Antarctic krill peptide having blood lipid-lowering and liver-protecting effects, and preparation method therefor and use thereof

Abstract

The present invention provides an Antarctic krill peptide or an Antarctic krill peptide mixture, and a preparation method therefor and a use thereof. Specifically, the Antarctic krill peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1-13, or a polypeptide having a homology (or identity) of 80% or higher with the amino acid sequence as shown in any one of SEQ ID NOs: 1-13. The Antarctic krill peptide or Antarctic krill peptide mixture of the present invention has excellent effects in the treatment and / or prevention of diseases related to lipid metabolism disorders, and can be used for the development of drugs, healthcare foods, foods for special medical purposes, and other products for diseases related to lipid metabolism disorders.

Description

Antarctic krill peptides with lipid-lowering and liver-protecting effects, their preparation methods and applications Technical Field

[0001] This invention relates to the field of functional peptides, specifically to Antarctic krill peptides with lipid-lowering and liver-protecting effects, their preparation methods, and applications. Background Technology

[0002] With global economic development, changing lifestyles, and upgraded dietary structures, chronic metabolic diseases such as obesity, fatty liver, and hyperlipidemia have become major global public health problems. Their incidence is continuously rising and increasingly affecting younger people, seriously threatening human health and increasing the burden on social healthcare. These diseases are centered on metabolic disorders, driving the development of metabolic syndrome and various serious complications.

[0003] Fatty liver has been redefined as metabolic dysfunction-associated fatty liver disease (MASLD), considered a hepatic manifestation of metabolic syndrome, closely related to dyslipidemia, central obesity, and insulin resistance. Hyperlipidemia refers to elevated levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C) or decreased levels of high-density lipoprotein cholesterol (HDL-C) in serum, reflecting systemic lipid metabolism disorders. Currently, interventions for chronic metabolic diseases mainly include lifestyle interventions and drug therapy. While lifestyle interventions (such as dietary control and exercise for weight loss) are fundamental, poor adherence and limited long-term effectiveness make them difficult to meet clinical needs. Traditional chemical drugs, although able to regulate weight, blood lipids, or improve hepatic steatosis to some extent, often have drawbacks such as single-target therapy, significant side effects, and insufficient safety with long-term use. They also cannot fundamentally repair the body's metabolic function, making it difficult to achieve synergistic improvement of multiple metabolic abnormalities. Furthermore, some drugs have inconvenient administration methods (such as insulin injection and smegglutide), limiting their clinical application.

[0004] Food-derived bioactive peptides, as a class of small molecules composed of amino acids, have advantages such as easy absorption, good biocompatibility, and high safety, and have become a research hotspot in the field of metabolic disease intervention. They are widely used in the food and pharmaceutical fields, including special diets, health foods, and medical foods.

[0005] Therefore, given the high incidence of chronic metabolic diseases such as obesity, fatty liver, and hyperlipidemia, and the limitations of existing intervention methods, developing a food-derived functional peptide with high activity, good safety, and the ability to synergistically improve glucose and lipid metabolism disorders has significant clinical value, social significance, and market prospects. It is also a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a food-derived polypeptide for the prevention and / or treatment of lipid metabolism disorders and a method for its preparation.

[0007] In a first aspect, there is an Antarctic krill peptide that lowers blood lipids and protects the liver, wherein the Antarctic krill peptide is selected from the group consisting of:

[0008] (a) A polypeptide having any of the amino acid sequences shown in SEQ ID NO:1 to 13;

[0009] (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 any of the amino acid sequences shown in SEQ ID NO: 1-13, and said polypeptide having the biological function shown in any of SEQ ID NO: 1-13; or

[0010] (c) A derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 to 3, more preferably 1 to 2) amino acid residues of any of the amino acid sequences shown in any of SEQ ID NO:1 to 13, and retaining the biological function shown in any of SEQ ID NO:1 to 13.

[0011] In another preferred embodiment, a polypeptide having any of the amino acid sequences shown in SEQ ID NO:1 to 5.

[0012] In another preferred embodiment, the Antarctic krill peptide consists of any of the amino acid sequences shown in SEQ ID NO:1 to 5.

[0013] In another preferred embodiment, the Antarctic krill peptide is bound to hydroxymethylglutaryl-CoA reductase.

[0014] In another preferred embodiment, the binding energy of the Antarctic krill peptide to hydroxymethylglutaryl-CoA reductase is ≤-7 kcal / mol, more preferably ≤-7.5 kcal / mol, and even more preferably ≤-8 kcal / mol.

[0015] In another preferred embodiment, the Antarctic krill peptide is prepared by the following method:

[0016] (s1) provides Antarctic krill solution; and

[0017] (s2) The protease is added to the Antarctic krill solution and reacted to obtain the Antarctic krill peptide;

[0018] The protease is selected from the group consisting of alkaline proteases, neutral proteases, flavor proteases, or combinations thereof.

[0019] A second aspect of the present invention provides a method for preparing Antarctic krill peptides as described in the first aspect of the present invention, comprising the steps of:

[0020] (s1) provides Antarctic krill solution; and

[0021] (s2) The protease is added to the Antarctic krill solution and reacted to obtain the Antarctic krill peptide;

[0022] The protease is selected from the group consisting of alkaline proteases, neutral proteases, flavor proteases, or combinations thereof.

[0023] In another preferred embodiment, the protease is derived from Bacillus or Aspergillus oryzae; preferably Bacillus chrysogenum, Bacillus amyloliquefaciens, or Aspergillus oryzae.

[0024] In another preferred embodiment, the protease is selected from the group consisting of: Novozymes neutral protease, Novozymes flavor protease, Novozymes alkaline protease, IFF alkaline protease, IFF neutral protease, Angel alkaline protease, or combinations thereof; preferably IFF alkaline protease and IFF neutral protease.

[0025] In another preferred embodiment, the mass ratio of the protease to Antarctic krill (based on protein content) is 0.001 to 10:100, more preferably 0.005 to 5:100, and even more preferably 0.1 to 3:100.

[0026] In another preferred embodiment, the protease includes neutral protease and alkaline protease.

[0027] In another preferred embodiment, in step (s2), the reaction is carried out at 40–75°C, preferably 45–70°C, more preferably 50–65°C, and even more preferably 50–60°C, for example, about 55°C.

[0028] In another preferred embodiment, the protease is added at 0.1% to 10% (w / w, based on the mass of Antarctic krill protein), more preferably 0.5% to 5%, more preferably 1% to 3%, for example, about 2.2% or 1.4%.

[0029] In another preferred embodiment, the mass ratio of the alkaline protease to the neutral protease is 1–5:1–5, more preferably 1–3:1–3, more preferably 1–2:1–2, for example, about 1.2:1.

[0030] In another preferred embodiment, 0.5% to 5% (w / w, based on the mass of Antarctic krill) alkaline protease and 0.5% to 5% (w / w, based on the mass of Antarctic krill) neutral protease are added; more preferably 1% to 3% alkaline protease and 0.5% to 3% neutral protease; even more preferably 1% to 3% IFF alkaline protease and 0.5% to 3% IFF neutral protease, for example, about 1.2% alkaline protease and 1% neutral protease.

[0031] In another preferred embodiment, the pH value of the Antarctic krill solution is 7 to 10, more preferably 7.5 to 9, more preferably 8 to 9, for example, about 8.5.

[0032] In another preferred embodiment, the Antarctic krill solution contains 50–500 mg / L, more preferably 80–300 mg / L, and even more preferably 100–200 mg / L.

[0033] In another preferred embodiment, the Antarctic krill solution is prepared from defatted Antarctic krill powder.

[0034] In another preferred embodiment, the Antarctic krill solution is a mixture of defatted Antarctic krill powder and water, with a material-to-liquid mass ratio of 1:5 to 10, more preferably 1:6 to 9, and even more preferably 1:7 to 9.

[0035] In another preferred embodiment, in step (s2), the reaction time is 3 to 8 hours, more preferably 4 to 7 hours, and even more preferably 4 to 6 hours.

[0036] In another preferred embodiment, the method further includes step (s3) of inactivating enzymes in the Antarctic krill peptides obtained in step (s2).

[0037] In another preferred embodiment, in step (s3), the enzyme inactivation is carried out at 80–100°C, preferably 80–95°C, more preferably 80–90°C, for example, about 85°C.

[0038] In another preferred embodiment, in step (s3), the enzyme inactivation treatment is performed for 1 to 10 minutes, more preferably 3 to 8 minutes, more preferably 4 to 6 minutes, for example, about 5 minutes.

[0039] In another preferred embodiment, the method further includes step (s4) defluorinating the enzyme-inactivated Antarctic krill peptides.

[0040] In another preferred embodiment, in step (s4), Antarctic krill peptides are defluorinated using calcium hydroxide, preferably 0.1-5% calcium hydroxide.

[0041] In a third aspect, the present invention provides an Antarctic krill peptide mixture comprising the Antarctic krill peptides as described in the first aspect of the present invention.

[0042] In another preferred embodiment, the fluorine content in the Antarctic krill peptide mixture is ≤5 mg / L, more preferably ≤3 mg / L, and even more preferably ≤2.8 mg / L.

[0043] In another preferred embodiment, the total nitrogen content in the Antarctic krill peptide mixture is 10-20 g / 100 g, more preferably 12-18 g / 100 g, and even more preferably 13-16 g / 100 g.

[0044] In another preferred embodiment, the ash content of the Antarctic krill peptide mixture is 1-10 g / 100 g, more preferably 3-8 g / 100 g, and even more preferably 5-8 g / 100 g.

[0045] In another preferred embodiment, the Antarctic krill peptide mixture does not contain inorganic arsenic.

[0046] In another preferred embodiment, the content of peptides with a molecular weight of less than 180 Da in the Antarctic krill peptide mixture is ≤20%, more preferably ≤15%, and even more preferably ≤10%.

[0047] In another preferred embodiment, the content of peptides with a molecular weight of 180 to 1000 Da in the Antarctic krill peptide mixture is ≥70%, more preferably ≥75%, and even more preferably ≥80%.

[0048] In another preferred embodiment, the Antarctic krill peptide mixture is in liquid, solid, or semi-solid form, with the solid form preferably being in powder form.

[0049] In another preferred embodiment, the Antarctic krill peptide mixture is prepared by the method of claim 2.

[0050] In a fourth aspect, the present invention provides the use of Antarctic krill peptides as described in the first aspect of the present invention or mixtures of Antarctic krill peptides as described in the third aspect of the present invention for the preparation of medicaments, pharmaceutical compositions, special medical foods, or health foods for the prevention and / or treatment of diseases selected from the group consisting of:

[0051] (a) Diseases related to lipid metabolism disorders;

[0052] (b) Liver injury.

[0053] In another preferred embodiment, the lipid metabolism disorder-related disease is selected from the group consisting of: hyperlipidemia, obesity, fatty liver, hepatomegaly, hypertension, coronary heart disease, pancreatitis, hyperglycemia, or combinations thereof.

[0054] In another preferred embodiment, the lipid metabolism disorder is caused by a high-fat diet.

[0055] In another preferred embodiment, the liver injury includes acute hepatitis, chronic hepatitis, or fatty liver.

[0056] In another preferred embodiment, the liver injury includes metabolic dysfunction-related fatty liver disease, metabolic dysfunction-related steatohepatitis, and / or liver fibrosis.

[0057] In another preferred embodiment, the metabolic dysfunction-related steatohepatitis is caused by a high-fat diet with choline deficiency.

[0058] In another preferred embodiment, the liver fibrosis is caused by a high-fat diet lacking choline and amino acids.

[0059] In another preferred embodiment, the liver injury is caused by a drug, preferably CCl4.

[0060] In another preferred embodiment, the drug or pharmaceutical composition is further used to select from the group consisting of:

[0061] (y1) Slows down the increase in weight and / or body fat;

[0062] (y2) Reduce the increase in liver and / or fat weight;

[0063] (y3) Improves abnormal serum lipid levels.

[0064] In another preferred embodiment, the abnormal serum lipid level is selected from the group consisting of: elevated serum triglyceride levels, elevated total cholesterol levels, decreased high-density lipoprotein levels, elevated low-density lipoprotein levels, or combinations thereof.

[0065] In another preferred embodiment, the drug or pharmaceutical composition is applied to a subject having characteristics selected from the group consisting of:

[0066] (x1) Have high cholesterol or are at risk of having high cholesterol;

[0067] (x2) High-fat diet;

[0068] (x3) Abnormal lipid levels in serum.

[0069] In another preferred embodiment, the increase in triglyceride content means that the triglyceride content X1 in the serum of the subject is ≥20% compared with the triglyceride content X0 in the serum of a healthy person ((X1-X0) / X0) ≥30%, more preferably ≥50%.

[0070] In another preferred embodiment, the increase in total cholesterol content means that the total cholesterol content Y1 in the serum of the subject is ≥50% compared with the total cholesterol content Y0 in the serum of a healthy person ((Y1-Y0) / Y0), preferably ≥80%, and more preferably ≥100%.

[0071] In another preferred embodiment, the reduction in high-density lipoprotein content means that the high-density lipoprotein content N1 in the serum of the subject is ≥30% compared with the high-density lipoprotein content N0 in the serum of healthy individuals ((N0-N1) / N0) ≥40%, more preferably ≥50%.

[0072] In another preferred embodiment, the increase in low-density lipoprotein content means that the low-density lipoprotein content M1 in the serum of the subject is ≥50% compared with the low-density lipoprotein content M0 in the serum of healthy individuals ((M1-M0) / M0) ≥80%, more preferably ≥100%.

[0073] In another preferred embodiment, the dosage of the drug or drug composition is 100-2000 mg / kg, more preferably 100-1500 mg / kg, and even more preferably 200-1000 mg / kg, for example, about 300 mg / kg or 800 mg / kg, based on Antarctic krill peptides.

[0074] In a fifth aspect, the present invention provides a medicament or pharmaceutical composition for the prevention and / or treatment of lipid metabolism disorder-related diseases and / or liver injury, comprising...

[0075] (a) Antarctic krill peptides as described in the first aspect of the invention or mixtures of Antarctic krill peptides as described in the third aspect of the invention; and

[0076] (b) A drug-acceptable carrier.

[0077] In another preferred embodiment, the lipid metabolism disorder-related disease is selected from the group consisting of: hyperlipidemia, obesity, fatty liver, hepatomegaly, hypertension, coronary heart disease, pancreatitis, hyperglycemia, or combinations thereof.

[0078] In another preferred embodiment, the lipid metabolism disorder is caused by a high-fat diet.

[0079] In a sixth aspect, the present invention provides a method for reducing cellular triglycerides, comprising the steps of:

[0080] Contacting cells with Antarctic krill peptides as described in the first aspect of the invention or a mixture of Antarctic krill peptides as described in the third aspect of the invention reduces the triglycerides in the cells.

[0081] In another preferred embodiment, the cells include hepatocytes, preferably AML12, Huh7, HepG2, or a combination thereof.

[0082] In another preferred embodiment, the concentration of the Antarctic krill peptide is 1–500 μM, more preferably 10–300 μM, and even more preferably 20–200 μM.

[0083] In another preferred embodiment, the cells have a high level of triglyceride expression.

[0084] In another preferred embodiment, the high triglyceride expression level in the cells is induced by oleic acid.

[0085] In another preferred embodiment, the high triglyceride expression level means that the triglyceride content Z1 of the cells is ≥50% ((Z1-Z0) / Z0) compared with Z0 of cells not induced by oleic acid. More preferably, it is ≥80% and even more preferably ≥100%.

[0086] In another preferred embodiment, the method is in vitro.

[0087] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0088] In a seventh aspect, the present invention provides a polynucleotide encoding an Antarctic krill peptide as described in the first aspect of the present invention.

[0089] In an eighth aspect, the present invention provides a carrier containing the polynucleotide as described in the seventh aspect of the present invention.

[0090] In a ninth aspect, the present invention provides a host cell containing a vector as described in the eighth aspect of the present invention or the genome of which an exogenous polynucleotide as described in the seventh aspect of the present invention is integrated.

[0091] In a tenth aspect, the present invention provides a method for preventing and / or treating lipid metabolism disorder-related diseases, comprising the steps of:

[0092] Administering a preventive or therapeutically effective amount of the Antarctic krill peptides of the first aspect of the present invention or a mixture of the Antarctic krill peptides of the third aspect of the present invention to subjects in need, thereby preventing and / or treating lipid metabolism disorder-related diseases.

[0093] In another preferred embodiment, the lipid metabolism disorder-related disease is selected from the group consisting of: hyperlipidemia, obesity, fatty liver, hepatomegaly, hypertension, coronary heart disease, pancreatitis, hyperglycemia, or combinations thereof.

[0094] In another preferred embodiment, the lipid metabolism disorder is caused by a high-fat diet.

[0095] In another preferred embodiment, the amount of Antarctic krill peptide applied is 100-2000 mg / kg, more preferably 100-1500 mg / kg, and even more preferably 200-1000 mg / kg, for example, about 300 mg / kg or 800 mg / kg.

[0096] In another preferred embodiment, the subject has characteristics selected from the following group:

[0097] (x1) Have high cholesterol or are at risk of having high cholesterol;

[0098] (x2) High-fat diet;

[0099] (x3) Abnormal lipid levels in serum.

[0100] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0101] Figure 1 shows a comparison of the molecular weights of Antarctic krill peptides under different enzymatic hydrolysis schemes.

[0102] Figure 2 shows that Antarctic krill peptides can control the increase in triglyceride levels in cells induced by oleic acid. A: Triglyceride content in AML12 cells after intervention with Antarctic krill peptides induced by oleic acid; B: Triglyceride content in Huh7 cells after intervention with Antarctic krill peptides induced by oleic acid; C: Triglyceride content in HepG2 cells after intervention with Antarctic krill peptides induced by oleic acid. 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.

[0103] Figure 3 shows the reduction of body weight and body fat gain induced by a high-fat diet in rats by high-dose T12 Antarctic krill peptide. Changes in body weight (A) and percentage change in body weight (B) after 6 weeks of different interventions in rats; liver weight (C) and its percentage of 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 to the normal diet group, *P < 0.05, **P < 0.01, ***P < 0.001 represent significant differences compared to the high-fat model group.

[0104] Figure 4 shows lipid accumulation in hyperlipidemic rats induced by a high-fat diet controlled by Antarctic krill peptides.

[0105] Figure 5 shows the control of abnormal serum lipid levels in obese rats induced by a high-fat diet using high-dose T12 Antarctic krill peptide. A: Serum triglycerides in rats; B: Serum total cholesterol in rats; C: Serum high-density lipoprotein cholesterol in rats; D: Serum low-density lipoprotein cholesterol in rats; E: Ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol in rats. Data are expressed as mean ± SEM, n = 5, #P < 0.05, ##P < 0.01, ###P < 0.001 represent significant differences compared to the normal diet group, *P < 0.05, **P < 0.01, ***P < 0.001 represent significant differences compared to the high-fat model group.

[0106] Figure 6 shows the reduction of CCl4-induced liver injury in mice by T12 Antarctic krill peptide. A: ALT levels in mice after 4 weeks of different interventions; B: AST levels in mice after 4 weeks of different interventions; C: H&E staining and Sirius red staining images of mouse liver sections. Data are expressed as mean ± SEM, n = 3–4. #P < 0.05 indicates a significant difference compared to the normal group, *P < 0.05 indicates a significant difference compared to the model group.

[0107] Figure 7 shows the high affinity of Antarctic krill peptides YDEVAR, IGKNTPSYT, and HMGCR. The interaction between atorvastatin and HMGCR is illustrated in 3D (A) and 2D (B) amino acid residue diagrams. The interaction between YDEVAR and HMGCR is illustrated in 3D (C) and 2D (D) amino acid residue diagrams. The interaction between IGKNTPSYT and HMGCR is illustrated in 3D (E) and 2D (F) amino acid residue diagrams.

[0108] Figure 8 shows that five peptides can control the increase in triglyceride and total cholesterol levels in cells induced by oleic acid / linolenic acid to varying degrees. (A) Triglyceride content after intervention with the five peptides in oleic acid / linolenic acid-induced AML12 cells. (B) Total cholesterol content after intervention with the five peptides in oleic acid / linolenic acid-induced AML12 cells. Data are expressed as mean ± SEM, n = 6, #P < 0.05 indicates a significant difference compared with the normal group, *P < 0.05 indicates a significant difference compared with the high-fat model group.

[0109] Figure 9 shows the effects of the Antarctic krill peptide mixture on body weight, food intake, and organ indices in CDA-HFD-induced mice. The function of krill peptides was evaluated using a CDA-HFD-induced mouse liver fibrosis model. (A) Mouse body weight. (B) Mouse food intake. (C) Ratio of mouse liver to body weight. Data are expressed as mean ± SEM. * indicates P < 0.05 compared to the model group and the control group, and # indicates P < 0.05 compared to the experimental group and the model group.

[0110] Figure 10 shows the effects of the Antarctic krill peptide mixture on CDA-HFD-induced transaminase, plasma, and liver lipids in mice. The function of the krill peptides was evaluated using a CDA-HFD-induced mouse liver fibrosis model. (A) H&E staining and Sirius red staining of mouse liver tissue. (B) Alanine aminotransferase (ALT) level in mouse liver. (C) Aspartate aminotransferase (AST) level in mouse. (D) Plasma triglyceride level in mouse. (E) Plasma total cholesterol level in mouse. (F) Triglyceride level in mouse liver. (G) Total cholesterol level in mouse liver. Data are expressed as mean ± SEM. * indicates P < 0.05 compared with the model group and the control group, and # indicates P < 0.05 compared with the experimental group and the model group. Detailed Implementation

[0111] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a polypeptide and a mixture of polypeptides derived from Antarctic krill that can lower blood lipids and protect the liver. These polypeptides possess any of the amino acid sequences shown in SEQ ID NO: 1-13. Cell experiments showed that Antarctic krill peptides significantly improved oleic acid-induced triglyceride accumulation. Animal experiments showed that Antarctic krill peptides improved body fat accumulation, abnormal serum lipids, hepatomegaly, liver damage, and diet-induced weight gain. The Antarctic krill peptides of this invention have excellent effects in treating and / or preventing diseases related to lipid metabolism disorders. Based on this, the present invention was completed.

[0112] the term

[0113] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0114] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0115] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition 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.).

[0116] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0117] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0118] As used in this article, the terms “oleic acid” and “OA” are used interchangeably.

[0119] As used in this article, the terms “atorvastatin” and “Ator” are used interchangeably.

[0120] As used herein, the terms “Antarctic krill peptide mixture of the present invention”, “LIPA144”, “T12 Antarctic krill peptide mixture” and “T12 Antarctic krill peptide” are used interchangeably.

[0121] "Identity" refers to the sequence matching between two polypeptides or two nucleic acids. "Identity" represents the percentage of identical residues in the polypeptide or nucleic acid sequence out of the total number of residues, and is calculated based on mutation type. Mutation types include insertions (extensions) at either end of a sequence, deletions (truncations) at either end of a sequence, substitutions of one or more amino acids / nucleotides, insertions within a sequence, and deletions within a sequence.

[0122] For example, in polypeptide sequences, 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 residue count is calculated based on the larger of the compared molecules. If the mutation type also includes insertions (extensions) or deletions (truncations) at either end or both ends of the sequence, the number of amino acids inserted or deleted at either end or both ends (e.g., less than 20 at either end) is not included in the total residue count. When calculating the percentage of identity, the sequences being compared are aligned in a manner that produces the maximum match between sequences, and gaps in the alignment (if present) are resolved using a specific algorithm.

[0123] protease

[0124] The protease of the present invention is used to enzymatically hydrolyze Antarctic krill solution to produce the Antarctic krill peptide of the present invention. The protease of the present invention is selected from the group consisting of alkaline proteases, neutral proteases, flavor proteases, or combinations thereof.

[0125] In a preferred embodiment, the protease is derived from Bacillus or Aspergillus oryzae; preferably Bacillus chrysogenum, Bacillus amyloliquefaciens, or Aspergillus oryzae.

[0126] In a preferred embodiment, the protease is selected from the group consisting of: Novozymes neutral protease, Novozymes flavor protease, Novozymes alkaline protease, IFF alkaline protease, IFF neutral protease, Angel alkaline protease, or combinations thereof; preferably IFF alkaline protease and IFF neutral protease.

[0127] 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.

[0128] Antarctic krill peptide of the present invention

[0129] As used herein, the terms "polypeptide of the present invention" and "Antarctic krill peptide of the present invention" are used interchangeably.

[0130] The Antarctic krill peptides of this invention are selected from the following group:

[0131] (a) A polypeptide having any of the amino acid sequences shown in SEQ ID NO:1 to 13;

[0132] (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 any of the amino acid sequences shown in SEQ ID NO:1-13, and said polypeptide having the biological function shown in any of SEQ ID NO:1-13;

[0133] (c) A derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 to 3, more preferably 1 to 2) amino acid residues of any of the amino acid sequences shown in any of SEQ ID NO:1 to 13, and retaining the biological function shown in any of SEQ ID NO:1 to 13.

[0134] Antarctic krill production is estimated at 1 billion tons, and it possesses the largest animal genome ever discovered. Antarctic krill protein contains abundant essential amino acids, exhibiting high digestibility and high biological value, effectively supporting human growth and development. Antarctic krill peptides possess significant antioxidant activity, blood pressure-lowering activity, blood sugar-lowering activity, DPP-IV inhibitory activity, joint inflammation relief, and liver protection, among other biological activities. These activities are related to the diversity of peptide chain structures, enabling them to regulate various biological processes.

[0135] Those skilled in the art will recognize examples and implementations of conserved amino acid substitutions. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., replacing another nonpolar amino acid residue with a nonpolar amino acid residue, replacing another polar uncharged amino acid residue with a polar uncharged amino acid residue, replacing another basic amino acid residue with a basic amino acid residue, and replacing another acidic amino acid residue with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitution, where an amino acid is replaced by another amino acid belonging to the same group, falls within the scope of this invention, provided that the substitution does not lead to inactivation of the polypeptide's biological activity. Therefore, the polypeptides of this invention can contain one or more conserved substitutions in their amino acid sequence, preferably generated by substitutions according to Table 1. Furthermore, this invention also covers polypeptides containing one or more other nonconservative substitutions, provided that such nonconservative substitutions do not significantly affect the desired function and biological activity of the polypeptides of this invention.

[0136] Conserved amino acid substitutions can occur at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing their biological activity, while “essential” amino acid residues are required for biological activity. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can occur in non-conserved regions of Cas enzymes. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity.

[0137] In some implementations, the selected group of amino acids considered to be mutually conserved substitutions includes:

[0138] The Antarctic krill peptides of the present invention can be artificially synthesized by chemical synthesis, biosynthesis, etc., preferably by solid-phase synthesis, liquid-phase synthesis, stepwise synthesis, enzymatic synthesis, gene expression, artificial grafting, and enzymatic hydrolysis.

[0139] The preparation method of Antarctic krill peptides of the present invention

[0140] The preparation method of Antarctic krill peptide of the present invention includes the following steps:

[0141] (s1) provides Antarctic krill solution; and

[0142] (s2) The protease is added to the Antarctic krill solution and reacted to obtain the Antarctic krill peptide;

[0143] The protease is selected from the group consisting of alkaline proteases, neutral proteases, flavor proteases, or combinations thereof.

[0144] In a preferred embodiment, the mass ratio of the protease to Antarctic krill is 0.01–10:100, more preferably 0.05–5:100, and even more preferably 0.1–3:100. In another preferred embodiment, the protease is added at 0.01%–10% (based on the protein content of Antarctic krill), more preferably 0.1%–5%, and even more preferably 0.2%–3%.

[0145] In a preferred embodiment, in step (s2), the reaction is carried out at 40–75°C, more preferably 45–70°C, more preferably 50–65°C, and even more preferably 50–60°C, for example, about 55°C. In a preferred embodiment, in step (s2), the reaction time is 3–8 hours, more preferably 4–7 hours, and even more preferably 4–6 hours.

[0146] In a preferred embodiment, the Antarctic krill solution contains 50–500 mg / L of Antarctic krill, more preferably 80–300 mg / L, and even 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, with a mass ratio of 1:5–10, more preferably 1:6–9, and even more preferably 1:7–9.

[0147] In a preferred embodiment, the method further includes step (s3) of inactivating the enzyme in the Antarctic krill peptides obtained in step (s2). In a preferred embodiment, the method further includes step (s4) of defluorinating the enzyme-inactivated Antarctic krill peptides.

[0148] An Antarctic krill peptide mixture containing the Antarctic krill peptides of the present invention can be prepared using the method of the present invention. In the Antarctic krill peptide mixture, the fluorine content is ≤5 mg / L, preferably ≤3 mg / L, more preferably ≤2.8 mg / L; the content of peptides less than 180 Da is ≤20%, preferably ≤15%, more preferably ≤10%; and the content of peptides from 180 to 1000 Da is ≥70%, preferably ≥75%, more preferably ≥80%.

[0149] The main advantages of this invention include:

[0150] 1. The Antarctic krill peptides or mixtures of Antarctic krill peptides of the present invention have excellent effects in treating lipid metabolism disorders, especially in slowing down weight gain, reducing body fat, improving abnormal serum lipid levels, and controlling liver enlargement.

[0151] 2. The Antarctic krill peptide or mixture of Antarctic krill peptides of the present invention has excellent effects in treating liver injury, and can increase the content of ALT and AST in serum and reduce liver tissue damage.

[0152] 3. The Antarctic krill peptide of the present invention interacts strongly with hydroxymethylglutaryl-CoA reductase (HMGCR), and has the same mechanism of action as atorovastatin, affecting the body's cholesterol synthesis by inhibiting HMGCR activity.

[0153] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0154] Materials and Instruments

[0155] Male SD rats aged 6-8 weeks were purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0156] Defatted krill meal 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 Preparations (Yichang) Co., Ltd.; alkaline protease FoodPro AP and neutral protease FoodPro PNL were purchased from International Flavors (China) Co., Ltd. - IFF. 60% kcal high-fat feed was purchased from Research Diets. Triglyceride, total cholesterol, LDL cholesterol, and HDL cholesterol assay kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0157] The high-speed multi-functional pulverizer (2500Y) was purchased from Yongkang Boou Hardware Products Co., Ltd.; the multi-functional microplate reader (Various Flash) was purchased from Thermo Fisher Scientific Ltd.; the ultrasonic instrument (SB25-12DT) was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; the TSKgel G2000SWXL gel filtration chromatography column was purchased from Tosoh (Shanghai) Biotechnology Co., Ltd.; the high-performance liquid chromatograph (Ultimate 3000) was purchased from Thermo Fisher Scientific Ltd.; and the high-speed refrigerated centrifuge (Centrifuge 5424) was purchased from Eppendorf (China) Co., Ltd.

[0158] Example 1: Preparation of Antarctic Krill Peptides

[0159] Defatted Antarctic krill powder was pulverized into a fine powder using a grinder. The powder was dissolved in deionized water at a material-to-liquid mass ratio of 1:8. The pH of the krill solution was adjusted to 8.5 using 0.1 mol / L hydrochloric acid. Protease was added according to Table 1, and the enzymatic hydrolysis temperature was controlled at 55℃ for 5 hours, with uniform stirring during hydrolysis. After hydrolysis, the hydrolysate was subjected to enzyme inactivation treatment by heating at 85℃ for 5 minutes. After cooling to room temperature, the hydrolysate was centrifuged at 4000 rpm for 20 minutes. The supernatant was collected, and the pH was adjusted to 7 using phosphoric acid. 0.5% (by weight of the supernatant) of calcium hydroxide (by weight of the supernatant) was added for defluorination treatment at 55℃ for 1 hour, with uniform stirring during defluorination. The defluorinated product was then centrifuged at 4000 rpm for 15 minutes. The defluorinated supernatant (i.e., the oligopeptide solution after hydrolysis) was spray-dried to obtain powdered krill peptides.

[0160] Table 1. Enzyme types and dosage group design:

[0161] Example 2: Determination of molecular weight distribution and fluorine content of krill oil peptide solution

[0162] 2.1 Method

[0163] A method for determining the molecular weight distribution of protein peptides (high-performance gel filtration chromatography) was established according to Appendix A of GB / T 22729-2008. 1 ml of Antarctic krill peptide solution prepared in Example 1 was filtered through a 0.22 μm filter membrane and injected, with each injection volume being 10 μl. The molecular weight distribution of the krill peptides was calculated. The fluorine content in the Antarctic krill peptides was determined using a fluoride ion electrode according to Method III of GB 5009.18 / -2003 (fluoride ion selective electrode method). The total nitrogen content in the Antarctic krill peptides was determined according to Method I of GB 5009.5-2016; the ash content was determined according to Method I of GB 5009.4-2016; and the inorganic arsenic content was determined according to Method I of Part II of GB 5009.11-2014.

[0164] 2.2 Results

[0165] Table 2. Molecular weight distribution of Antarctic krill peptides under different enzymatic hydrolysis conditions.

[0166] Figure 1 and Table 2 show the molecular weight distribution of Antarctic krill peptides prepared under enzymatic hydrolysis conditions T10, T12, T18, and T19. Bioactive peptides in the 180-1000 Da range may have higher bioavailability and activity. The T12, T18, and T19 samples showed a higher proportion of peptides in the 180-1000 Da range, while the T10 sample had a higher proportion of peptides smaller than 180 Da, indicating that the T10 sample contained more free amino acids and had a lower proportion of polypeptides than the other three samples. Therefore, the T12, T18, and T19 enzymatic hydrolysis conditions were selected for further research.

[0167] Table 3. Results of physicochemical properties determination of peptides from T12 Antarctic krill

[0168] As shown in Table 3, the fluoride content in Antarctic krill peptides before defluorination was 117.9 mg / L, and after defluorination, the fluoride content was 2.62 mg / L, with a defluorination rate exceeding 97%, and the fluoride content meeting safety standards. The total nitrogen content in T12 krill peptides was 14.4 g / 100g, and the calculated protein content was approximately 90%. The ash content was 6.7 g / 100g, and inorganic arsenic was not detected.

[0169] Example 3: Antarctic krill peptides can control the increase in triglyceride levels in cells induced by oleic acid.

[0170] 3.1 Method

[0171] AML12, Huh7, and HepG2 cells were divided into 11 groups (Table 4) and cultured in DMEM high-glucose medium. Group 1 cells received no intervention, while groups 2-12 cells were treated with oleic acid (OA) to induce cell modeling. On the second day after modeling, groups 3-12 underwent different interventions: groups 3-11 cells were given high, medium, and low doses of T12, T18, and T19 krill peptide solutions, respectively, while group 12 cells were given atorvastatin as a positive control. Triglyceride levels in each group were measured using a triglyceride assay kit.

[0172] Table 4 Cell grouping and intervention regimen

[0173] 3.2 Results

[0174] In all three cell lines, the triglyceride content in the high-lipid model group 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 induced by oleic acid, with the high-dose krill peptide group showing the best control (Figure 2A). In the Huh7 cell line, except for the medium-dose T19 krill peptide group, all other krill peptide intervention groups significantly controlled the increase in triglyceride levels induced by oleic acid (Figure 2B). In the HepG2 cell line, all krill peptide intervention groups significantly controlled the increase in triglyceride levels induced by oleic acid (Figure 2C). These findings indicate that Antarctic krill peptides can control the increase in triglyceride levels induced by oleic acid in cells.

[0175] Example 4: Lipid-lowering function test of Antarctic krill peptides

[0176] 4.1 Methods

[0177] After one week of acclimatization, rats were randomly divided into 9 groups (Table 5). Group 1 rats were fed a normal diet, while groups 2-9 rats were fed a 60% high-fat diet for 6 weeks. At week 2, groups 1-9 underwent 4 weeks of different gavage treatments: groups 1-2 rats were administered physiological saline daily by gavage; groups 3-8 rats were administered high and low doses of T12, T18, and T19 krill peptide solutions daily by gavage, respectively; and group 9 rats were administered atorvastatin daily as a positive control.

[0178] Table 5. Grouping and intervention protocols for rats

[0179] The body weight, liver, epididymal fat, and serum parameters of the mice were measured.

[0180] (1) The weight of the mice was measured and recorded every week.

[0181] (2) After anesthesia with an overdose of isoflurane, blood was collected in an anticoagulant tube using the apical sampling method. The tube was centrifuged at 3000 r / min for 15 min at 4℃, and the supernatant was collected and stored at -80℃. The liver and epididymal fat of the rats were collected, weighed, and stored at -80℃.

[0182] (3) The total cholesterol content in rat serum was determined using a triglyceride, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol detection kit.

[0183] 4.2 Results

[0184] (1) High-dose T12 Antarctic krill peptide slowed down the increase in body weight and body fat induced by a high-fat diet in rats.

[0185] The high- and low-dose T19 krill peptide groups and the high-dose T18 krill peptide groups showed greater weight changes than other groups throughout the intervention. The low-dose T12 krill peptide, low-dose T18 krill peptide, and atorvastatin groups showed slightly lower weight changes than the high-fat model group throughout the intervention, falling within the middle range. The high-dose T12 krill peptide group showed significantly lower overall weight changes than the high-fat model group throughout the intervention (Figure 3A and B).

[0186] In conclusion, high-dose T12 krill peptide intervention can control weight gain in rats fed a high-fat diet.

[0187] (2) High-dose T12 Antarctic krill peptide controlled the increase in liver weight, liver index and fat weight in rats.

[0188] After 6 weeks of intervention, rats were sacrificed for organ index analysis. Results showed that the liver weight and liver index (liver weight as a percentage of body weight) in the high-fat diet 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 all controlled the increase in liver weight and liver index to varying degrees compared to the high-fat diet group (Figure 3, C and D). Morphologically, the high-fat diet group showed significantly more lipid accumulation in the liver compared to the normal diet group. The low-dose T12 krill peptide group, high-dose T12 krill peptide group, and low-dose T18 krill peptide group showed varying degrees of improvement in liver lipid accumulation and reduced hepatic fatty 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 all controlled the epididymal fat weight to varying degrees compared to the high-fat diet group (Figure 3, 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, the high-dose T12 krill peptide group, the low-dose T18 krill peptide group, and the atorvastatin group all controlled the white fat weight in rats to varying degrees compared with the high-fat diet group (F in Figure 3).

[0189] In conclusion, a diet high in T12 krill peptides can control the significant increase in liver weight, liver index, and fat weight in rats.

[0190] (3) High-dose T12 Antarctic krill peptide can control abnormal serum lipid levels in obese rats induced by a high-fat diet.

[0191] As shown in Figure 5A, the serum triglyceride level in the high-fat diet group was 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 serum triglyceride level in rats to varying degrees.

[0192] As shown in Figure 5B, the serum total 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-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 serum total cholesterol in rats to varying degrees.

[0193] As shown in Figure 5C, 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 all controlled the reduction of serum high-density lipoprotein in rats to varying degrees.

[0194] As shown in Figure 5D, 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 of serum low-density lipoprotein in rats to varying degrees.

[0195] As shown in Figure 5E, the serum HDL / LDL ratio 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 all controlled the reduction of serum low-density lipoprotein in rats to varying degrees.

[0196] In conclusion, T12 Antarctic krill peptide can control abnormal serum lipid levels induced by a high-fat diet.

[0197] Example 5: Functional test of Antarctic krill peptides in reducing liver damage

[0198] 5.1 Method

[0199] (1) Animal grouping and model construction

[0200] Mice were randomly divided into 4 groups after one week of acclimatization (Table 6). All groups of mice were fed a normal diet and underwent experimental intervention for 4 weeks. All mice were given intraperitoneal injections twice a week, with the injection volume being 10 μl × mouse body weight (g). Group 1 mice were injected with olive oil, and groups 2-4 mice were injected with olive oil containing 10% CCl4. All mice were administered silygrafts daily; group 1-2 mice were administered PBS solution daily, group 3 mice were administered T12 krill peptide solution (600 mg / kg), and group 4 mice were administered silymarin solution (30 mg / kg).

[0201] Table 6. Mouse grouping and intervention regimen

[0202] (2) Detection indicators

[0203] Transaminase levels: The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in mouse serum were measured using a detection kit.

[0204] Liver section indicators: Mouse livers were collected, sectioned, and stained with H&E (hematoxylin-eosin) and Sirius red, respectively. The sections were then observed and photographed under a microscope for analysis.

[0205] 5.2 Results

[0206] As shown in Figure 6, the ALT and AST levels 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 of serum ALT and AST levels in mice to varying degrees (Figure 6, A and B). Liver section staining results showed that the model group had obvious liver damage compared with the normal group, and the T12 krill peptide group and the positive control group could alleviate liver damage in mice to varying degrees compared with the model group (Figure 6, C).

[0207] Example 6: Peptide screening of Antarctic krill peptides

[0208] (1) Molecular sequence identification of Antarctic krill peptides

[0209] Antarctic krill peptides were ultrafiltered using a 1000 Da membrane, and fractions smaller than 1000 Da were collected. The ultrafiltered T12 Antarctic krill peptides were then sequenced by LC-MS / MS using an Orbitrap Exploris 480 ultraresolution mass spectrometer. Parameter settings included: No-Enzyme (Unspecific) mode, maximum allowable number of undigested fragments during digestion of 2, primary mass spectrometry precision of 10 ppm, secondary mass spectrometry precision of 0.02 Da, peptide FDR ≤ 0.01, and the Unique Peptide method for quantification. Sequence alignment was performed with previously reported Antarctic krill protein databases in Uniprot; high-confidence peptides were identified as Antarctic krill protein peptides.

[0210] (2) Virtual screening of highly active peptides from Antarctic krill

[0211] The identified Antarctic krill peptide sequences were plotted and their structures optimized using Chemdraw and Chem3D software to obtain the three-dimensional structures of the peptides. HMG-CoA reductase (PDB:1HWK), a lipid-lowering target, was selected as the receptor protein, and the Antarctic krill peptides were selected as the ligand molecules. Molecular docking was performed using Autodock Vina. All identified Antarctic krill peptides were virtually screened based on Affinity scores. 3D and 2D diagrams of the interaction between the receptor and ligand were plotted using Pymol and Discovery Studio 2019, respectively, to analyze the interaction mechanisms of key amino acid residues.

[0212] (3) Antarctic krill peptides YDEVAR, IGKNTPSYT have a high affinity for HMGCR.

[0213] The T12 krill peptides after 1000 Da ultrafiltration were sequenced using LC-MS / MS technology, and a total of 41 peptides were identified. Krill peptides with up to ten peptides were screened and molecularly docked with HMGCR.

[0214] Table 7. The top ten peptides with the strongest binding energy to HMGCR in T12 Antarctic krill peptides.

[0215] The positive control atorvastatin had a molecular docking binding energy of -8.9 kcal / mol with HMGCR. Table 7 shows five peptides in T12 krill peptides with an abundance greater than 5% and a binding energy less than -7 kcal / mol. These first five peptides likely play an important role in T12 Antarctic krill peptides, exhibiting stable binding to HMGCR and high affinity.

[0216] According to the molecular docking results (Figure 7), the main interacting amino acid residues of atorvastatin (red part) and HMGCR (purple part) are GLU559, ARG590, ASN755, and LEU853 (Figure 7, A and B), indicating that atorvastatin may interact with these sites on HMGCR to exert an inhibitory effect, thereby affecting cholesterol synthesis. YDEVAR (yellow part) and IGKNTPSYT (green part) show significant overlap in the amino acid residues interacting with HMGCR (Figure 7, C, D, E, and F), suggesting that YDEVAR and IGKNTPSYT may share the same mechanism of action as atorvastatin on HMGCR, thus affecting cholesterol synthesis.

[0217] Example 7: Efficacy Verification of Key Lipid-Lowering Peptides from Antarctic Krill Peptides

[0218] 7.1 Method

[0219] The five peptides with the lowest binding energy for molecular docking, i.e., the top five peptides with high affinity for HMGCR (YDEVAR, IGKNTPSYT, ERDWPEGRG, VNEEDQLR, and RDWPEGR), were selected for efficacy verification. AML12 cells were divided into 8 groups (Table 8) and cultured in DMEM high-glucose medium. Group 1 cells received no intervention, while groups 2-8 cells were treated with oleic acid (OA) / palmitic acid (PA) to induce the model. Simultaneously, groups 3-8 underwent different preventative interventions: groups 3-7 cells were given different krill peptide solutions, and group 8 cells were given atorvastatin as a positive control. The triglyceride and total cholesterol levels in each group were measured using triglyceride and total cholesterol assay kits.

[0220] Table 8

[0221] 7.2 Conclusion

[0222] As shown in Figure 8, AML12 cells were treated with oleic acid / palmitic acid to induce a lipid accumulation cell model, and different groups were then treated with krill peptides. The high-lipid model group showed significantly higher levels of triglycerides and total cholesterol than the normal group, indicating successful model establishment. The krill peptide intervention groups were able to control the increase in triglyceride levels induced by oleic acid / palmitic acid to varying degrees, with the YDEVAR peptide showing the strongest improvement in triglyceride levels (Figure 8, A). The krill peptide intervention groups were also able to control the increase in total cholesterol levels induced by oleic acid / palmitic acid to varying degrees, with the YDEVAR peptide and atorvastatin groups showing significant improvements (Figure 8, B). This demonstrates that the five peptides identified based on molecular docking results can control the abnormal increase in lipid levels induced by oleic acid / palmitic acid to varying degrees.

[0223] Example 8: Evaluation of the Functional Improvement of MASH by Antarctic Krill Peptide Mixture

[0224] 8.1 Method

[0225] In this embodiment, LIPA144 is a mixture of T12 krill peptides.

[0226] 1) Animal grouping and model construction

[0227] After one week of acclimatization, mice were randomly divided into 5 groups (Table 9). Group 1 mice were fed a normal diet, while groups 2-5 mice were fed a choline-deficient, high-fat, amino acid-deficient diet (CDA-HFD) and underwent a 10-week experimental intervention (6 weeks for modeling + 4 weeks for gavage). Starting from week 7, all mice were administered gavage daily. Group 1-2 mice were administered PBS solution daily, group 3 mice were administered krill peptide (LIPA144) solution (600 mg / kg), group 4 mice were administered resmetirom solution (3 mg / kg), and group 5 mice were administered silymarin solution (30 mg / kg).

[0228] Table 9: Mouse grouping and intervention regimens

[0229] 2) Detection indicators

[0230] ① Weight and food intake.

[0231] ② Organ index: Record liver weight, epididymal fat, and abdominal fat weight, and calculate liver-to-body ratio and fat-to-body ratio.

[0232] ③ Transaminase levels: The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in mouse plasma were measured using a detection kit.

[0233] ④ Liver section indicators: Mouse livers were taken, sectioned, and stained with H&E (hematoxylin-eosin) and Sirius red, respectively. The sections were then observed and photographed for analysis under a microscope.

[0234] ⑤ Liver and plasma fat: The triglyceride and total cholesterol content in mouse liver and plasma were determined using a test kit.

[0235] 8.2 Results

[0236] 1) Krill peptides can improve CDA-HFD-induced liver injury and fibrosis in mice.

[0237] During the interventions in different experimental groups, there was no significant difference in body weight among the groups (model specificity), and all were lower than normal mice (Figure 9A). The food intake of mice in the krill peptide group was significantly higher than that in the model group (Figure 9B), which may be related to the differential peptides in oligopeptides that affect appetite.

[0238] After 10 weeks of intervention, mice were sacrificed for organ index analysis. The results showed that the liver-to-body ratio in the model group was significantly higher than that in the normal diet group. Compared with the model group, the krill peptide group and the positive drug group could control the increase in the liver-to-body ratio of mice to varying degrees (Figure 9C).

[0239] Observation of stained liver sections revealed significantly more lipid accumulation and liver fibrosis in the model group mice compared to the normal diet group. The krill peptide group and the positive drug group showed varying degrees of improvement in liver lipid accumulation, reducing hepatic steatosis and fibrosis symptoms (Figure 10A).

[0240] After 10 weeks of intervention in different experimental groups, mouse plasma was collected and analyzed. The ALT and AST levels in the model group were significantly higher than those in the normal group. Compared with the model group, the krill peptide group and the positive control group could control the increase in plasma ALT and AST levels in mice to varying degrees (Figure 10, B and C). Krill peptide and Resmetirom significantly improved the increase in liver ALT and AST levels induced by CDA-HFD and alleviated liver damage symptoms in mice.

[0241] After 10 weeks of intervention in different experimental groups, mouse plasma and liver tissue were collected to analyze lipid levels in plasma and liver. The plasma lipid levels in the model group showed no significant difference compared to the normal diet group, consistent with the CDA-HFD model creation criteria. Compared to the model group, the krill peptide group and the positive control group showed further reductions in plasma lipid levels to varying degrees (D and E in Figure 10). Krill peptide significantly reduced plasma triglyceride and total cholesterol levels.

[0242] In mice modeled with CDA-HFD, impaired lipid transport in the liver led to lipid accumulation, resulting in significantly higher levels of triglycerides and total cholesterol in the liver compared to the normal diet group. Both the krill peptide group and the positive control drug group, compared to the model group, effectively controlled the elevated liver lipid levels to varying degrees (F and G in Figure 10). Specifically, krill peptide significantly improved the abnormally elevated liver triglycerides, while Resmetirom significantly improved the abnormally elevated liver triglycerides and total cholesterol.

[0243] In summary, krill peptides can improve CDA-HFD-induced liver injury and fibrosis in mice.

[0244] discuss

[0245] This application is the first to discover the effect of T12 Antarctic krill peptide, prepared by combined enzymatic hydrolysis of alkaline protease and neutral protease, on controlling lipid metabolism abnormalities in diet-induced hyperlipidemic rats. Specifically, it slows down the weight gain and fat accumulation in hyperlipidemic rats, controls abnormal serum lipid levels, and controls liver enlargement. T12 krill peptide outperforms other T18 and T19 krill peptides, especially in controlling weight, body fat, liver enlargement, and blood lipids. It was also found that T12 Antarctic krill peptide can reduce liver tissue damage and has a hepatoprotective effect.

[0246] For the first time, molecular docking technology was used to discover potential key lipid-lowering peptides in Antarctic krill peptides. The study found that (1) peptides YDEVAR, IGKNTPSYT, ERDWPEGRG, VNEEDQLR, and RDWPEGR have strong binding energies to the lipid-lowering target HMGCR, and are the peptides in T12 Antarctic krill peptides that have strong interactions with HMGCR. (2) Analysis of the amino acid sites that interact with HMGCR suggests that YDEVAR and IGKNTPSYT may have the same mechanism of action as atorvastatin, affecting the body's cholesterol synthesis by inhibiting the activity of HMGCR. (3) The efficacy of the top five peptides with the lowest binding energy (i.e., high binding affinity) screened by molecular docking was verified, and it was found that the five peptides could control the abnormal increase in cellular lipid levels caused by oleic acid / palmitic acid to varying degrees.

[0247] The Antarctic krill peptide and Antarctic krill peptide mixture of the present invention can be used to control and improve disorders of glucose and lipid metabolism such as obesity, fatty liver, and hyperlipidemia.

[0248] The Antarctic krill peptide and Antarctic krill peptide mixture of the present invention can be applied to the development of food, health food, special dietary food, special medical food, pharmaceuticals and other products.

[0249] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A lipid-lowering Antarctic krill peptide, characterized in that, The Antarctic krill peptides were selected from the following group: (a) A polypeptide having any of the amino acid sequences shown in SEQ ID NO: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 any of the amino acid sequences shown in SEQ ID NO: 1-13, and said polypeptide having the biological function shown in any of SEQ ID NO: 1-13; or (c) A derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1 to 3, more preferably 1 to 2) amino acid residues of any of the amino acid sequences shown in any of SEQ ID NO:1 to 13, and retaining the biological function shown in any of SEQ ID NO:1 to 13.

2. A method for preparing the Antarctic krill peptide as described in claim 1, characterized in that, Including the following steps: (s1) provides Antarctic krill solution; and (s2) The protease is added to the Antarctic krill solution and reacted to obtain the Antarctic krill peptide; The protease is selected from the group consisting of alkaline proteases, neutral proteases, flavor proteases, or combinations thereof.

3. The method as described in claim 2, characterized in that, The mass ratio of the protease to Antarctic krill (based on protein) is 0.001–10:100, preferably 0.005–5:100, and more preferably 0.1–3:

100.

4. The method as described in claim 2, characterized in that, The proteases include neutral proteases and alkaline proteases.

5. The method as described in claim 4, characterized in that, The mass ratio of alkaline protease to neutral protease is 1–5:1–5.

6. A mixture of Antarctic krill peptides, characterized in that, The Antarctic krill peptide mixture comprises the Antarctic krill peptide as described in claim 1.

7. The use of the Antarctic krill peptide as described in claim 1 or the Antarctic krill peptide mixture as described in claim 6, characterized in that, Used for the preparation of medicines, pharmaceutical compositions, special medical foods, or health foods for the prevention and / or treatment of diseases selected from the following group; (a) Diseases related to lipid metabolism disorders; (b) Liver injury.

8. A medicament or pharmaceutical composition for the prevention and / or treatment of lipid metabolism disorders and / or liver injury, characterized in that, Include (a) the Antarctic krill peptide as claimed in claim 1 or the Antarctic krill peptide mixture as claimed in claim 6; and (b) A drug-acceptable carrier.

9. A method for reducing cellular triglycerides, characterized in that, Including the following steps: Contacting cells with the Antarctic krill peptides as described in claim 1 or the Antarctic krill peptide mixture as described in claim 6 reduces the triglycerides in the cells.

10. A polynucleotide, characterized in that, The polynucleotide encodes the Antarctic krill peptide as described in claim 1.

11. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 10.

12. A host cell, characterized in that, The host cell contains the vector as described in claim 11 or its genome having an exogenous polynucleotide as described in claim 10 integrated therein.

13. A method for preventing and / or treating lipid metabolism disorder-related diseases, comprising the steps of: Administering a preventive or therapeutically effective amount of the Antarctic krill peptide as described in claim 1 or a mixture of the Antarctic krill peptides as described in claim 6 to subjects in need, thereby preventing and / or treating lipid metabolism disorder-related diseases.

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