Composition for treating neurodegenerative diseases comprising whey protein hydrolysate or whey protein hydrolysate-derived peptide

Abstract

The present invention relates to a composition for treating neurodegenerative diseases, comprising a whey protein hydrolysate or a whey protein hydrolysate-derived peptide. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a whey protein hydrolysate comprising same, of the present invention, can be used to effectively prevent, treat and ameliorate neurodegenerative diseases.

Description

Composition for treating neurodegenerative diseases comprising whey protein hydrolysate or whey protein hydrolysate-derived peptide

[0001] The present invention was made under the support of the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea under the research number 1545026767, and the research management specialized institution of the said project is the National Institute of Agricultural Science and Technology Planning and Evaluation, the research project name is "Development of high value-added food technology", the research project name is "Establishment of mass production technology of milk-derived functional peptides and development of utilization products", the main institution is Neocrema Co., Ltd., and the research period is from January 1, 2023 to December 31, 2023.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2024-0021379, filed with the Korean Intellectual Property Office on February 14, 2024, the disclosure of which is incorporated herein by reference.

[0003] The present invention relates to a composition for treating a neurodegenerative disease comprising whey protein hydrolysate or a peptide derived from whey protein hydrolysate.

[0004]

[0005] Recent advances in medicine and life sciences have led to an aging population, leading to increased interest in degenerative diseases. Cognitive dysfunction, a representative degenerative disease that causes memory impairment, is called cognitive impairment. Alzheimer's disease is the most common form of cognitive impairment and has emerged as a significant social problem. Cognitive impairment is characterized by cognitive decline and language impairment, which interfere with daily life. The exact cause of cognitive impairment is unknown, and no consensus hypothesis exists. This is due to the simultaneous involvement of multiple factors. Reported mechanisms of cognitive impairment include neuronal death due to oxidative stress, the accumulation of senile plaques composed of amyloid beta protein and neurofibrillary tangles composed of hyperphosphorylated tau protein, and a decrease in the neurotransmitter acetylcholine (Ach) caused by acetylcholinesterase (AchE). Currently, antioxidants such as vitamin E and selegiline, as well as AChE inhibitors such as tacrine and Aricept, are used as treatments. These treatments do not completely cure dementia, but rather slow or prevent its progression. Furthermore, they can cause side effects such as temporary hepatotoxicity, gastrointestinal distress, and cardiac bradycardia, necessitating the development of new treatments.

[0006]

[0007] The inventors of the present invention have conducted extensive research to develop an effective treatment for neurodegenerative diseases. As a result, they have discovered that whey protein hydrolysate or whey protein hydrolysate-derived peptides can effectively treat neurodegenerative diseases, thereby completing the present invention.

[0008] Accordingly, an object of the present invention is to provide a peptide comprising an amino acid sequence represented by SEQ ID NO: 1.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0011] Another object of the present invention is to provide a food composition for preventing or improving a degenerative neurological disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0012] Another object of the present invention is to provide a food composition for preventing or improving a degenerative neurological disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0013] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0014] The present invention provides the following inventions 1 to 17.

[0015] 1. A peptide consisting of an amino acid sequence represented by sequence number 1.

[0016] 2. In 1, the peptide has the characteristics of reducing the expression level of apoptotic factors, reducing the concentration of intracellular calcium ions, or a combination thereof.

[0017] 3. A nucleic acid molecule comprising a nucleotide sequence encoding a peptide of 1 or 2.

[0018] 4. A recombinant vector comprising a nucleic acid molecule of 3.

[0019] 5. A host cell containing the recombinant vector of 4.

[0020] 6. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0021] 7. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising any one of 1 to 5 peptides, a recombinant vector, and a host cell.

[0022] 8. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by sequence number 1, in 6 or 7.

[0023] 9. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the whey protein hydrolysate has the properties of increasing cell viability, alleviating oxidative stress, decreasing intracellular calcium ion concentration, decreasing apoptotic factor expression, regulating neurotransmitter expression, improving cognitive ability, decreasing tau protein phosphorylation, or a combination thereof, in any one of 6 to 8.

[0024] 10. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the content of the peptide is 0.5 mg / g to 40 mg / g based on the total weight of whey protein hydrolysate in any one of 6 to 9.

[0025] 11. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the whey protein hydrolysate is obtained by first hydrolyzing whey protein with an endo protease derived from Bacillus licheniformis and second hydrolyzing whey protein with an exo protease derived from Aspergillus oryzae.

[0026] 12. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the endo protease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof; and the exo protease derived from Aspergillus oryzae is Flavorzyme.

[0027] 13. A pharmaceutical composition for preventing or treating a degenerative neurological disease, wherein the mixed enzyme is a mixture of Alcalase and Protamex in a weight ratio of 1:0.5 to 1:2 in any one of 6 to 12.

[0028] 14. A food composition for preventing or improving a degenerative neurological disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0029] 15. A food composition for preventing or improving a neurodegenerative disease, comprising any one of 1 to 5 peptides, a recombinant vector, and a host cell.

[0030] 16. A food composition for preventing or improving a degenerative neurological disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by sequence number 1, in accordance with 14 or 15.

[0031] 17. A method for preventing, improving, or treating a neurodegenerative disease, comprising administering to a subject in need of treatment any one of the peptides, recombinant vectors, host cells, or compositions of 1 to 5; or any one of the compositions of 6 to 13.

[0032] In one aspect of the present invention, the present invention provides a peptide comprising an amino acid sequence represented by SEQ ID NO: 1.

[0033] The inventors of the present invention have conducted extensive research to develop effective treatments for neurodegenerative diseases. As a result, they have discovered that whey protein hydrolysate or whey protein hydrolysate-derived peptides can effectively treat neurodegenerative diseases.

[0034] As used herein, the peptide may comprise at least one additional amino acid at the C-terminus and / or N-terminus of the peptide. The additional amino acid residues may be added individually or collectively for purposes such as improving productivity, purification, stabilization in vivo or in vitro, coupling, or detection of the complex. For example, the peptide may additionally comprise a cysteine ​​residue at the C-terminus and / or N-terminus of the peptide. The additional amino acid residue may provide a "tag" for purification or detection of the peptide, for example, for interaction of the tag with a specific antibody. In the case of a His6 tag, a tag such as a His6 tag, a (HisGlu)3 tag ("HEHEHE" tag), a "myc" (c-myc) tag, or a "FLAG" tag may be provided for immobilized metal affinity chromatography (IMAC).

[0035] The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention is interpreted to include the amino acid sequence of SEQ ID NO: 1, and also include a sequence showing substantial identity with the sequence of SEQ ID NO: 1. The substantial identity means a sequence showing preferably at least 80% homology, more preferably at least 85% homology, even more preferably at least 90% homology, and most preferably at least 95% homology when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403-10(1990)) is accessible from NCBI (National Center for Biological Information) and can be used in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx on the Internet.

[0036] As a peptide used in the present invention, a biologically functional equivalent, which is an amino acid sequence variant that exhibits the same biological activity as the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention, may also be used. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0037] When introducing mutations, the hydrophobicity index of an amino acid can be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobicity index of ±2, more preferably ±1, or even ±0.5.

[0038] Meanwhile, it is also well known that substitutions between amino acids having similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing mutations with reference to hydrophilicity values, substitutions are preferably made between amino acids that exhibit a difference in hydrophilicity values ​​of within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0039] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H.Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0040] In one embodiment of the present invention, the whey protein of the present invention may be normal whey powder, demineralized whey powder, whey protein concentrate, or whey protein isolate.

[0041] In one embodiment of the present invention, the peptide has the properties of reducing the expression of a cell death factor, reducing the concentration of intracellular calcium ions, or a combination thereof.

[0042] The term "expression" as used herein means expression of a protein or expression of a gene encoding the protein, and "gene expression" may refer to transcription of a gene into a polynucleotide, translation of a gene into a polypeptide, or modification of a polynucleotide and / or polypeptide (including, for example, post-translational modification of a polypeptide). An "expressed gene" includes a gene that is transcribed as mRNA and then translated into a polypeptide, or a gene that is transcribed into RNA but not translated into a polypeptide (e.g., tRNA and rRNA).

[0043] As used herein, the term "inhibition or reduction in gene or protein expression" refers to a case where the quantitative expression value of a gene or protein in a test group is measurably reduced compared to the control group. For example, this may mean, but is not limited to, a case where the expression level of a gene or protein in a test group is 90% or less, 80% or less, or 70% or less compared to the control group.

[0044]

[0045] In one aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a peptide composed of amino acids represented by SEQ ID NO: 1.

[0046] In this specification, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0047] It is sufficient for the nucleotide sequence encoding the peptide of the present invention to be a nucleotide sequence encoding a peptide consisting of an amino acid sequence represented by the above sequence number 1, and it is obvious to those skilled in the art that it is not limited to any specific nucleotide sequence.

[0048] This is because even if a mutation occurs in the nucleotide sequence, when the mutated nucleotide sequence is expressed as a protein, there are cases where the protein sequence does not change. This is called codon degeneracy. Therefore, the nucleotide sequence includes a nucleotide sequence that includes functionally equivalent codons, codons that encode the same amino acid (for example, due to codon degeneracy, there are six codons for arginine or serine), or codons that encode biologically equivalent amino acids.

[0049] A nucleic acid molecule comprising a nucleotide sequence encoding a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 is interpreted to also include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The substantial identity refers to a sequence that exhibits at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology, when the nucleotide sequence of the present invention described above and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0050]

[0051] In one aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0052] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, including plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0053] According to a specific embodiment of the present invention, a nucleic acid molecule comprising a nucleotide sequence encoding a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 in the vector of the present invention is operatively linked to a promoter of the vector.

[0054] As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0055] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0056] The vector of the present invention can typically be constructed as a vector for gene cloning or a vector for protein expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0057] For example, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, beta actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, which generally have a polyadenylation sequence as a transcription termination sequence.

[0058] The vector of the present invention may be fused with other sequences to facilitate the purification of peptides or proteins expressed therefrom. Examples of such fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6-His (hexahistidine; Qiagen, USA).

[0059] Meanwhile, the expression vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0060]

[0061] In one aspect of the present invention, the present invention provides a host cell comprising the recombinant vector. In one specific embodiment, the host cell is transformed with the recombinant vector.

[0062] Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell can be used. For example, suitable eukaryotic host cells for the vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK-293 cells.

[0063] As used herein, the terms "transformed", "transduced" or "transfected" refer to a process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transformed", "transduced" or "transfected" cell is a cell that has been transformed, transduced or transfected with an exogenous nucleic acid, and the cell includes the cell and progeny cells resulting from passage thereof.

[0064]

[0065] In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0066] The term "prevention" as used herein refers to the prevention or protective treatment of a disease or disease state. The term "treatment" as used herein refers to the reduction, suppression, alleviation, or eradication of a disease state. For example, it refers to minimizing the spread or worsening of a disease caused by administering a therapeutic agent to a subject.

[0067] The term "subject" or "patient" in this specification refers to an animal, preferably a mammal, including a human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat.

[0068] The pharmaceutical composition of the present invention may be formulated as a powder, granule, tablet, coated tablet, pill, dragee, capsule, liquid, suspension, gel, syrup, slurry, suppository, enema, emulsion, paste, ointment, cream, lotion, powder, spray or suspension.

[0069] The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions. Examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, mannitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

[0070] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and the excretion rate, the treatment period, concurrently used drugs, and other factors well known in the medical field. The amount of the composition used may vary depending on the patient's age, sex, and weight, but the peptide can be administered once or several times daily in an amount sufficient to achieve a blood concentration useful for the treatment of cancer.

[0071] The dosage of the above composition may be increased or decreased depending on the route of administration, severity of the disease, gender, weight, age, etc. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0072] The dosage of the pharmaceutical composition of the present invention is preferably 0.001 to 100 mg / kg (body weight) per day, and the pharmaceutical composition at the dosage described above can be applied locally to the desired area, depending on the purpose of application.

[0073] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including intracerebral administration, oral ingestion, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration. Preferably, the method is intravenous injection.

[0074] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0075] In this specification, 'degenerative neurological disease' refers to abnormalities in motor control ability, cognitive function, perception function, sensory function, and autonomic nervous system function caused by decreased function of nerve cells or death of nerve cells, and is used with the same meaning as 'degenerative brain disease'.

[0076] Neurodegenerative diseases can be classified by clinical characteristics. Diseases that present with progressive cognitive impairment as a major symptom include Alzheimer's disease, dementia (frontotemporal dementia, Lewy body dementia), and corticobasal degeneration. Diseases that present with progressive ataxia include Parkinson's disease, multiple system atrophy, Huntington's disease, and progressive supranuclear palsy. Neurodegenerative diseases that present with symptoms of muscle weakness and atrophy include amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).

[0077] In one embodiment of the present invention, the above-described neurodegenerative disease may be any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Lewy body dementia, progressive supranuclear palsy, and frontotemporal dementia, but is not limited thereto.

[0078]

[0079] In one embodiment of the present invention, the pharmaceutical composition comprises a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0080] In one embodiment of the present invention, the whey protein hydrolysate has the properties of increasing cell viability, alleviating oxidative stress, reducing intracellular calcium ion concentration, reducing apoptotic factor expression, regulating neurotransmitter expression, improving cognitive ability, reducing tau protein phosphorylation, or a combination thereof.

[0081] In one embodiment of the present invention, the content of the peptide is 0.5 mg / g to 40 mg / g based on the total weight of whey protein hydrolysate.

[0082] More specifically, the content of the peptide consisting of the amino acid sequence of the above sequence number 1 is 0.5 mg / g to 40 mg / g, 0.5 mg / g to 25 mg / g, 0.5 mg / g to 15 mg / g, 0.5 mg / g to 10 mg / g, 0.5 mg / g to 8 mg / g, 0.5 mg / g to 7 mg / g, 0.5 mg / g to 6 mg / g, 1.0 mg / g to 40 mg / g, 1.0 mg / g to 25 mg / g, 1.0 mg / g to 15 mg / g, 1.0 mg / g to 10 mg / g, 1.0 mg / g to 8 mg / g, 1.0 mg / g to 7 mg / g, 1.0 mg / g to 6 mg / g, 1.5 mg / g to 40 mg / g, 1.5 mg / g to 25 mg / g, 1.5 mg / g to 15 mg / g, 1.5 mg / g to 10 mg / g, 1.5 mg / g to 8 mg / g, 1.5 mg / g to 7 mg / g, 1.5 mg / g to 6 mg / g, 2.0 mg / g to 40 mg / g, 2.0 mg / g to 25 mg / g, 2.0 mg / g to 15 mg / g, 2.0 mg / g to 10 mg / g, 2.0 mg / g to 8 mg / g, 2.0 mg / g to 7 mg / g, 2.0 mg / g to 6 mg / g, 3.0 mg / g to 40 mg / g, 3.0 mg / g to 25 mg / g, 3.0 mg / g to 15 mg / g, 3.0 It may be from 3.0 mg / g to 10 mg / g, from 3.0 mg / g to 8 mg / g, from 3.0 mg / g to 7 mg / g, from 3.0 mg / g to 6 mg / g, from 4.0 mg / g to 40 mg / g, from 4.0 mg / g to 25 mg / g, from 4.0 mg / g to 15 mg / g, from 4.0 mg / g to 10 mg / g, from 4.0 mg / g to 8 mg / g, from 4.0 mg / g to 7 mg / g, or from 4.0 mg / g to 6 mg / g.

[0083] In one embodiment of the present invention, the whey protein hydrolysate is whey protein that is first hydrolyzed with an endo protease derived from Bacillus licheniformis and secondarily hydrolyzed with an exo protease derived from Aspergillus oryzae.

[0084] In one embodiment of the present invention, the endo protease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof; and the exo protease derived from Aspergillus oryzae is Flavorzyme.

[0085] In one embodiment of the present invention, the mixed enzyme is a mixture of Alcalase and Protamex in a weight ratio of 1:0.5 to 1:2. More specifically, the mixed enzyme may be a mixture of Alcalase and Protamex in a weight ratio of 1:0.5 to 1:2, 1:0.5 to 1:1.75, 1:0.5 to 1:1.5, 1:0.5 to 1:1.25, 1:0.5 to 1:1, 1:0.75 to 1:2, 1:0.75 to 1:1.75, 1:0.75 to 1:1.5, 1:0.75 to 1:1.25, 1:0.75 to 1:1, 1:1 to 1:2, 1:1 to 1:1.75, 1:1 to 1:1.5, or 1:1 to 1:1.25.

[0086]

[0087] In one aspect of the present invention, the present invention provides a food composition for preventing or improving a degenerative neurological disease, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0088] The food composition of the present invention can be manufactured in the form of powder, granules, tablets, capsules, or beverages. Examples include various food products such as candies, beverages, gum, tea, vitamin complexes, or health supplements.

[0089] The food composition of the present invention includes processed forms of all natural ingredients, such as foods, functional foods, nutritional supplements, health foods, and food additives. The above-mentioned types of food compositions can be prepared in various forms according to conventional methods known in the art. For example, health foods can be prepared and consumed in the form of tea, juice, and drinks, or can be granulated, encapsulated, or powdered for consumption. In addition, as food, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.) can be prepared by adding a peptide composed of an amino acid represented by SEQ ID NO: 1 of the present invention or a whey protein hydrolysate including a peptide composed of an amino acid represented by SEQ ID NO: 1. In addition, in order to use the peptide consisting of the amino acid represented by sequence number 1 of the present invention or the whey protein hydrolysate containing the peptide consisting of the amino acid represented by sequence number 1 of the present invention in the form of a food additive, it can be manufactured and used in the form of a powder or concentrate.

[0090] The food composition of the present invention can be taken for a long period of time.

[0091] The mixing amount of the active ingredient of the food composition of the present invention can be appropriately determined depending on its purpose of use (prevention, improvement, or therapeutic treatment). Generally, the whey hydrolysate of the present invention can be added in an amount of 0.1 to 70 wt%, preferably 2 to 50 wt%, based on 100 wt% of the raw material of the food or beverage during the manufacture of the food or beverage. The effective dosage of the peptide of the present invention can be used in accordance with the effective dosage of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it can be below the above range, and since the active ingredient does not have any problem in terms of safety, it can also be used in an amount above the above range.

[0092] The food composition of the present invention may include ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to the peptide consisting of the amino acid represented by sequence number 1 of the present invention or the whey protein hydrolysate containing the peptide consisting of the amino acid represented by sequence number 1, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0093] In one embodiment of the present invention, the food composition comprises a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1.

[0094]

[0095] In one aspect of the present invention, the present invention provides a method for preventing, improving, or treating a neurodegenerative disease, comprising administering a composition comprising a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 to a subject in need of treatment.

[0096] The method for preventing, improving, or treating a neurodegenerative disease of the present invention is similar to the content of the peptide or composition in that it involves administering a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or a composition containing the same, and description of common details is omitted to prevent excessive redundant description.

[0097]

[0098] The features and advantages of the present invention are summarized as follows:

[0099] (a) The present invention provides a peptide consisting of an amino acid sequence represented by sequence number 1.

[0100] (b) The present invention provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0101] (c) The present invention provides a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0102] (d) The present invention provides a food composition for preventing or improving a degenerative neurological disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0103] (e) The present invention provides a food composition for preventing or improving a degenerative neurological disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

[0104] (f) When a peptide consisting of an amino acid sequence represented by sequence number 1 of the present invention or a whey protein hydrolysate containing the same is used, degenerative neurological diseases can be effectively prevented, treated, and improved.

[0105]

[0106] Figure 1a shows the Q-Sepharose HiLOad 16 / 10 column chromatography of whey protein hydrolysate. Figure 1b shows that fractions Ex1 to Ex7 are Ca 2+ The effect on the influx is shown in Figure 1c, size exclusion chromatography (SEC) of Ex4. Figure 1d, fractions S14 to S21 show the Ca 2+ Figure 1e shows the MS / MS spectrum of peptide LDIQK (SEQ ID NO: 1), and Figure 1f shows the amino acid sequence and chemical structure of peptide LDIQK (SEQ ID NO: 1).

[0107] Figure 2 shows the HPLC chromatograms of peptide LDIQK (SEQ ID NO: 1) (A) and whey protein hydrolysate (B).

[0108] Figure 3 shows the effect of WPC hydrolysate on cell viability in HT22 cells. NOR, normal; CON, control; WPC, whey protein concentrate. Data are expressed as mean ± SD. Different symbols indicate significant differences (*** p < 0.001) in the NOR group and (### p < 0.001) in the CON group.

[0109] Figure 4 shows the effects of whey protein hydrolysate on ROS production in HT22 cells oxidatively damaged by H2O2. NOR, normal; CON, control; WPC, whey protein concentrate. Data are expressed as mean ± SD. Different symbols indicate significant differences (*** p < 0.001) in the NOR group and ### p < 0.001 in the CON group. One-way analysis of variance (ANOVA) was performed followed by a post hoc Tukey test.

[0110] Figure 5 shows the Ca of whey protein hydrolysate in HT22 cells subjected to oxidative damage by H2O2. 2+ The effects on NOR, normal; CON, control; WPC, whey protein concentrate. Data are presented as mean ± SD. Different symbols indicate significant *** p < 0.001 vs. NOR group, ### p < 0.001 vs. CON group.

[0111] Figure 6 shows the analysis of the effect of peptide LDIQK (SEQ ID NO: 1) on intracellular calcium concentration. NOR, normal; CON, control. Data are expressed as mean ± SD. Different symbols indicate ***p<0.001 for the NOR group, #p<0.05, ###p<0.001 for the CON group.

[0112] Figure 7 shows the effects of whey protein hydrolysate on H2O2-induced apoptosis in HT22 cells. NOR, normal; CON, control; WPC, whey protein concentrate. Data are expressed as mean ± SD. Different symbols indicate *p<0.05, **p<0.01, ***p<0.001 versus the NOR group, and #p<0.05, ##p<0.01, ###p<0.001 versus the CON group.

[0113] Figure 8 shows the effect of peptide LDIQK (SEQ ID NO: 1) on H2O2-induced apoptosis in HT22 cells. NOR, normal; CON, control. Data are expressed as mean ± SD. Different symbols indicate ***p<0.001 versus the NOR group, #p<0.05, ###p<0.001 versus the CON group.

[0114] Figure 9 shows the effect of whey protein hydrolysate on the expression of neurotransmitters.

[0115] Figure 10 shows the effect of whey protein hydrolysate on the expression of antioxidant-related factors.

[0116] Figure 11 shows the effects of whey protein hydrolysate and treadmill exercise on improving cognitive ability.

[0117] Figure 12 shows the effects of whey protein hydrolysate and treadmill exercise on ROS and MDA. NOR, normal; CON, control; WPC, whey protein concentrate. Data are expressed as mean ± SE. Different symbols indicate significant differences: *p < 0.05, **p < 0.01. ***p < 0.001 vs. NOR group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CON group.

[0118] Figure 13 shows the effects of whey protein hydrolysate and treadmill exercise on acetylcholine content, AChE activity, and ChAT protein levels. NOR, normal; CON, control; WPC, whey protein concentrate. Data are expressed as mean ± SE. Different symbols indicate significant differences: *p < 0.05, **p < 0.01. ***p < 0.001 vs. NOR group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CON group.

[0119] Figure 14 shows the effect of whey protein hydrolysate on the expression of apoptotic factors.

[0120] Figure 15 shows the results of analyzing the effect of whey protein hydrolysate treatment on the MAPK pathway.

[0121] Figures 16 to 18 (a to c) show the effects of whey protein hydrolysate and treadmill exercise on the gut microbiota. NOR, normal; CON, control; WPC, whey protein concentrate. Data are expressed as mean ± SE. Different symbols indicate significant differences: *p < 0.05, **p < 0.01. ***p < 0.001 vs. NOR group, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. CON group.

[0122]

[0123] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0124]

[0125] Example

[0126] Example 1: Materials and Methods

[0127] 1-1. Reagents and Materials

[0128] Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), L-glutamate, 0.25% trypsin-EDTA, and penicilin-streptomycin (P / S) used as cell culture media were purchased from Welgene (Welgene, Gyeonsan-si, Korea). H2O2 was purchased from Sigma (St. Louis, USA).

[0129]

[0130] 1-2. Manufacturing of whey protein hydrolysate

[0131] Enzymes for hydrolyzing whey proteins were used as primary enzymes, 0.2% Alcalase and 0.2% Protamex, and hydrolyzed at 50–55°C for 4 hours to obtain a primary hydrolysate. The primary hydrolysate was hydrolyzed at 50–55°C for 15 hours with 0.2% Flavorzyme to obtain a secondary hydrolysate. The secondary hydrolysate was cooled to room temperature after inactivating the enzymes at 90°C for 10 minutes. The secondary hydrolysate was filtered through a 1 μm housing filter, sterilized at 90°C for 30 minutes, and spray-dried at an inlet temperature of 190±10°C and an outlet temperature of 95±5°C. The spray-dried product was packaged after removing metallic foreign substances with an 8,000 GAUS magnetic bar and used as a sample for analysis.

[0132]

[0133] 1-3. Amino acid composition analysis

[0134] The amino acid composition was measured using an amino acid auto-analyzer after hydrolyzing whey protein hydrolysate using the acid hydrolysis method. 25 mg of whey protein hydrolysate was precisely weighed into a cap tube, 2.5 mL of 6 N HCl was added, and hydrolyzed at 110°C for 24 hours. The filtrate, after removing unhydrolyzed substances using a 3G-4 glass filter, was completely evaporated from the solvent using a rotary vacuum evaporator (N-1110, EYELA, Tokyo, Japan) at 50°C, and diluted to 25 mL with 0.01 N HCl to use as a sample for amino acid analysis. Amino acid analysis was performed by injecting 40 uL of the sample solution and analyzing it using an amino acid auto-analyzer (Biochrom 30, Cambridge, UK).

[0135]

[0136] 1-4. Separation and purification of whey protein hydrolysate

[0137] Whey protein hydrolysate peptides were separated and purified stepwise using anion exchange chromatography, gel filtration chromatography, and reversed phase chromatography. Whey protein peptides were separated by homogenization and elution with 25 mM Tris-Cl (pH 8.0) containing 0.6 M NaCl using a Q-Sepharose HiLoad 16 / 10 ion chromatography column. A total of seven fractions were obtained. Among them, 1 mL of the EX-4 fraction was injected onto a Superdex peptide 10 / 30 column, eluted with HPLC-grade distilled water, and peaks eluting at 280 nm and 220 nm were detected. The active fraction EX-4 / S16 obtained from gel filtration chromatography was completely evaporated in a speed vac, dissolved in acetonitrile solvent containing 100 to 300 uL of 0.1% TFA on a source 5 RPC ST column (4.6x150, 4.5 μm), and the peptides were separated by detecting the peaks eluting at 280 nm and 220 nm.

[0138]

[0139] 1-5. HT22 cell culture and cell viability evaluation

[0140] Mouse hippocampus-derived neuronal cells (HT22) were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in an incubator (5% CO2, 37°C) using Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P / S), and 1% L-glutamate.

[0141] To evaluate the anti-apoptotic effect of whey protein hydrolysate on H2O2, 1X10 4Cells were seeded at a concentration of 10 cells / mL and treated with whey protein concentrate (WPC) and skim milk hydrolysate at various concentrations (50, 100, 200, 400, and 800 μg / mL). Two hours after sample treatment, 300 μM H2O2 was treated to induce oxidative stress. Cell viability was evaluated 24 hours after H2O2 treatment using the WST-1 assay.

[0142]

[0143] 1-6. ROS (Reactive Oxygen Species) Analysis (Cells)

[0144] To measure the ROS scavenging activity of whey protein hydrolysate, a 2,7-dichlorofluorescin diacetate (DCF-DA) assay was performed using HT22 cells. HT22 cells were seeded in 6-well plates at a density of 1 X 10 5 The cells were aliquoted at 100 cells / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. WPC hydrolysate was treated at various concentrations (50, 100, and 200 μg / mL) and cultured for 2 hours, then 300 μM H2O2 was treated and cultured for 24 hours. To measure the amount of ROS, 10 μM DCF-DA reagent diluted in DMSO was treated and cultured for 30 minutes. The supernatant was transferred to a fluorescence plate and the fluorescence intensity (excitation 485 nm, emission 535 nm) was measured using a fluorescence microplate reader.

[0145]

[0146] 1-7. Intracellular Ca 2+ measurement

[0147] Intracellular Ca present in HT22 cells 2+ The concentration of Ca 2+The measurement was performed using Fura-2AM, a fluorescent substance that reacts sensitively to HT22 cells. HT22 cells were seeded in 6-well plates at 1 Х 10 5 Cells were seeded at 100 cells / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. Whey protein hydrolysate (50, 100, and 200 μg / mL) and LDIQK peptide (1.25, 2.5, 5, and 10 μg / mL) were treated and cultured for 2 hours, followed by treatment with 300 μM H2O2 and cultured for 24 hours. HT22 cells were then administered 10 μL of 20 μM Fura-2AM and incubated for 1 hour in a CO2 incubator at 37°C and 5% CO2. After 1 hour of reaction, the medium was removed, washed three times with PBS, and then lysed with 1.0% Triton X-100 at 37°C for 15 minutes. The dissolved solution was transferred to a 96-well plate, and the fluorescence intensity (excitation 340 nm, emission 510 nm) was measured using a fluorescence microplate reader.

[0148]

[0149] 1-8. Western blot (cell experiment)

[0150] HT22 cell line was seeded in 6-well plates at 1 x 10 5 The cells were aliquoted at 10 cells / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. Whey protein hydrolysate was treated at various concentrations (50, 100, and 200 μg / mL) and cultured for 2 hours, then 300 μM H2O2 was treated and cultured for 24 hours. Afterwards, 600 μL of lysis buffer [200 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1% NP40, 1 mM PMSF, 1 mM Na3VO4, protease inhibitor cocktail] was added, homogenized, and centrifuged (12,000 rpm, 5 min, 4°C) to recover the supernatant.

[0151] After protein quantification using the BCA method, 30 μg of protein was electrophoresed on 10% SDS-PAGE. After transfer to PVDF membrane, it was blocked for 1 hour with 5% skim milk and bovine serum albumin, and then incubated with primary antibodies [Beta-actin (Cell Signaling Technology, Inc., Cat.#4967), BAX (Cell Signaling Technology, Inc., Cat.#2772), BCL-2 (Cell Signaling Technology, Inc., Cat.#2876), Nrf2 (Cell Signaling Technology, Inc., Cat.#4399), HO-1 (Cell Signaling Technology, Inc., Cat.#70081), PARP (Cell Signaling Technology, Inc., Cat.#9553), BDNF (Cell Signaling Technology, Inc., Cat.#47808), phosphor-tau (Cell Signaling Technology, Inc., Cat.#29957), tau (Cell Signaling Technology, Inc., Cat.#4019), ChAT (abcam, ab183591)] were reacted at 4°C for 16 hours. After washing three times with TBST, secondary antibody (Anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Inc., Cat.#7074) was treated and reacted at room temperature for 2 hours. Afterwards, SuperSignal TMAfter dispensing Western Blot Enhancer (Thermo. Scientific, Cat. #46641), protein bands were identified using the FluorChem M Fluorescent Western Imaging System (Protein Simple, California, USA). Antibodies were diluted in 5% skim milk and bovine serum albumin according to the data sheet for each antibody.

[0152]

[0153] 1-9. Laboratory Animals and Animal Experiment Design

[0154] Four-week-old male mice (C57BL6 strain) weighing 20–25 g and free of specific pathogens were purchased from Orient Bio (Seongnam, Korea). The animals were kept in a breeding environment set at temperature 23±3℃, relative humidity 50±10%, ventilation rate 10–15 times / h, lighting time 12 h (08:00–20:00), and light intensity 150–300 Lux. During a one-week adaptation period, the animals were allowed free access to solid food for laboratory animals (Cargil Agri Purina, Inc., Seongnam, Korea) and water. After the one-week adaptation period, they were divided into seven experimental groups (control group, scopolamine administration group, scopolamine + exercise group, scopolamine + WPH administration group, scopolamine + exercise + WPH administration group), and eight experimental animals were used in each experimental group. The whey protein hydrolysate (WPH) administration group was administered orally 100 mg or 200 ng of WPH per kg of body weight per day based on body weight and food intake. Treadmill exercise was performed five times a week for 30 minutes at a speed of 15 m / min over a 4-week experimental period. To induce memory impairment, scopolamine (Sigma-Aldrich Co., St. Louis, MO, USA) was administered intraperitoneally at 1 mg per kg of body weight 30 minutes before the cognitive test. All animal experiments in the present invention were performed under the approval of the Animal Experiment Ethics Committee of Korea University (KIACUC-2022-0076).

[0155]

[0156] 1-10. Y-maze experiment

[0157] The Y-maze animal behavioral experiment was conducted to evaluate short-term memory-type spatial recognition. The maze used was a white plastic maze consisting of three branches, each 50 cm long, 20 cm high, and 10 cm wide, and the three branches folded at a 120° angle. After designating each branch as A, B, and C, a mouse was placed at the beginning and allowed to freely roam the maze for 60 seconds. The number of times and the order in which it entered each maze were measured to assess its ability to change behavior. One point was awarded for sequential entry into different areas of the three branches, while non-consecutive entries were not recognized as points. The test administrator established basic conditions for evaluating learning and memory abilities and verified the test method by considering its rationality, accuracy, and reproducibility.

[0158]

[0159] 1-11. Novel Object Recognition Test

[0160] The novel object recognition test was conducted in a box (30х30х30 cm) made of white polyvinyl plastic. To acclimate the experimental animals to the box, the mice were allowed to freely explore it for 10 minutes for 2 days. On the third day of the experiment, WPH was administered at concentrations of 100 and 200 mg / kg 1 hour before placing the mice in the box. All experimental groups, except the control group, were intraperitoneally administered scopolamine (1 mg / kg) dissolved in 0.9% saline solution 30 minutes before the start of the experiment. After placing them in the center of the box, identical objects were presented at an equal distance (5 cm) from the two diagonal corners of the box and allowed to freely explore the objects for 5 minutes. After 24 hours of exploration, only one of the objects in the box was replaced with a novel object of a different shape, and the mice were again allowed to freely explore the objects for 5 minutes. The time taken by mice to engage in exploratory behaviors, such as touching, sniffing, and licking, toward familiar and novel objects for 5 minutes was measured. The object preference ratio and discrimination index were calculated, with a higher discrimination index indicating a higher level of recognition of the novel object.

[0161]

[0162] 1-12. Measurement of acetylcholine content

[0163] The amount of acetylcholine in the brain was measured. The method of Vincent and Newsom-Davis was modified. The extracted brain tissue was homogenized in 1 mL of PBS and centrifuged (12,000 rpm, 10 min), and the supernatant was used for analysis. Alkaline hydroxylamine reagent [3.5 N sodium hydroxide and 2 M hydroxylamine in HCl] was added to the supernatant and reacted at room temperature for 1 min. After adding 0.5 N HCl (pH 1.2) and 0.37 M FeCl3 in 0.1 N HCl, the absorbance was measured at a wavelength of 540 nm.

[0164]

[0165] 1-13. Analysis of acetylcholinesterase activity

[0166] Acetylcholinesterase activity was measured using an acetylcholinesterase activity assay kit (BM-ACH-100, BIOMAX, Seoul, South Korea). The brain tissue samples used in the experiment were homogenized in 1 mL of PBS containing 1% Triton X-100 (Sigma Aldrich), centrifuged (12,000 rpm, 10 min), and the supernatant was obtained. 50 μL of the supernatant was mixed with assay buffer, enzyme mix, substrate solution, and reaction mix containing the probe. The mixture was incubated at 37°C for 30 min under light-blocking conditions, and the absorbance was measured at 570 nm using a microplate reader in kinetic mode.

[0167]

[0168] 1-14. ROS and MDA (Malondialdehyde) Analysis (Animal Experiment)

[0169] 1 mL of 40 mM Tris-HCl buffer was added to 100 mg of tissue, the tissue was ground, centrifuged, and the supernatant was collected. 500 μL of 40 mM Tris-HCl buffer and 10 μL of 10 μM DCF-DA (2`,7`-dichlorofluorescein diacetate) were added to 50 μL of the supernatant, and the mixture was incubated at 37°C for 30 minutes. The fluorescence was measured at excitation = 482 nm, emission = 535 nm, and the ROS content in the tissue was quantified by comparison with the ROS standard curve.

[0170] MDA content in brain tissue was analyzed using the Oxitec™ TBARS Assay kit (BIOMAX Co, Ltd., UK). 1 mL of PBS was added to 100 mg of skin tissue, the tissue was ground, centrifuged, and the supernatant was collected. 200 μL of indicator solution was added to 200 μL of the supernatant, and the reaction was performed at 65°C for 45 minutes. The absorbance was then measured at 450 nm and compared with an MDA standard curve to quantify the MDA content in the tissue.

[0171]

[0172] 1-15. Western blot (animal experiment)

[0173] Approximately 50 mg of brain tissue was homogenized by adding 1000 μL of dissolution buffer [200 mM Tris (pH8.0), 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1% NP40, 1 mM PMSF, 1 mM Na3VO4, protease inhibitor cocktail], and then centrifuged (12,000 rpm, 5 min, 4°C) to collect the supernatant.

[0174] After protein quantification using the BCA method, 30 μg of protein was electrophoresed on 10% SDS-PAGE. After transfer to PVDF membrane, blocking was performed with 5% skim milk and bovine serum albumin for 1 hour, and then primary antibodies [Alpha-tubulin (Cell Signaling Technology, Inc., Cat.#2144), BAX (Cell Signaling Technology, Inc., Cat.#2772), BCL-2 (Cell Signaling Technology, Inc., Cat.#2876), PARP (Cell Signaling Technology, Inc., Cat.#9553), BDNF (Cell Signaling Technology, Inc., Cat.#47808), phosphor-tau (Cell Signaling Technology, Inc., Cat.#29957), tau (Cell Signaling Technology, Inc., Cat.#4019), ChAT (abcam, ab183591)] were reacted at 4°C for 16 hours. After washing three times with TBST, the secondary antibody (Anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, Inc., Cat.#7074) was treated and reacted at room temperature for 2 hours. After that, SuperSignal TM After dispensing Western Blot Enhancer (Thermo. Scientific, Cat. #46641), protein bands were identified using the FluorChem M Fluorescent Western Imaging System (Protein Simple, California, USA). Antibodies were diluted in 5% skim milk and bovine serum albumin according to the data sheet for each antibody.

[0175]

[0176] 1-16. Intestinal Microbiota Analysis

[0177] DNA extraction and 16S rRNA gene sequencing dsDNA was extracted from 100 mg of cecum using the QIAamp Power Fecal Pro DNA Kit (QIAGEN, Frederick, MD, USA) according to the manufacturer's protocol. The DNA concentration of all samples was adjusted to 5 ng / μL, and the uniformly concentrated DNA was subjected to a two-step PCR using the 341F and 806R primer sets to amplify V3-4 of the variable region of the 16S rRNA gene. Illumina MiSeq (Illumina) library construction was performed according to the manufacturer's protocol, and sequencing was performed by Macrogen (Seoul, Korea).

[0178]

[0179] 1-17. Statistical processing

[0180] Experimental data were analyzed using the SPSS program (SPSS 12.0 for Windows, SPSS Inc., Chicago, IL, USA). Each experimental data was expressed as percentage and mean ± SD. All measurements were subjected to one-way ANOVA, and significance was verified using the Turkey's test. The significance level for determining statistical significance was set at p<0.05.

[0181]

[0182] Example 2: Analysis of amino acid composition of whey protein hydrolysate

[0183] The amino acid composition of whey protein hydrolysate was analyzed.

[0184] The results are shown in Table 1.

[0185] As shown in Table 1, the total amino acid content of whey protein hydrolysate was 80.11±2.41 g / 100 g, which was slightly higher than that of bovine whey protein (74.09 g / 100 g) and significantly higher than that of egg, wheat, and soybean proteins (64.45 g / 100 g, 60.53 g / 100 g, and 61.57 / 100 g, respectively). In particular, the contents of aspartic acid, glutamic acid, lysine, and branched chain amino acids (isoleucine, leucine, and valine) were confirmed to be high.

[0186] Amino acid composition of whey protein hydrolysate (g / 100g) hydrolysateAsp8.61±0.28Thr5.61±0.16Ser4.28±0.14Glu14.49±0.45Pro4.11±0.01Gly1.51±0.05Ala3.90±0.11Cys0.69±0.02Val4.47±0.16Met1.69±0.0 6Ile4.85±0.15Leu8.59±0.26Tyr2.45±0.08Phe2.84±0.08His1.53±0.11Lys7. 29±0.28NH31.10±0.04Arg2.08±0.05Trp-Ile+Leu+Val17.91Total80.11±2.41

[0187] Data are presented as mean±SD.

[0188]

[0189] Example 3: Isolation and purification of whey protein

[0190] Whey protein was separated and purified to obtain fractions.

[0191] The results are shown in Figures 1a to 1f.

[0192] As shown in Fig. 1a, whey protein peptides were separated by elution with 25 mM Tris-Cl (pH 8.0) containing 0.6 M NaCl using a Q-Sepharose HiLoad 16 / 10 ion chromatography column, and a total of seven fractions were obtained. As shown in Fig. 1b, among the obtained fractions, Ca from HT22 cells 2+ The fraction that showed the greatest decrease in concentration was the EX-4 fraction. As shown in Fig. 1c, 1 mL was injected into a Superdex peptide 10 / 30 column and eluted with HPLC-grade distilled water to obtain the S14~S21 fractions. As shown in Fig. 1d, among the fractions, Ca in HT22 cells 2+ The fraction with the greatest decrease in concentration was identified as S16. As shown in Fig. 1e, the S16 fraction was completely evaporated from the solvent in a speed vac, and each fraction was dissolved in acetonitrile containing 100 to 300 μL of 0.1% TFA, and peptide RPC2 was isolated under reversed-phase chromatography analysis conditions. As shown in Fig. 1e and Fig. 1f, the amino acid sequence of the representative peptide of the second fraction EX4 / S16 / RPC2 was identified as LDIQK with 99% confidence. LDIQK (SEQ ID NO: 1) corresponds to f26 to 30 of β-lactoglobulin. The chromatograms of whey protein hydrolysate and LDIQK peptide are shown in Fig. 2.

[0193]

[0194] Additionally, the content of LDIQK in whey protein hydrolysate was measured by HPLC analysis.

[0195] The results are shown in Table 2.

[0196] Peptide LDIQK was detected at a retention time of 10.4 min. The LDIQK content was measured three times using whey protein hydrolysate, and the LDIQK content was 14.2±0.7 mg / g.

[0197]

[0198] Analysis of LDIQK in whey protein hydrolysate (mg / g)TrialRT, minLDIQK-mg / g-sample113.6513.6213.7514.0313.9614.9AVE13.7914.2±0.7RSD, %1.164.51

[0199]

[0200] Example 4: Cytotoxicity and cytoprotective effects of whey protein hydrolysate

[0201] HT22 cells were treated with whey protein hydrolysate at various concentrations (50, 100, 200, 400, and 800 μg / mL), and cell viability and cytotoxicity by H2O2 treatment were evaluated using the WST assay method.

[0202] The results are shown in Fig. 3.

[0203] As shown in Fig. 3A, when whey protein hydrolysate was treated to HT22 cells, cell viability significantly increased compared to the normal control group (p<0.001), and no cytotoxicity was observed at any concentration. As shown in Fig. 3B, whey protein hydrolysate was treated to HT22 cells induced by oxidation with H2O2 to confirm whether it protected them from oxidative toxicity. As a result, it was confirmed that the treatment group treated with H2O2 and whey protein simultaneously significantly increased cell viability compared to the control group (p<0.001), and that it had the effect of protecting cells from oxidative toxicity induced by H2O2.

[0204]

[0205] Example 5: Measurement of intracellular ROS production

[0206] DCF-DA ((2',7'-dichlorodihydrofluorescein diacetate) passes through the cell membrane and is deacetylated into DCFH ((2',7'-dichlorodihydrofluorescein) by esterase within the cell, and is then oxidized by reactive oxygen species to DCF (2',7'-dichlorofluorescein) that exhibits strong fluorescence. Therefore, the DCF-DA fluorescence emission of each treatment group was compared.

[0207] The results are shown in Fig. 4.

[0208] As shown in Fig. 4, when cells were treated with H2O2, the amount of ROS production, which increased by 185.01% compared to the NOR group, was confirmed to decrease by 162.06, 126.57, and 118.02%, respectively, when whey protein hydrolysate was treated to cells at concentrations of 50, 100, and 200 μg / mL.

[0209]

[0210] Example 6: Measurement of intracellular calcium ions

[0211] Intracellular Ca in HT22 cells induced by H2O2 apoptosis with whey protein hydrolysate and LDIQK 2+ To evaluate the effect on ion concentration, the fluorescent dye Fura-2AM was used.

[0212] The results are shown in Figures 5 and 6.

[0213] As shown in Figure 5, when cells were treated with H2O2, Ca increased by 221.12% compared to the NOR group. 2+ When this whey protein hydrolysate was treated to cells at concentrations of 50, 100, and 200 μg / mL, it was confirmed that there was a significant reduction of 146.52, 149.11, and 121.38%, respectively (p<0.001).

[0214] In addition, as shown in Fig. 6, it was confirmed that when the peptide LDIQK obtained by separation and purification from whey protein hydrolysate was treated at concentrations of 1.25, 2.5, 5, and 10 ㎍ / mL, it was significantly reduced by 134.40, 135.01, 136.40, and 101.43%, respectively (p<0.001).

[0215]

[0216] Example 7: Analysis of expression levels of apoptosis-related factors

[0217] To observe changes in the apoptosis mechanism of cells caused by whey protein hydrolysate, the expression level of Bcl-2 family proteins related to mitochondrial function regulation was measured.

[0218] The results are shown in Figures 7 and 8.

[0219] As shown in Figure 7, the BAX / BCL-2 ratio was significantly increased in the CON group treated with H2O2 compared to the NOR group (p<0.001). The BAX / BCL-2 ratio in the high-concentration whey protein hydrolysate administration group significantly decreased compared to the CON group (p<0.05), and the protein expression level of PARP was significantly increased in the WPC200 group compared to the CON group (p<0.05).

[0220] As shown in Figure 8, when a peptide (LDIQK) purified from whey protein was administered to HT22 cells, it was confirmed that the expression level of BAX / Bcl-2 was significantly reduced compared to the CON group treated with H2O2 (p<0.001).

[0221]

[0222] Example 8: Analysis of expression levels of neurotransmitter-related factors

[0223] The effects of whey protein hydrolysate on neurotransmitters were evaluated through the protein expression levels of ChAT, BDNF, p-Tau, and Tau.

[0224] The results are shown in Fig. 9.

[0225] As shown in Fig. 9, the protein expression levels of BDNF and ChAT in HT22 cells were measured and significantly decreased in the CON group compared to the NOR group (p<0.001), and the phosphorylation level of Tau was significantly increased in the CON group compared to the NOR group (p<0.001). When whey protein (WPC) hydrolysate was treated to HT22 cells induced with oxidative stress by H2O2, the protein expression levels of BDNF and ChAT were significantly increased in the WPC200 group (p<0.01, p<0.05 respectively), and the expression level of p-Tau was significantly decreased in the WPC200 group compared to the CON group (p<0.001).

[0226]

[0227] Example 9: Analysis of expression levels of antioxidant-related factors

[0228] The effects of whey protein hydrolysate on antioxidant-related factors were evaluated through the protein expression levels of Nrf2 and HO-1.

[0229] The results are shown in Fig. 10.

[0230] As shown in Fig. 10, it was confirmed that the expression levels of Nrf2 and HO-1 in the CON group treated with H2O2 in HT22 cells were significantly reduced compared to the NOR group (p<0.001, p<0.05). In addition, when whey protein hydrolysate was administered to the cells at a high concentration, it was confirmed that the expression levels of Nrf2 and HO1 were significantly increased compared to the CON group (p<0.001).

[0231]

[0232] In the present invention, the neuroprotective effect of whey protein hydrolysate was evaluated in HT22 cells induced with oxidative stress by H2O2. In the experiment, it was confirmed that whey protein hydrolysate suppressed protein expression of BAX and p-Tau, intracellular ROS and Ca2+, and increased the expression of Bcl-2, BDNF, ChAT, and PARP in HT22 cells induced with oxidative stress by H2O2, thereby inhibiting apoptosis. In addition, an active peptide (LDIQK) with an anti-apoptotic function was identified in whey protein hydrolysate.

[0233] One of the most important pathological factors that induces neuronal death in neurodegeneration is oxidative stress, and the activation of apoptotic signaling pathways by excessive production of ROS has been proven to be the most important mechanism for inducing neuronal death. H2O2 acts as a signaling substance (regulating cell movement or function), but due to its strong oxidizing power, it can be converted into hydroxyl radicals, and when it accumulates excessively in the human body, it causes oxidative stress, generates ROS, and increases intracellular Ca. 2+ Increases the level. Therefore, H2O2 is one of the substances widely used in vitro and cellular systems to monitor various pharmacological activities under conditions of oxidative stress. Neuronal damage due to oxidative stress is caused by reactive oxygen species causing damage to major cellular components (lipids, proteins, nucleic acids, etc.) and ultimately inducing cell death. In the present invention, it was confirmed that whey protein hydrolysate significantly reduced the increased ROS and Ca2+ due to H2O2 treatment.

[0234] Bcl-2 family proteins, which are composed of proteins that regulate the promotion and inhibition of apoptosis, play a pivotal role in determining the progression of apoptosis. Among them, Bax, a representative apoptosis promoter, is located in the mitochondrial outer membrane and induces the release of pro-apoptotic factors by promoting mitochondrial permeability transition (MPT) or weakening the MMT barrier function. Conversely, anti-apoptotic proteins such as Bcl-2 are essential for maintaining mitochondrial permeability and membrane barrier stability to suppress the release of pro-apoptotic factors. Therefore, the expression balance between Bax and Bcl-2 family proteins serves as a critical determinant of apoptosis induction and inhibition. In this experiment, we confirmed that whey protein hydrolysate and the peptide LDIQK significantly decreased Bax protein expression and significantly increased Bcl-2 protein expression in H2O2-induced oxidative stress-induced HT22 cells.

[0235] HO-1 is a protein that plays a major role in the antioxidant system, and many studies have shown that the mechanism for HO-1 protein expression is most directly related to the nuclear transcription of nuclear factor-E2-related factor2 (Nrf2). Nrf2, as a transcription factor, binds to the Antioxidant Response Element (ARE) present in the genes of antioxidant proteins such as HO-1, thereby regulating the expression of these genes and promoting protein expression, thereby playing a central role in the biological defense mechanism against oxidative stress. In the present invention, it was confirmed that the administration of whey protein hydrolysate increased the expression of HO-1 and Nrf2, which were reduced by oxidative stress caused by H2O2 in HT22 cells.

[0236] Factors that have been suggested for cognitive dysfunction include not only hippocampal cell death, but also a decrease in BDNF (brain driven neurotrophic factor), hyperphosphorylation of tau protein, and decreased acetylcholine production. BDNF (brain driven neurotrophic factor) is a type of nerve growth factor that plays an important role in the growth, development, plasticity, and survival of nerve cells. In the hippocampal region of the brain, a decrease in neuroplasticity and a decrease in neurogenesis with age lead to a decline in learning, memory, and cognitive functions, and further induce neurodegenerative diseases such as Alzheimer's disease. In the present invention, it was confirmed that the administration of whey protein hydrolysate significantly reduced the expression of BDNF in HT22 cells induced by oxidative stress.

[0237] Tau is a microtubule-associated protein (MAP) primarily expressed in the central nervous system, particularly in the axons of neurons, and plays a role in stabilizing microtubules (MTs) (Drechsel et al., 1992). Tau expressed in humans has six isoforms resulting from alternative splicing of exons 2, 3, and 10 of a single gene (Goedent et al., 1989). Specifically, splicing of exon 10 results in isoforms containing three or four microtubule-binding repeats. Although the cause of tau aggregation is not precisely understood, hyperphosphorylation of tau observed in all tauopathies has been reported to weaken MT binding and increase aggregation (Lonso et al., 1996; Aonso et al., 1994). Hyperphosphorylation of tau causes loss of function (LOF) of the tau protein, leading to its detachment from MTs and weakening their stability. This leads to abnormalities in axonal transport of neurons, leading to degeneration. In this study, we confirmed that administration of whey protein hydrolysate significantly reduced tau hyperphosphorylation in HT22 cells.

[0238] Acetylcholine (ACh) is a neurotransmitter used by all cholinergic neurons and plays an important role in the peripheral and central nervous systems. It is degraded by AChE in the synaptic cleft, and choline acetyltransferase (ChAT) is an enzyme that synthesizes the neurotransmitter acetylcholine and is a factor that can observe the functional status of cholinergic neurons (Ferreira-Vieira et al., 2016; Lim et al., 2016). In AD, the loss of choline acetyltransferase activity in the cerebral cortex and hippocampus is related to the degree and duration of dementia, and thus serves as the basis for treatment. Increasing ChAT activity in the forebrain cortex and hippocampus is a key factor in preventing the transition of mild cognitive impairment to AD. In the present invention, it was confirmed that the administration of whey protein hydrolysate significantly increased the protein expression of ChAT in HT22 cells.

[0239] LDIQK, the active ingredient in whey protein hydrolysate, inhibited Ca influx and showed a regulating effect on Bax and Bcl-2, which are cell death-related factors. In other words, LDIQK contained in whey protein was found to improve cognitive function.

[0240]

[0241] Example 10: Verification of the effect of improving cognitive ability

[0242] The memory improvement efficacy was measured in vivo using the Y-maze test and novel object recognition test (NORT) in mice with memory impairment induced by scopolamine.

[0243] The results are shown in Figures 11A and 11B.

[0244] As shown in Fig. 11A, to confirm the effect of whey protein hydrolysate (WPH) administration in the cognitive dysfunction model induced by scopolamine administration, a behavioral analysis was performed using a Y-maze. As a result, the behavioral change ability of the normal NOR group was 79.99%, whereas the behavioral change ability of the CON group administered scopolamine decreased to 42.255%. After administering WPH at 100 and 200 mg / kg, the experimental group administered scopolamine showed a concentration-dependent increase of 54.1% and 62.7%, and in the EXR group that performed treadmill exercise and the EWPHL_EWPHH group that combined treadmill exercise and WPH administration, the behavior ability significantly increased by 61.4%, 66.1%, and 64.7% compared to the CON group, respectively (p<0.001). Additionally, there was no significant difference in the total number of entries into each branch across all experimental groups (Figure 11B). Combining whey protein hydrolysate with exercise tended to increase spontaneous alternation.

[0245]

[0246] To confirm the improvement effect of whey protein hydrolysate administration on long-term memory and recognition memory in a model of cognitive dysfunction induced by scopolamine administration, a novel object recognition test was conducted.

[0247] The results are shown in C of Fig. 11.

[0248] As shown in Fig. 11C, by analyzing the time to explore a novel object and an existing object, the discrimination index (time taken to explore an existing object or a novel object / total experimental time * 100) was calculated. As a result, the CON group administered scopolamine showed a significant decrease in the object discrimination index compared to the normal group (p<0.05), and it was confirmed that recognition memory was damaged as there was no significant difference in the time to explore a novel object and an existing object. On the other hand, the discrimination index was significantly increased in the EXR, WPHL, WPHH, EWPL, and EWPH groups compared to the CON group, confirming that the recognition memory damage induced by scopolamine was significantly recovered (p<0.001).

[0249]

[0250] Example 11: Antioxidant effect analysis

[0251] To verify the antioxidant effect and cognitive function improvement effect of whey protein hydrolysate, the ROS content in brain tissue was measured using DCF-DA fluorescent dye, the amount of ROS produced, and the MDA content.

[0252] The results are shown in Fig. 12.

[0253] As shown in Fig. 12, the measurement results confirmed that the ROS content increased by 185.6% in the CON group administered scopolamine compared to the NOR group. It was confirmed that the ROS content was reduced by 132.1% and 129.4% when WPH was administered at 100 and 200 mg / kg. In the EXR group that only performed treadmill exercise and the EWPHL and EWPHH groups that combined exercise with whey protein hydrolysate, the ROS content was significantly reduced by 124.0%, 129.6%, and 109.8% compared to the CON group, respectively (p<0.001). These results indicate that WPH administration alleviates oxidative stress caused by ROS.

[0254] As shown in Figure 12, the MDA content of the CON group administered scopolamine was 52.6 uM, which was significantly higher than that of the NOR group at 12.1 uM (p<0.001). Conversely, the MDA content of the EXR, WPHH, EWPHL, and EWPHH groups administered exercise and WPH was 27.1, 34.6, 29.9, and 24.8 uM, which were significantly reduced compared to the CON group (p<0.001). The above results indicate that whey protein hydrolysate has an oxidative stress-relieving effect and a cognitive function-improving effect. In addition, a synergistic effect could be expected when whey protein hydrolysate administration was combined with exercise.

[0255]

[0256] Example 12: Verification of acetylcholine activity

[0257] Changes in acetylcholine activity following administration of whey protein hydrolysate were confirmed.

[0258] The results are shown in Fig. 13.

[0259] As shown in Figure 13, in the brain tissue of the CON group mice administered scopolamine, the acetylcholine concentration was significantly reduced by 0.6 times compared to the NOR group, the activity of the acetylcholine decomposition enzyme Ache was increased by 2.7 times, and the protein expression of the acetylcholine synthase ChAT was reduced by 3.3 times. These results indicate that scopolamine administration induced cognitive dysfunction by acting as an antagonist of the acetylcholine receptor. However, administration of WPH restored the acetylcholine lowered by scopolamine administration. The acetylcholine concentration was significantly increased by 1.3 times in the EXR group, WPH_H group, and EWPH_H group compared to the CON group (p<0.01). The activity of Ache was significantly reduced by 0.8-fold, 0.8-fold, 0.5-fold, 0.5-fold, and 0.4-fold in the EXR, WPHL, WPHH, EWPHL, and EWPHH groups (p<0.001). The protein expression of ChaT was confirmed to be significantly increased by 309.12% in the EWPHH group compared to the CON group (p<0.05). These results indicate that WPH administration increases the lowered acetylcholine concentration.

[0260]

[0261] Example 13: Analysis of the effect of whey protein hydrolysate on cell death

[0262] The effect of whey protein hydrolysate on cell death was analyzed.

[0263] The results are shown in Figures 14A and 14B.

[0264] As shown in Fig. 14A and Fig. 14B, the CON group administered scopolamine showed a significantly increased BAX / BCL2 ratio compared to the NOR group. However, the EXR group that performed treadmill exercise, the WPH_L and WPH_H groups administered WPH, and the EWPH_L and EWPH_H groups administered exercise and WPH showed a significant decrease in the BAX / BCL2 ratio (31.6%, 32.7%, 31.3%, 41.6%, and 27.6%, respectively; p<0.001, fig.). These results indicate that WPH administration can inhibit scopolamine-induced apoptosis.

[0265]

[0266] Example 14: Analysis of the Effect of Whey Protein Hydrolysate on Neuronal Function

[0267] The effects of whey protein hydrolysate on neuronal function were analyzed.

[0268] The results are shown in Figures 14A, 14C and 14D.

[0269] As shown in Figures 14A, 14C, and 14D, whey protein hydrolysate regulated regulatory factors (p-tau and BDNF) related to neuronal function. Administration of scopolamine significantly increased (3.4-fold) the p-tau / tau protein ratio, a neurodegenerative marker in the brain, compared to the NOR group. When treadmill exercise and WPH (100 and 200 mg / kg) were administered together with exercise and WPH (100 and 200 mg / kg), the p-tau / tau ratio decreased in a dose-dependent manner (33.4%, 32.5%, 24.6%, 41.9%, and 28.9%, respectively).

[0270] Scopolamine significantly reduces brain-derived neurotrophic factor (BDNF) expression, thereby disrupting neuroplasticity. However, when WPH (100 and 200 mg / kg) was administered concurrently with treadmill exercise, BDNF expression was significantly reduced (150.8%, 151.1%, 153.3%, and 168.3%, respectively, p<0.05, fig.). These results suggest that WPH administration can improve impaired neuronal function.

[0271]

[0272] Example 15: Analysis of the effect of whey protein hydrolysate on the MAPK pathway.

[0273] The effects of whey protein hydrolysate on the MAPK signaling pathway were evaluated in brain tissue.

[0274] The results are shown in Fig. 15.

[0275] As shown in Figure 15, administration of scopolamine significantly increased the protein expression of p-JNK, p-ERK, and p-P38, which are key components of the MAPK signaling pathway, compared to the NOR group (2.3, 2.8, and 1.9 times, respectively, p<0.01). However, in the EXR group that performed treadmill exercise, the WPH_L and WPH_H groups that were administered WPH, and the EWPH_L and EWPH_H groups that were administered both exercise and WPH, the expression of p-JNK was reduced by 53.3%, 50.9%, 42.4%, 41.1%, and 39.8%, respectively, compared to the CON group, the expression of p-ERK was reduced by 50.7%, 71.8%, 50.3%, 57.2%, and 51.1%, respectively, and the expression of p-P38 was reduced by 70.2%, 78.2%, 75.3%, 59.5%, and 47.3%, respectively. These results indicate that exercise and administration of whey protein hydrolysate inhibit the MAPK signaling pathway activated by oxidative stress.

[0276]

[0277] Example 16: Analysis of the Effects of Whey Protein Hydrolysate on Intestinal Microflora

[0278] The effects of whey protein hydrolysate on intestinal microflora were evaluated in cecum tissue.

[0279] The results are shown in Figs. 16, 17 and 18a to c.

[0280] As shown in Fig. 16, the Shannon index representing the diversity of the microbiome and the CHAO index representing the richness tended to decrease in the CON group compared to the NOR group. When treadmill exercise and WPH (100 and 200 mg / kg) administration and exercise and WPH (100 and 200 mg / kg) were administered together, the Shannon index increased by 112%, 112%, 114%, 108%, and 110%, respectively, compared to the CON group, and the CHAO index increased by 123%, 114%, 120%, 113%, and 116%, respectively. When PcoA analysis was performed in relation to beta diversity, it was confirmed that the microbiome community structure was different in the CON and NOR groups administered scopolamine, and it was confirmed that the administration of treadmill exercise and WPH transformed it into a different community structure from the CON group.

[0281] As shown in Figure 17, the proportion of Verrucomicrobiota that increased in the CON group was confirmed to significantly decrease in the EXR group that exercised, the WPH_L and WPH_H groups that were administered WPH, and the EWPH_L and EWPH_H groups that were administered exercise and WPH, and the proportion of Actinomycetota in the EWPH_H group was confirmed to significantly increase compared to the CON group (p<0.01).

[0282] As shown in Figures 18 a to c, at the genus level, the proportion of Lactobacillus tended to increase compared to the CON group when exercise and 200 mg / mL WPH were simultaneously treated, and Eubacterium was confirmed to significantly increase compared to the CON group when 100 mg / kg WPH was administered and when exercise and 200 mg / kg WPH were simultaneously treated (p<0.01). Additionally, Clostridium was confirmed to significantly increase compared to the CON group when 200 mg / kg WPH was treated (p<0.01). These results indicate that treadmill exercise alters the gut microbiota, which is altered by scopolamine.

[0283]

[0284] In the present invention, the inventors studied the effects of whey protein hydrolysate (WPH) on scopolamine-induced cognitive dysfunction. Scopolamine, a muscarinic cholinergic receptor antagonist, has been reported to induce memory impairment by inducing cholinergic dysfunction and oxidative stress in the brain. Furthermore, scopolamine-administered animal models are widely used to evaluate the memory-enhancing efficacy of materials for the prevention and treatment of AD and to elucidate their mechanisms.

[0285] In the present invention, the cognitive function-improving effects of whey protein hydrolysate, treadmill exercise, and their combination on scopolamine-induced memory impairment were measured using the Y-maze and Novel object recognition (NOR) tests. The experimental animals were placed in the Y-maze and allowed to move freely, and the number of times they entered the Y-maze was sequentially measured. It was confirmed that the spontaneous change behavior, expressed as a percentage, was significantly reduced in the CON group compared to the NOR group, confirming that the memory and cognitive impairment model was well established. In addition, it was confirmed that the EXR group that performed treadmill exercise, the WPH_L and WPH_H groups administered 100 and 200 mg / kg of WPH, and the EWPH_L and EWPH_H groups that were simultaneously treated with exercise and 100 and 200 mg / kg of WPH recovered to a level similar to the NOR group, confirming that memory was improved. In the NOR test, there was no significant difference in the time taken to explore a familiar object compared to a novel object when scopolamine was administered. These results suggest that the discrimination index was significantly reduced in the animal group administered scopolamine, which induces cholinergic dysfunction and oxidative stress, and that learning and cognitive processes were impaired. However, in the EXR, WPH_L, WPH_H, EWPH_L, and EWPH_H groups, the time taken to explore a novel object was significantly increased compared to the time taken to explore a familiar object. This suggests that exercise and WPH administration alleviate the cognitive impairment induced by scopolamine.

[0286] The cholinergic nervous system is a representative nervous system related to cognition and memory, and scopolamine increases the level of AChE and decreases the levels of ACh and ChAT. Acetylcholine (Ach) is known as a major neurotransmitter and regulator of the nervous system, and is known to play an important role in cognitive functions such as learning and memory in the neuromuscular junction and parasympathetic nervous system. Increased activity of enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain breaks down the neurotransmitter acetylcholine into choline and acetyl CoA, inducing cholinergic dysfunction, which is known to affect memory and cognitive function impairment. In the present invention, it was confirmed that administration of WPH significantly decreased the activity of AChE and significantly increased the protein expression of ChAT, thereby increasing the concentration of acetylcholine. These results, similar to the previous behavioral analysis results, indicate that treadmill exercise and WPH protect cognitive function by regulating the cholinergic nervous system.

[0287] Acetylcholine, which is involved in cognitive function, is closely related to oxidative stress in the brain. Brain tissue is rich in unsaturated fatty acids and is therefore sensitive to oxidative stress. Increased reactive oxygen species (ROS) lead to lipid peroxide accumulation, protein denaturation, DNA oxidation, and cell damage, inhibiting physiological activity. This oxidative stress leads to peroxidation of the unsaturated fatty acids surrounding neurons, which increases the activity of acetylcholinesterase (AChE), accelerating the breakdown of acetylcholine, disrupting neurotransmission, and ultimately leading to decreased memory and learning abilities. The results of this study confirmed that exercise and whey protein hydrolysate (WPH) administration significantly reduced scopolamine-induced ROS and MDA. These results suggest that treadmill exercise and WPH reduce ROS and MDA, inhibiting the breakdown of acetylcholine, and improving cognitive function.

[0288] Furthermore, the Bax / Bcl-2 ratio, which increases due to oxidative stress, is an important factor in regulating apoptosis. Bcl-2 and Bcl-xL inhibit apoptosis, while Bax, Blk, and Bad promote apoptosis. Bax translocates into the mitochondria to become an activated monodimer, promoting apoptosis, and this Bax-induced cell death is inhibited by heterodimerization with Bcl-2. Previous studies on exercise, synaptic plasticity, and cell proliferation have shown that exercise increases the expression of proteins involved in synaptic efficacy and learning in key brain regions, as well as the expression of Bcl-2, an anti-apopotosis marker. In contrast, it has been shown to alleviate cognitive dysfunction by suppressing the expression of Bax and the caspase family, which are major promoters of apoptosis as downstream pro-apoptotic pathway members. In the present invention, we confirmed that treadmill exercise and WPH administration at concentrations of 100 and 200 mg / kg significantly lowered the BAX / Bcl-2 ratio. These results suggest that WPH exerts a positive effect on cognitive function by inhibiting apoptosis.

[0289] Oxidative stress, which is involved in cognitive function, is caused by the deposition of Aβ and tau proteins in the brain. Hyperphosphorylation of tau protein is reported to be associated with neurodegenerative diseases, causing synaptic loss and neuronal cell death. In the present invention, we confirmed that 4 weeks of moderate-intensity treadmill exercise and administration of whey protein hydrolysate at concentrations of 100 and 200 mg / kg significantly inhibited tau hyperphosphorylation.

[0290] Under oxidative stress, mitogen-activated protein kinases (MAPKs) are signaling molecules that regulate cell death, with JNK and p38 MAP kinase playing a pivotal role. Activation of the MAPk pathway (extracellular-signal regulated kinase 1 / 2 (ERK1 / 2), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase) is known to be a symptom observed in Alzheimer's disease patients. We confirmed that moderate-intensity treadmill exercise and whey protein hydrolysate administration significantly reduced activated MAPk. These results suggest that whey protein hydrolysate is effective in improving cognitive function.

[0291] Gut microbes comprise 95% of the human microbiome and form a two-way microbiome-gut axis that communicates through cytokines, immune systems, hormones, and neural signals. Gut microbes, which coexist with human life, lose their diversity with aging, primarily causing gastrointestinal problems such as decreased digestive function. Recently, various studies have reported that gut microbes and their metabolites affect the brain, and this correlation is explained as the gut-brain axis. Currently, gut microbes known to be associated with cognitive function include verrucomicrobiota, which are significantly increased in AD patients and the elderly; Actinobacterota, known to inhibit the activity of AchE, an enzyme that breaks down acetylcholine; and eubacterium, known as beneficial bacteria with anti-inflammatory properties. In the present invention, species richness and evenness were investigated using the Chao index and Shannon index based on α-diversity, respectively. As a result, it was confirmed that treadmill exercise and WPH administration increased the species richness and evenness that were reduced by scopolamine administration. In addition, when treadmill exercise and 200 mg / kg WPH were administered simultaneously, the proportion of verrucomicrobiota significantly decreased, and the proportions of actinobacterota and eubacterium significantly increased. These results indicate that WPH is effective in improving cognitive function by regulating intestinal microorganisms related to cognitive function.

Claims

1. A peptide consisting of an amino acid sequence represented by sequence number 1.

2. A peptide according to claim 1, wherein the peptide has the properties of reducing the expression of a cell death factor, reducing the concentration of intracellular calcium ions, or a combination thereof.

3. A nucleic acid molecule comprising a nucleotide sequence encoding the peptide of claim 1.

4. A recombinant vector comprising the nucleic acid molecule of paragraph 3.

5. A host cell containing the recombinant vector of paragraph 4.

6. A pharmaceutical composition for preventing or treating a degenerative neurological disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

7. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by sequence number 1, in accordance with paragraph 6.

8. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the whey protein hydrolysate in paragraph 7 has the properties of increasing cell viability, alleviating oxidative stress, decreasing intracellular calcium ion concentration, decreasing apoptotic factor expression, regulating neurotransmitter expression, improving cognitive ability, decreasing tau protein phosphorylation, or a combination thereof.

9. A pharmaceutical composition for preventing or treating a degenerative neurological disease, wherein the content of the peptide in paragraph 7 is 0.5 mg / g to 40 mg / g based on the total weight of whey protein hydrolysate.

10. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the whey protein hydrolyzate in paragraph 7 is obtained by first hydrolyzing whey protein with an endo protease derived from Bacillus licheniformis and second hydrolyzing whey protein with an exo protease derived from Aspergillus oryzae.

11. A pharmaceutical composition for preventing or treating a neurodegenerative disease, wherein the endo protease derived from Bacillus licheniformis is Alcalase, Protamex, or a mixed enzyme thereof; and the exo protease derived from Aspergillus oryzae is Flavorzyme.

12. A pharmaceutical composition for preventing or treating a degenerative neurological disease, wherein the mixed enzyme in claim 11 is a mixture of Alcalase and Protamex in a weight ratio of 1:0.5 to 1:

2.

13. A food composition for preventing or improving a degenerative neurological disease, comprising a peptide consisting of an amino acid sequence represented by sequence number 1.

14. A food composition for preventing or improving a degenerative neurological disease, comprising a whey protein hydrolysate comprising a peptide consisting of an amino acid sequence represented by sequence number 1, in accordance with paragraph 13.

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