group 500 μM OA + 50 μM T19 krill peptide 10 Medium-dose T19 krill peptide group 500 μM OA + 100 μM T19 krill peptide 11 High-dose T19 krill peptide group 500 μM OA + 200 μM T19 krill peptide 12 Atorvastatin group 500 μM OA + 20 μM atorvastatin

[0169] 3.2 Results

[0170] In the three cell lines, the triglyceride content in the high-fat model group cells was significantly higher than that in the normal group. In the AML12 cell line, all krill peptide intervention groups significantly controlled the increase in triglyceride levels in oleic acid-induced cells, and the high-dose krill peptide group had the best control effect ( Figure 2In Huh7 cell line, except for the medium-dose T19 krill peptide group, the other krill peptide intervention groups significantly controlled the increase in triglyceride levels in oleic acid-induced cells ( Figure 2 In B). In HepG2 cell line, all krill peptide intervention groups significantly controlled the increase in triglyceride levels in oleic acid-induced cells ( Figure 2 In C). This indicates that Antarctic krill peptide can control the increase in triglyceride levels in oleic acid-induced cells.

[0171] Example 4 Hypolipidemic function test of Antarctic krill peptide

[0172] 4.1 Method

[0173] After 1 week of adaptive feeding, the rats were randomly divided into 9 groups (Table 5). The rats in Group 1 were given normal feed, and the rats in Groups 2-9 were given 60% high-fat feed for 6 weeks. Among them, different gavage treatments were carried out on Groups 1-9 for 4 weeks starting from the 2nd week: the rats in Groups 1-2 were gavaged with normal saline daily, the rats in Groups 3-8 were gavaged with high- and low-dose T12, T18, and T19 krill peptide solutions daily, and the rats in Group 9 were gavaged with atorvastatin as a positive control.

[0174] Table 5 Rat grouping and intervention plan

[0175]

[0176]

[0177] The body weight, liver, epididymal fat, and serum indicators of the mice were detected:

[0178] (1) The body weight of the mice was measured every week, and the data was recorded.

[0179] (2) After anesthesia with excessive isoflurane, blood was collected from the apex of the heart into an anticoagulant tube. Centrifuge at 3000 r / min for 15 min at 4°C, take the supernatant, and store it at -80°C. The liver and epididymal fat of the rats were taken and weighed, and stored at -80°C.

[0180] (3) The total cholesterol content in the serum of the rats was measured using triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol detection kits.

[0181] 4.2 Results

[0182] (1) High-dose T12 Antarctic krill peptide slowed down the increase in body weight and body fat in rats induced by high-fat diet

[0183] The weight changes in the high-dose T19 krill peptide, high-dose T18 krill peptide, and high-dose T12 krill peptide groups were higher than those in other groups throughout the intervention period. The weight changes in the low-dose T12 krill peptide, low-dose T18 krill peptide, and atorvastatin groups were slightly lower than those in the high-fat model group throughout the intervention period and were at a medium level. The overall weight change in the high-dose T12 krill peptide group was significantly lower than that in the high-fat model group throughout the intervention period( Figure 3 in Figures A and B).

[0184] In summary, high-dose T12 krill peptide intervention can control the weight gain of rats fed a high-fat diet.

[0185] (2) High-dose T12 Antarctic krill peptide controls the increase in rat liver weight, liver index, and fat weight

[0186] After 6 weeks of intervention, the rats were sacrificed for organ index analysis. The results showed that the liver weight and liver index (the percentage of liver weight to body weight) in the high-fat model group were significantly higher than those in the normal diet group; the high-dose T12 krill peptide group, low-dose T19 krill peptide group, and atorvastatin group could all control the increase in rat liver weight and liver index to varying degrees compared with the high-fat diet group( Figure 3 in Figures C and D). From the observation of liver morphology, there was significantly more lipid accumulation in the livers of rats in the high-fat model group compared with the normal diet group. The lipid accumulation in the livers of rats in the low-dose T12 krill peptide group, high-dose T12 krill peptide group, and low-dose T18 krill peptide group was improved to varying degrees, reducing liver fat infiltration( Figure 4 ). The epididymal fat weight in the high-fat diet group was significantly higher than that in the normal diet group. The high-dose T12 krill peptide group, low-dose T18 krill peptide group, and atorvastatin group could all control the epididymal fat weight in rats to varying degrees compared with the high-fat diet group( Figure 3 in Figure E). The white fat weight in the high-fat diet group was significantly higher than that in the normal diet group. The low-dose T12 krill peptide group, high-dose T12 krill peptide group, low-dose T18 krill peptide group, and atorvastatin group could all control the white fat weight in rats to varying degrees compared with the high-fat diet group( Figure 3 in Figure F).

[0187] In summary, the high-dose T12 krill peptide diet can control the significant increase in rat liver weight, liver index, and fat weight.

[0188] (3) High-dose T12 Antarctic krill peptide can control abnormal lipid levels in the serum of obese rats induced by a high-fat diet

[0189] As Figure 5 shown in Figure A, the serum triglyceride in the high-fat diet group was higher than that in the normal diet group. The high-dose T12 krill peptide group, high-dose T18 krill peptide group, low-dose T19 krill peptide group, and atorvastatin group could all control the increase in the serum triglyceride content in rats to varying degrees compared with the high-fat diet group.

[0190] As Figure 5 shown in B, the total serum cholesterol in the high-fat diet group was significantly higher than that in the normal diet group. Compared with the high-fat diet group, the high- and low-dose T12 krill peptide groups, the high-dose T18 krill peptide group, the low-dose T19 krill peptide group, and the atorvastatin group could all control the increase in the total serum cholesterol content of rats to varying degrees.

[0191] As Figure 5 shown in C, the serum high-density lipoprotein in the high-fat diet group was significantly lower than that in the normal diet group. Compared with the high-fat diet group, the high-dose T12 krill peptide group, the high-dose T18 krill peptide group, the high- and low-dose T19 krill peptide groups, and the atorvastatin group could all control the decrease in the serum high-density lipoprotein content of rats to varying degrees.

[0192] As Figure 5 shown in D, the serum low-density lipoprotein in the high-fat diet group was significantly higher than that in the normal diet group. Compared with the high-fat diet group, the high-dose T12 krill peptide group, the high-dose T18 krill peptide group, the low-dose T19 krill peptide group, and the atorvastatin group could all control the increase in the serum low-density lipoprotein content of rats to varying degrees.

[0193] As Figure 5 shown in E, the serum HDL / LDL in the high-fat diet group was significantly lower than that in the normal diet group. Compared with the high-fat diet group, the high-dose T12 krill peptide group, the high-dose T18 krill peptide group, the high- and low-dose T19 krill peptide groups, and the atorvastatin group could all control the decrease in the serum low-density lipoprotein content of rats to varying degrees.

[0194] In summary, T12 Antarctic krill peptide can control the abnormal lipid levels in serum induced by high-fat diet.

[0195] Example 5 Function Test of Antarctic Krill Peptide in Alleviating Liver Injury

[0196] 5.1 Method

[0197] (1) Animal Grouping and Model Establishment

[0198] After 1 week of adaptive feeding, the mice were randomly divided into 4 groups (Table 6). All groups of mice were given normal diet and subjected to 4 weeks of experimental intervention. All mice were intraperitoneally injected twice a week, and the intraperitoneal injection volume was the body weight of the mouse (g) × 10 (μl). The mice in Group 1 were injected with olive oil, and the mice in Groups 2-4 were injected with olive oil containing 10% CCl 4 . All mice were gavaged daily. The mice in Groups 1-2 were gavaged with PBS solution daily, the mice in Group 3 were gavaged with T12 krill peptide solution (600 mg / kg), and the mice in Group 4 were gavaged with silybin solution (30 mg / kg).

[0199] Table 6 Mouse Grouping and Intervention Scheme

[0200] Group number Grouping Intervention plan Quantity 1 Normal group Intraperitoneal injection of olive oil + gavage with PBS solution 3 rats 2 Model group <![CDATA[Intraperitoneal injection of 10% CCl 4 -olive oil + intragastric administration of PBS solution]]> 3 rats 3 T12 krill peptide group <![CDATA[Intraperitoneal injection of 10% CCl 4 -olive oil + intragastric administration of 600 mg / kg T12 krill peptide solution]]> 4 rats 4 Positive control group <![CDATA[Intraperitoneal injection of 10% CCl 4 -olive oil + intragastric administration of 30 mg / kg silybin solution]]> 4 rats

[0201] (2) Detection indicators

[0202] Transaminase indicators: The contents of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the serum of mice were measured using a detection kit.

[0203] Liver section indicators: The livers of mice were taken, sliced, and then stained with H&E (hematoxylin-eosin) and Sirius red respectively, and observed and photographed under a microscope for analysis.

[0204] 5.2 Results

[0205] As Figure 6 shown, the levels of ALT and AST in the model group were significantly higher than those in the normal group. Compared with the model group, the T12 krill peptide group and the positive control group could control the increase in the contents of ALT and AST in the serum of mice to varying degrees ( Figure 6 A and B in). The results of liver section staining showed that there was obvious liver injury in the model group compared with the normal group, and the T12 krill peptide group and the positive control group could reduce the liver injury of mice to varying degrees ( Figure 6 C in).

[0206] Example 4 Peptide segment screening of Antarctic krill peptide

[0207] (1) Molecular sequence identification of Antarctic krill peptide

[0208] The Antarctic krill peptide was ultrafiltered with a filter membrane pore size of 1000 Da, and the fraction less than 1000 Da was collected. The LC-MS / MS protein sequence identification of the ultrafiltered T12 Antarctic krill peptide was performed using an ultra-high resolution mass spectrometer Orbitrap Exploris 480. Parameter settings: Select the state No-Enzyme (Unspecific), the maximum allowable missed cleavage number during enzymatic digestion is 2, the primary mass spectrometry accuracy is 10 ppm, the secondary mass spectrometry accuracy is 0.02 Da, Peptide FDR≤0.01, and the quantification method uses Unique peptide. Sequence alignment was performed with the reported Antarctic krill protein database in Uniprot, and the highly confident peptide segments are the Antarctic krill protein peptide segments.

[0209] (2) Virtual screening of highly active peptide segments of Antarctic krill

[0210] The identified Antarctic krill peptide sequences were drawn and structurally optimized using Chemdraw and Chem3D software to obtain the three-dimensional structures of the peptides. The lipid-lowering target HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) (PDB: 1HWK) was selected as the receptor protein, and the Antarctic krill peptide was the ligand small molecule. Molecular docking was performed using Autodock Vina. All identified Antarctic krill peptides were virtually screened based on the Affinity score, and 3D and 2D maps of the interaction between the ligand and receptor were drawn using Pymol and DiscoveryStudio 2019 respectively to analyze the interaction mode of the key amino acid residues between the two.

[0211] (3) The Antarctic krill peptides YDEVAR and IGKNTPSYT have a high affinity for HMGCR.

[0212] The LC-MS / MS technique was used to identify the sequences of the T12 krill peptides after ultrafiltration through 1000 Da. A total of 41 peptides were identified, and the krill peptides within decapeptides were screened for molecular docking with HMGCR.

[0213] Table 7 The top ten peptides with the lowest binding energy to HMGCR in T12 Antarctic krill peptides

[0214]

[0215]

[0216] The molecular docking binding energy of the positive control atorvastatin to HMGCR was -8.9 kcal / mol. Five peptides in T12 krill peptides with an abundance ratio greater than 5% and a binding energy less than -7 kcal / mol are shown in Table 7. The first 5 peptide segments may play an important role in T12 Antarctic krill peptides. They bind stably to HMGCR and have a high affinity.

[0217] Discussion

[0218] This application first discovered the role of T12 Antarctic krill peptides prepared by complex enzymatic hydrolysis of alkaline protease and neutral protease in controlling lipid metabolism disorders in diet-induced hyperlipidemic rats. Specifically, it is manifested as (1) slowing down the weight gain and body fat accumulation of hyperlipidemic rats; (2) controlling abnormal serum lipid levels; (3) controlling hepatomegaly. T12 krill peptides have better effects than other T18 and T19 krill peptides, especially in controlling body weight, body fat, hepatomegaly and blood lipids. It was also found that T12 Antarctic krill peptides can reduce liver tissue damage and have a liver-protecting effect.

[0219] The molecular docking technology was first adopted to mine the potential key lipid-lowering peptide segments in Antarctic krill peptides. The research found that (1) the peptide segments YDEVAR and IGKNTPSYT had low binding energies with the lipid-lowering target HMGCR, and were the peptide segments in T12 Antarctic krill peptides with strong interactions with HMGCR. (2) Analyzing from the amino acid sites interacting with HMGCR, YDEVAR and IGKNTPSYT might have the same mechanism of action as atorvastatin, and affect the synthesis of body cholesterol by inhibiting the activity of HMGCR.

[0220] The Antarctic krill peptides and the Antarctic krill peptide mixture of the present invention can be used for the control and improvement of glycolipid metabolism disorder diseases such as obesity, fatty liver, and hyperlipidemia.

[0221] The Antarctic krill peptides and the Antarctic krill peptide mixture of the present invention can be applied to the development of products such as food, health food, special dietary food, special medical food, and medicine.

[0222] All the documents mentioned in the present invention are cited in this application as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An Antarctic krill peptide for lowering blood lipids, characterized in that: The Antarctic krill peptide is selected from the following group: (a) a polypeptide having an amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; (b) a polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% homology (or identity) with the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13, and the polypeptide has the biological function shown in any one of SEQ ID NOs: 1 to 13; or (c) A derivative polypeptide formed by replacing, deleting or adding one or more (preferably 1 to 3, more preferably 1 to 2) amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13, and retaining the biological function shown in any one of SEQ ID NOs: 1 to 13.

2. A method for preparing the Antarctic krill peptide according to claim 1, characterized in that: Includes steps: (s1) providing Antarctic krill solution; and (s2) adding protease to the Antarctic krill solution to react and obtain the Antarctic krill peptide; Wherein, the protease is selected from the following group: alkaline protease, neutral protease, flavor protease, or a combination thereof.

3. The method according to claim 2, characterized in that The mass ratio of the protease to Antarctic krill is 0.001-10:100, preferably 0.005-5:100, and more preferably 0.1-3:

100.

4. An Antarctic krill peptide mixture, characterized in that: The Antarctic krill peptide mixture comprises the Antarctic krill peptide according to claim 1.

5. The use of the Antarctic krill peptide according to claim 1 or the Antarctic krill peptide mixture according to claim 4, characterized in that: For preparing a drug, a pharmaceutical composition, a special medical food, or a health food for preventing and / or treating a disease selected from the following group; (a) Diseases related to lipid metabolism disorders; (b) Liver damage.

6. A drug or pharmaceutical composition for preventing and / or treating diseases related to lipid metabolism disorders and / or liver damage, characterized in that: Include (a) the Antarctic krill peptide according to claim 1 or the Antarctic krill peptide mixture according to claim 4; and (b) a pharmaceutically acceptable carrier.

7. A method for reducing cellular triglycerides, characterized in that: Includes steps: The Antarctic krill peptide according to claim 1 or the Antarctic krill peptide mixture according to claim 4 is contacted with cells to reduce triglycerides in the cells.

8. A polynucleotide, characterized in that The polynucleotide encodes the Antarctic krill peptide according to claim 1.

9. A carrier, characterized in that The vector contains the polynucleotide according to claim 8.

10. A host cell, characterized in that The host cell contains the vector according to claim 9 or the exogenous polynucleotide according to claim 8 is integrated into its genome.

Images