Euphausia superba peptide with hypolipidemic and liver-protective effects, and preparation method and application thereof

Abstract

The present application provides a euphausia superba peptide or a euphausia superba peptide mixture, a preparation method and application thereof. Specifically, the euphausia superba peptide has an amino acid sequence shown in any one of SEQ ID NO: 1-13 or a polypeptide with homology (or identity) of >80% to the amino acid sequence shown in any one of SEQ ID NO: 1-13. The euphausia superba peptide or the euphausia superba peptide mixture of the present application has excellent effects in treating and / or preventing diseases related to lipid metabolism disorder, and can be used for developing drugs, health foods, special medical foods and the like for diseases related to lipid metabolism disorder.

Description

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] Hyperlipidemia is a chronic disease characterized primarily by abnormal lipid metabolism. The prevention and treatment of hyperlipidemia has attracted widespread attention worldwide. Currently, statins are the most commonly used lipid-lowering drugs in clinical practice. Although statins are generally safe, a significant number of patients experience varying degrees of intolerance, and many patients are concerned about the adverse reactions of statins. Therefore, clarifying the key regulatory mechanisms of lipid metabolism in the body and discovering safe and effective means and methods for regulating blood lipids is particularly important.

[0003] Therefore, there is an urgent need in this field to develop a dietary peptide that lowers blood lipids. Summary of the Invention

[0004] 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.

[0005] In a first aspect, there is a lipid-lowering Antarctic krill peptide, wherein the Antarctic krill peptide is selected from the group consisting of: (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.

[0006] In another preferred embodiment, a polypeptide having any of the amino acid sequences shown in SEQ ID NO: 1 to 5.

[0007] In another preferred embodiment, the Antarctic krill peptide consists of any of the amino acid sequences shown in SEQ ID NO: 1 to 5.

[0008] In another preferred embodiment, the Antarctic krill peptide is bound to hydroxymethylglutaryl-CoA reductase.

[0009] 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.

[0010] In another preferred embodiment, the Antarctic krill peptide is prepared by the following method: (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.

[0011] 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: (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.

[0012] In another preferred embodiment, the protease is derived from Bacillus or Aspergillus oryzae; preferably Bacillus chrysogenum, Bacillus amyloliquefaciens, or Aspergillus oryzae.

[0013] 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.

[0014] In another preferred embodiment, the mass ratio of the protease to Antarctic krill is 0.001 to 10:100, more preferably 0.005 to 5:100, and even more preferably 0.1 to 3:100.

[0015] 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.

[0016] In another preferred embodiment, the protease is added at 0.1% to 10% (w / w, based on the mass of Antarctic krill), more preferably 0.5% to 5%, more preferably 1% to 3%, for example, about 2.2% or 1.4%.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In another preferred embodiment, the Antarctic krill solution is prepared from Antarctic krill powder.

[0022] In another preferred embodiment, the Antarctic krill solution is a mixture of 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.

[0023] In another preferred embodiment, in step (s2), the reaction time is 3-8 hours, more preferably 4-7 hours, and even more preferably 4-6 hours.

[0024] In another preferred embodiment, the method further includes step (s3) of inactivating enzymes in the Antarctic krill peptides obtained in step (s2).

[0025] 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.

[0026] 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.

[0027] In another preferred embodiment, the method further includes step (s4) defluorinating the enzyme-inactivated Antarctic krill peptides.

[0028] In another preferred embodiment, in step (s4), Antarctic krill peptides are defluorinated using calcium hydroxide, preferably 0.1-5% calcium hydroxide.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] In another preferred embodiment, the ash content of the Antarctic krill peptide mixture is 1~10g / 100g, more preferably 3~8g / 100g, and even more preferably 5~8g / 100g.

[0033] In another preferred embodiment, the Antarctic krill peptide mixture does not contain inorganic arsenic.

[0034] 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%.

[0035] In another preferred embodiment, the content of peptides with a molecular weight of 180~1000 Da in the Antarctic krill peptide mixture is ≥70%, more preferably ≥75%, and even more preferably ≥80%.

[0036] 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.

[0037] In another preferred embodiment, the Antarctic krill peptide mixture is prepared by the method described in the second aspect of the present invention.

[0038] 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 or medicament compositions for the prevention and / or treatment of diseases selected from the group consisting of; (a) Diseases related to lipid metabolism disorders; (b) Liver injury.

[0039] 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.

[0040] In another preferred embodiment, the lipid metabolism disorder is caused by a high-fat diet.

[0041] In another preferred embodiment, the liver injury includes acute hepatitis, chronic hepatitis, or fatty liver.

[0042] In another preferred embodiment, the liver injury is caused by a drug, preferably CCl4.

[0043] In another preferred embodiment, the drug or pharmaceutical composition is further used to select from the group consisting of: (y1) Slows down the increase in weight and / or body fat; (y2) Reduce the increase in liver and / or fat weight; (y3) Improves abnormal serum lipid levels.

[0044] 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.

[0045] In another preferred embodiment, the drug or pharmaceutical composition is applied to a subject having characteristics selected from the group consisting of: (x1) Have high cholesterol or are at risk of having high cholesterol; (x2) High-fat diet; (x3) Abnormal lipid levels in serum.

[0046] 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 healthy individuals ((X1-X0) / X0) ≥30%, more preferably ≥50%.

[0047] 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%.

[0048] 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% ((N0-N1) / N0) compared with the high-density lipoprotein content N0 in the serum of healthy individuals. This reduction is more preferably ≥40% and even more preferably ≥50%.

[0049] 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%.

[0050] In another preferred embodiment, the dosage of the drug or drug composition is 100-2000 mg / kg, more preferably 100-1500 mg / kg, more preferably 200-1000 mg / kg, for example, about 300 mg / kg or 800 mg / kg, based on Antarctic krill peptides.

[0051] 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... (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 (b) A drug-acceptable carrier.

[0052] 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.

[0053] In another preferred embodiment, the lipid metabolism disorder is caused by a high-fat diet.

[0054] In a sixth aspect, the present invention provides a method for reducing cellular triglycerides, comprising the steps of: 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.

[0055] In another preferred embodiment, the cells include hepatocytes, preferably AML12, Huh7, HepG2, or a combination thereof.

[0056] 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.

[0057] In another preferred embodiment, the cells have a high level of triglyceride expression.

[0058] In another preferred embodiment, the high triglyceride expression level in the cells is induced by oleic acid.

[0059] 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 that have not been induced by oleic acid, preferably ≥80%, and more preferably ≥100%.

[0060] In another preferred embodiment, the method is in vitro.

[0061] In another preferred embodiment, the method is obtained without diagnosis or treatment.

[0062] 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.

[0063] In an eighth aspect, the present invention provides a carrier containing the polynucleotide as described in the seventh aspect of the present invention.

[0064] 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.

[0065] In a tenth aspect, the present invention provides 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 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.

[0066] 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.

[0067] In another preferred embodiment, the lipid metabolism disorder is caused by a high-fat diet.

[0068] In another preferred embodiment, the amount of Antarctic krill peptide applied is 100-2000 mg / kg, more preferably 100-1500 mg / kg, more preferably 200-1000 mg / kg, for example, about 300 mg / kg or 800 mg / kg.

[0069] In another preferred embodiment, the subject has characteristics selected from the following group: (x1) Have high cholesterol or are at risk of having high cholesterol; (x2) High-fat diet; (x3) Abnormal lipid levels in serum.

[0070] 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

[0071] Figure 1 The molecular weights of Antarctic krill peptides under different enzymatic hydrolysis methods are compared.

[0072] Figure 2This study showed that Antarctic krill peptides can control the increase in triglyceride levels in cells induced by oleic acid. A: Triglyceride levels in AML12 cells after intervention with Antarctic krill peptides; B: Triglyceride levels in Huh7 cells after intervention with Antarctic krill peptides; C: Triglyceride levels in HepG2 cells after intervention with Antarctic krill peptides. Data are expressed as mean ± SEM, n=5, # P<0.05, ## P<0.01, ### P<0.001 indicate a significant difference compared to the normal diet group. P<0.05, P<0.01, P<0.001 indicates a significant difference compared to the high-fat model group.

[0073] Figure 3 This study showed that high-dose T12 Antarctic krill peptides slowed the increase in body weight and body fat induced by a high-fat diet in rats. 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 indicate a significant difference compared to the normal diet group. P<0.05, P<0.01, P<0.001 indicates a significant difference compared to the high-fat model group.

[0074] Figure 4 The study demonstrated that Antarctic krill peptides controlled lipid accumulation in hyperlipidemic rats induced by a high-fat diet.

[0075] Figure 5 This study demonstrates that high-dose T12 Antarctic krill peptide can control abnormal serum lipid levels in obese rats induced by a high-fat diet. 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 indicate a significant difference compared to the normal diet group. P<0.05, P<0.01, P<0.001 indicates a significant difference compared to the high-fat model group.

[0076] Figure 6 This study demonstrates how T12 Antarctic krill peptide alleviates CCl4-induced liver injury in mice. A: ALT levels in mice after 4 weeks of different interventions; B: AST levels in mice after 4 weeks of different interventions; C: Images of H&E staining and Sirius red staining 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. Detailed Implementation

[0077] 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. The polypeptides have the amino acid sequences shown in any of 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, 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.

[0078] the term 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.

[0079] 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.

[0080] 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.).

[0081] 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).

[0082] 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.

[0083] As used in this article, the terms “oleic acid” and “OA” are used interchangeably.

[0084] As used in this article, the terms “atorvastatin” and “Ator” are used interchangeably.

[0085] "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.

[0086] 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.

[0087] protease 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.

[0088] In a preferred embodiment, the protease is derived from Bacillus or Aspergillus oryzae; preferably Bacillus chrysogenum, Bacillus amyloliquefaciens, or Aspergillus oryzae.

[0089] 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.

[0090] Angel basic protein is produced by Bacillus licheniformis ( Baclicus lincheniformis This enzyme preparation is refined through deep liquid fermentation, extraction, multiple filtrations, concentration, and purification. IFF alkaline protease is a protease produced by fermentation with Bacillus licheniformis. Novozymes alkaline protease is produced by Bacillus licheniformis (…). Baclicus lincheniformis It is made through fermentation. Novozymes flavor protease is made from Aspergillus oryzae. (Aspergillus oryzae It is produced through fermentation. Novozymes neutral protease is made from Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens It is made through fermentation.

[0091] Antarctic krill peptide of the present invention As used herein, the terms "polypeptide of the present invention" and "Antarctic krill peptide of the present invention" are used interchangeably.

[0092] The Antarctic krill peptides of this invention are 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; (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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] In some implementations, the selected group of amino acids considered to be mutually conserved substitutions includes: 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.

[0097] The preparation method of Antarctic krill peptides of the present invention The preparation method of Antarctic krill peptide of the present invention includes 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.

[0098] 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 mass of Antarctic krill), more preferably 0.1%~5%, and even more preferably 0.2%~3%.

[0099] 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, 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, preferably 4-7 hours, and even more preferably 4-6 hours.

[0100] In a 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. 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.

[0101] 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.

[0102] 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%.

[0103] The main advantages of this invention include: 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Materials and Instruments Male SD rats aged 6-8 weeks were purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0108] 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.

[0109] 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.

[0110] Example 1: Preparation of Antarctic krill peptides 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 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.

[0111] Table 1. Enzyme type and dosage group design: Example 2: Molecular weight distribution and fluorine content determination of krill oil peptide solution 2.1 Methods 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 into the solution in 10 µl injections. 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.

[0112] 2.2 Results Table 2. Molecular weight distribution of Antarctic krill peptides under different enzymatic hydrolysis conditions Figure 1Table 2 shows 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.

[0113] Table 3. Results of physicochemical properties determination of peptides from T12 Antarctic krill As shown in Table 3, the fluoride content in Antarctic krill peptides before defluorination was 117.9 mg / L, and the fluoride content after defluorination 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.

[0114] Example 3: Antarctic krill peptides can control the increase in triglyceride levels in cells induced by oleic acid. 3.1 Methods 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.

[0115] Table 4 Cell grouping and intervention regimen 3.2 Results 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 effect. Figure 2 (A). 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 in cells induced by oleic acid ( Figure 2 (B). In the HepG2 cell line, all krill peptide intervention groups significantly controlled the increase in triglyceride levels in cells induced by oleic acid (B). Figure 2(C). This indicates that Antarctic krill peptides can control the oleic acid-induced increase in triglyceride levels in cells.

[0116] Example 4: Test of the lipid-lowering function of Antarctic krill peptides 4.1 Methods 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 different gavage treatments for 4 weeks: groups 1-2 rats were gavaged daily with physiological saline, groups 3-8 rats were gavaged daily with high and low doses of T12, T18, and T19 krill peptide solutions, respectively, and group 9 rats were gavaged daily with atorvastatin as a positive control.

[0117] Table 5. Rat grouping and intervention protocols The body weight, liver, epididymal fat, and serum parameters of the mice were measured. (1) The weight of the mice was measured and recorded every week.

[0118] (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℃.

[0119] (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.

[0120] 4.2 Results (1) High-dose T12 Antarctic krill peptides slowed down the increase in body weight and body fat induced by a high-fat diet in rats. 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 3 (A and B in the middle).

[0121] In conclusion, high-dose T12 krill peptide intervention can control weight gain in rats fed a high-fat diet.

[0122] (2) High-dose T12 Antarctic krill peptide controlled the increase of liver weight, liver index and fat weight in rats. After 6 weeks of intervention, rats were sacrificed for organ index analysis. The 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. Compared with the high-fat diet group, the high-dose T12 krill peptide group, the low-dose T19 krill peptide group, and the atorvastatin group could all control the increase in liver weight and liver index in rats to varying degrees. Figure 3 (C and D). Morphological observation of the liver showed that the high-fat diet group rats had significantly more lipid accumulation in their livers 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 all 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, the low-dose T18 krill peptide group, and the atorvastatin group all controlled the epididymal fat weight in rats 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. Figure 3 (Middle F).

[0123] In conclusion, a diet high in T12 krill peptides can control the significant increase in liver weight, liver index, and fat weight in rats.

[0124] (3) High-dose T12 Antarctic krill peptide can control abnormal serum lipid levels in obese rats induced by a high-fat diet. like Figure 5 As shown in Figure A, serum triglycerides in the high-fat diet group were higher than those 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 all controlled the increase in serum triglyceride levels in rats to varying degrees.

[0125] like Figure 5 As shown in Figure B, 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- and low-dose T12 krill peptide groups, the high-dose T18 krill peptide groups, the low-dose T19 krill peptide groups, and the atorvastatin group all controlled the increase in serum total cholesterol in rats to varying degrees.

[0126] like Figure 5 As shown in Figure 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 all controlled the reduction of serum high-density lipoprotein in rats to varying degrees.

[0127] like Figure 5 As shown in Figure 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 all controlled the increase in serum low-density lipoprotein in rats to varying degrees.

[0128] like Figure 5 As shown in Figure E, 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 content in rats to varying degrees.

[0129] In conclusion, T12 Antarctic krill peptide can control abnormal serum lipid levels induced by a high-fat diet.

[0130] Example 5: Functional test of Antarctic krill peptides in reducing liver damage 5.1 Methods (1) Animal grouping and model construction 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).

[0131] Table 6. Mouse grouping and intervention protocols (2) Detection indicators Transaminase levels: The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in mouse serum were measured using a detection kit.

[0132] 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.

[0133] 5.2 Results like Figure 6 As shown, 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 in 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 significant liver damage compared to the normal group. The T12 krill peptide group and the positive control group could reduce liver damage in mice to varying degrees compared with the model group. Figure 6 (C)

[0134] Example 4: Peptide screening of Antarctic krill peptides (1) Molecular sequence identification of Antarctic krill peptides 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.

[0135] (2) Virtual screening of highly active peptides from Antarctic krill 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.

[0136] (3) Antarctic krill peptides YDEVAR, IGKNTPSYT have a high affinity for HMGCR. 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.

[0137] Table 7. The ten peptides with the lowest binding energy to HMGCR in T12 Antarctic krill peptides. 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.

[0138] discuss 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 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.

[0139] 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 and IGKNTPSYT have low 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.

[0140] 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.

[0141] 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.

[0142] 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 mixture, characterized in that, The Antarctic krill peptide mixture was obtained by enzymatic hydrolysis of Antarctic krill solution with protease and contains the polypeptides shown in SEQ ID NO: 1~13; The Antarctic krill peptide mixture contains fluorine content ≤5 mg / L, total nitrogen content of 10~20g / 100g, ash content of 1~10g / 100g, and does not contain inorganic arsenic. Furthermore, the content of peptides with a molecular weight less than 180 Da in the Antarctic krill peptide mixture is ≤10%, and the content of peptides with a molecular weight of 180~1000 Da is ≥75%. The Antarctic krill peptide mixture is prepared by including the following steps: (s1) Provide 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 an alkaline protease and a neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 1-3:1-3; and the mass ratio of the protease to Antarctic krill is 0.001-10:

100.

2. The Antarctic krill peptide mixture as described in claim 1, characterized in that, The Antarctic krill solution is a mixture of Antarctic krill powder and water, with a material-to-liquid mass ratio of 1:5~10.

3. The Antarctic krill peptide mixture as described in claim 1, characterized in that, The mass ratio of alkaline protease to neutral protease is 1~2:1~2.

4. The Antarctic krill peptide mixture as described in claim 1, characterized in that, The proteases are IFF alkaline protease and IFF neutral protease.

5. The Antarctic krill peptide mixture as described in claim 1, characterized in that, The mass ratio of the protease to Antarctic krill is 0.005 to 5:

100.

6. The Antarctic krill peptide mixture as described in claim 1, characterized in that, The mass ratio of the protease to Antarctic krill is 0.1~3:

100.

7. The Antarctic krill peptide mixture as described in claim 1, characterized in that, The protease was added at a rate of 0.1 w / w % to 10 w / w %, based on the mass of Antarctic krill.

8. The Antarctic krill peptide mixture as described in claim 1, characterized in that, In step (s2), the reaction is carried out at 40~75°C.

9. The use of the Antarctic krill peptide mixture as described in claim 1, characterized in that, Used to prepare medicines for the prevention and / or treatment of diseases selected from the following group; (a) Diseases related to lipid metabolism disorders; (b) Liver injury; The lipid metabolism disorder-related diseases are selected from the following group: hyperlipidemia, obesity, fatty liver, or a combination thereof; The liver damage was caused by medication.

10. 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 mixture as described in claim 1; and (b) A drug-acceptable carrier.

11. A method for preparing the Antarctic krill peptide mixture 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 an alkaline protease and a neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 1-3:1-3; and the mass ratio of the protease to Antarctic krill is 0.001-10:

100.

12. The method as described in claim 11, characterized in that, The protease is an IFF alkaline protease and an IFF neutral protease; the protease is added at a rate of 0.1 w / w % to 10 w / w % based on the mass of Antarctic krill.

13. The method as described in claim 11, characterized in that, The mass ratio of alkaline protease to neutral protease is 1~2:1~2.

14. The method as described in claim 11, characterized in that, In step (s2), the reaction is carried out at 40~75°C.

15. The method as described in claim 11, characterized in that, The Antarctic krill solution is a mixture of Antarctic krill powder and water, with a material-to-liquid mass ratio of 1:5~10.

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