CN108939039B - Application of spirulina protein zymolyte and preparation method thereof - Google Patents
Application of spirulina protein zymolyte and preparation method thereof Download PDFInfo
- Publication number
- CN108939039B CN108939039B CN201810897805.9A CN201810897805A CN108939039B CN 108939039 B CN108939039 B CN 108939039B CN 201810897805 A CN201810897805 A CN 201810897805A CN 108939039 B CN108939039 B CN 108939039B
- Authority
- CN
- China
- Prior art keywords
- protein
- spirulina
- adjusting
- spirulina protein
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 102
- 240000002900 Arthrospira platensis Species 0.000 title claims abstract description 102
- 229940082787 spirulina Drugs 0.000 title claims abstract description 101
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 96
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 94
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 230000002776 aggregation Effects 0.000 claims abstract description 21
- 238000004220 aggregation Methods 0.000 claims abstract description 21
- 108010040003 polyglutamine Proteins 0.000 claims abstract description 17
- 206010029350 Neurotoxicity Diseases 0.000 claims abstract description 11
- 206010044221 Toxic encephalopathy Diseases 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 11
- 231100000228 neurotoxicity Toxicity 0.000 claims abstract description 11
- 230000007135 neurotoxicity Effects 0.000 claims abstract description 11
- 231100000189 neurotoxic Toxicity 0.000 claims abstract description 9
- 230000002887 neurotoxic effect Effects 0.000 claims abstract description 9
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 18
- 239000003531 protein hydrolysate Substances 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000000284 extract Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 11
- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- 102000003802 alpha-Synuclein Human genes 0.000 claims description 10
- 108090000185 alpha-Synuclein Proteins 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 10
- 230000008014 freezing Effects 0.000 claims description 10
- 239000012460 protein solution Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000004365 Protease Substances 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- 238000010257 thawing Methods 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 6
- 230000006269 (delayed) early viral mRNA transcription Effects 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 108091005658 Basic proteases Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 108090000526 Papain Proteins 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- 229940055729 papain Drugs 0.000 claims description 5
- 235000019834 papain Nutrition 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 230000001988 toxicity Effects 0.000 claims description 4
- 231100000419 toxicity Toxicity 0.000 claims description 4
- 230000003301 hydrolyzing effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 24
- 201000010099 disease Diseases 0.000 abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 12
- 229940079593 drug Drugs 0.000 abstract description 6
- 229920000155 polyglutamine Polymers 0.000 abstract description 6
- 208000032859 Synucleinopathies Diseases 0.000 abstract description 5
- 239000002537 cosmetic Substances 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 3
- 241000244206 Nematoda Species 0.000 description 34
- 238000012360 testing method Methods 0.000 description 32
- 241000244203 Caenorhabditis elegans Species 0.000 description 29
- 230000009261 transgenic effect Effects 0.000 description 28
- 230000004083 survival effect Effects 0.000 description 15
- 230000032683 aging Effects 0.000 description 10
- 238000005406 washing Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000010998 test method Methods 0.000 description 7
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 230000036542 oxidative stress Effects 0.000 description 6
- 230000004845 protein aggregation Effects 0.000 description 6
- 208000024827 Alzheimer disease Diseases 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- 230000003712 anti-aging effect Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000003399 chemotactic effect Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 4
- 229960002949 fluorouracil Drugs 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000019419 proteases Nutrition 0.000 description 4
- 230000035939 shock Effects 0.000 description 4
- 239000013543 active substance Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 208000023105 Huntington disease Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000035605 chemotaxis Effects 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000008642 heat stress Effects 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010067889 Dementia with Lewy bodies Diseases 0.000 description 1
- QSJXEFYPDANLFS-UHFFFAOYSA-N Diacetyl Chemical group CC(=O)C(C)=O QSJXEFYPDANLFS-UHFFFAOYSA-N 0.000 description 1
- 241000218671 Ephedra Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 201000002832 Lewy body dementia Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 230000006933 amyloid-beta aggregation Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000031902 chemoattractant activity Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 235000004626 essential fatty acids Nutrition 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000004558 lewy body Anatomy 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000001508 potassium citrate Substances 0.000 description 1
- 229960002635 potassium citrate Drugs 0.000 description 1
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 1
- 235000011082 potassium citrates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 239000009537 qingyi Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 208000002320 spinal muscular atrophy Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/195—Proteins from microorganisms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Birds (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Dermatology (AREA)
- Immunology (AREA)
- Hospice & Palliative Care (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Molecular Biology (AREA)
- Psychiatry (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to application of a spirulina protein zymolyte and a preparation method thereof. The spirulina protein zymolyte has high biological activity, can obviously delay senility, has obvious effect of inhibiting the aggregation of neurotoxic protein or relieving neurotoxicity, has certain relieving and treating effect on polyglutamine disease and alpha-synucleinopathy, can be used for preparing medicines, health-care products and cosmetics, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of a spirulina protein zymolyte and a preparation method thereof.
Background
With the increasing aging of population, the pressure of aging and related diseases in the aging process on society is increasing, researchers are gradually aware that not only the life is prolonged, but also the related diseases in the aging process are prevented and treated, and the life quality of the old is improved, so that the prolonging of the health life becomes the key for researching the anti-aging field.
Polyglutamine (polyQ) diseases are common neurodegenerative diseases in the aging process, and the diseases are caused by the fact that the coding protein of the gene, namely polyQ protein, generates polyglutamine extension mutation due to CAG trinucleotide repeat extension mutation of the coding region of a pathogenic gene, wherein typical diseases comprise Huntington chorea (HD), bulbar spinal muscular atrophy (SBMA) and the like. Alpha-synucleinopathies are diseases with pathological features of alpha-synuclein, including Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease with Lewy body variants, etc., and the common pathological feature is that alpha-synuclein selectively accumulates in vulnerable neurons and glial cells to form inclusion bodies. At present, the treatment means for the two types of neurological diseases are quite limited, only symptoms can be improved but the disease progress cannot be stopped, and the research progress of related treatment medicines is very slow.
Spirulina is a kind of lower prokaryote, is composed of single cells or multiple cells, contains rich active substances such as protein, trace elements, essential amino acids, essential fatty acids, carotenoids, vitamins and the like, has the functions of reducing cholesterol, regulating blood sugar, enhancing immune system, protecting intestines and stomach, resisting tumor and the like, and is widely applied to the research fields of food, medicines and the like at present. For example, chinese patent application CN101928743A discloses an enzymatic hydrolysate of phycocyanin of spirulina and its application, wherein the enzymatic hydrolysate is prepared by using trypsin, lichenibacillus protease, chymotrypsin, pepsin or their combination for enzymolysis, and can be applied to reduce the level of free radicals or enhance the individual antioxidant system. Chinese patent application CN106923337A discloses a preparation method and application of spirulina full-active substance, which adopts methods of solvent extraction with different polarities (from small to large), compound protease enzymolysis and the like to extract and prepare the spirulina full-active substance, and can be used for reducing blood sugar, blood fat and the like. The two patent applications carry out enzymolysis preparation on the spirulina, and the obtained product has good effects of resisting oxidation, reducing blood fat, reducing blood sugar and the like, but the application of spirulina zymolyte in the aspects of resisting aging, inhibiting neurotoxic protein aggregation, relieving neurotoxicity and the like is not mentioned.
Therefore, there is an urgent need to develop the use of spirulina protein zymolyte for delaying aging, inhibiting the aggregation of neurotoxic protein and relieving neurotoxicity and the preparation method thereof.
Disclosure of Invention
The invention aims to provide the application of spirulina protein zymolyte in delaying senility, inhibiting neurotoxic protein aggregation and relieving neurotoxicity and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
use of spirulina protein zymolyte in preparing medicine or health product for inhibiting neurotoxic protein aggregation or relieving neurotoxicity is provided.
Further, the neurotoxic protein includes polyQ protein and α -synuclein.
Furthermore, the medicine or health care product can relieve toxicity caused by aggregation of the A beta protein.
In addition, the invention also provides a polyQ protein aggregation inhibitor, which comprises effective amount of spirulina protein zymolyte.
In addition, the invention also provides an alpha-synuclein aggregation inhibitor, which comprises an effective amount of spirulina protein zymolyte.
In addition, the invention also provides an A beta protein toxicity relieving agent, which comprises effective amount of spirulina protein zymolyte.
In addition, the invention also provides the application of the spirulina protein zymolyte in preparing anti-aging medicaments, health-care products or cosmetics.
In addition, the invention also provides an anti-aging preparation which comprises effective amount of spirulina protein zymolyte.
In addition, the invention also provides an anti-aging cosmetic which contains spirulina protein zymolyte.
In addition, the invention also provides a preparation method of the spirulina protein zymolyte, which comprises the following steps:
s1, wall breaking: preparing suspension with the weight volume percentage of 8-12% by using Tris-HCl buffer solution, adjusting the pH to 7.0-7.5, soaking for 1-3 days, freezing at-20-40 ℃ for 12-24 h, then unfreezing at 4-25 ℃, repeatedly freezing and thawing for 3-5 times, and centrifuging at 5000-8000 rpm for 10-30 min to obtain supernatant;
s2, protein isolate: taking the supernatant obtained in the step S1, slowly adding NH4SO4 until the saturation degree is 50%, continuously stirring, standing overnight at 4 ℃, centrifuging at 5000-8000 rpm for 10-30 min, taking the precipitate to redissolve in primary water, placing in a dialysis belt of 3.5KDa, dialyzing for 2-3 d with flowing water, dialyzing for 3-5 times with deionized water, changing the deionized water every 12h, taking the dialyzed protein solution, and freeze-drying to obtain the spirulina protein extract;
s3, enzymolysis: dissolving the spirulina protein extract obtained in the step S2 in primary water to prepare a protein solution with the weight volume percentage of 15-30%, adjusting the pH to 7.0-8.0 by using 0.5mol/L NaOH solution, adding alkaline protease according to 4000-4500U/g, adjusting the temperature to 45-60 ℃, stirring and performing enzymolysis for 120-180 min, cooling to room temperature, adjusting the pH to 6.0-7.0 by using 1mol/L HCl, adding papain according to the weight percentage of 3.5-5.0% of enzyme and substrate, adjusting the temperature to 55-65 ℃, stirring and hydrolyzing for 180-250 min, placing in a constant-temperature water bath kettle at 95-100 ℃ for 10-15 min, cooling and freeze-drying to obtain the spirulina protein extract.
The application and the preparation method of the spirulina protein zymolyte of the invention lead the cell wall to be fully cracked by repeated freeze thawing and the cell content to be dissolved out; the ammonium sulfate precipitation method is adopted to carry out coarse separation on the protein, and dialysis is carried out to remove salt, so that the influence of impurities on the subsequent biological activity test is avoided; then, the obtained spirulina protein is subjected to stepwise enzymolysis by adopting a two-step enzymolysis method, so that the spirulina protein zymolyte with the effects of delaying senility, inhibiting neurotoxic protein aggregation and relieving neurotoxicity can be obtained. The experimental examples 1-3 show that the spirulina protein zymolyte can obviously improve the survival rate of the caenorhabditis elegans under natural conditions, heat shock conditions and oxidative stress conditions, and the experimental example 4 shows that the spirulina protein zymolyte prepared by the method can reduce the aggregation of caenorhabditis elegans lipofuscin and reduce the formation of color spots, which indicates that the spirulina protein zymolyte prepared by the method can effectively prolong the service life and delay aging; test example 5 shows that the spirulina protein zymolyte prepared by the invention can obviously inhibit the aggregation of polyQ protein and has certain relieving and treating effects on polyglutamine disease; the test example 6 shows that the spirulina protein zymolyte prepared by the invention can obviously inhibit the aggregation of alpha-synuclein, and the test example 7 shows that the spirulina protein zymolyte prepared by the invention can effectively relieve the neurotoxicity caused by the aggregation of Abeta protein, which indicates that the spirulina protein zymolyte prepared by the invention has certain relieving and treating effects on alpha-synucleinopathy, in particular to Alzheimer disease.
The invention has the following advantages:
(1) the spirulina protein zymolyte prepared by the preparation method of the spirulina protein zymolyte has high biological activity, can obviously delay senility, has obvious neuroprotective effect, has certain relieving and treating effect on polyglutamine disease and alpha-synucleinopathy, and can obviously improve the later life quality of the sick old.
(2) The preparation method of the spirulina protein zymolyte firstly breaks the wall, then separates the protein and then carries out enzymolysis, has simple steps and simple and convenient operation, is suitable for industrialized large-scale production, and the obtained product can be widely applied to the fields of medicines, foods and cosmetics and has wide application prospect.
Drawings
FIG. 1 is a graph showing the effect of spirulina protein zymolyte on the survival rate of caenorhabditis elegans under natural conditions.
FIG. 2 is a graph showing the effect of Spirulina protein zymolyte on the survival rate of C.elegans under heat shock conditions.
FIG. 3 is a graph showing the effect of Spirulina protein hydrolysate on the survival rate of C.elegans under oxidative stress.
FIG. 4 is a graph showing the effect of Spirulina protein hydrolysate on the accumulation of lipofuscin in C.elegans.
FIG. 5 is a graph showing the effect of Spirulina protein hydrolysate on polyQ protein aggregation in transgenic nematode AM 141.
FIG. 6 is a graph of the effect of Spirulina protein zymolyte on alpha-synuclein aggregation in transgenic nematode NL 5901.
FIG. 7 is a graph of the effect of Spirulina protein zymolyte on chemotactic capacity of transgenic nematode CL 2355.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
Wherein, the spirulina powder is purchased from biological development industry limited company of Qingyi spirulina in Yunnan, Escherichia coli NA22 and caenorhabditis elegans (including wild type and transgenic type) are purchased from the genetic information collection center of caenorhabditis elegans of Minnesota university, and other reagents are common reagents and can be purchased from conventional reagent production enterprises.
S basic solution: weighing 5.8g NaCl, 1.3g K2HPO4·3H2O and 6.0g KH2PO4And (3) placing the mixture into a reagent bottle, adding 750ml of deionized water, fully dissolving, then fixing the volume to 1L, sterilizing for 30min, and cooling to 40-50 ℃ for later use.
S Medium solution: adding 1L sterilized S Basal solution dropwise into 1ml cholesterol ethanol solution with concentration of 5.0mg/ml, shaking while adding to dissolve completely (the solution changes from colorless transparency to microemulsion transparency without obvious precipitation), and sequentially adding 3ml CaCl with concentration of 1mol/L2Solution, 3ml MgSO 1mol/L MgSO4The solution, 10ml of potassium citrate solution (pH 6.0) with the concentration of 1mol/L and 10ml of trace element solution are mixed evenly and placed at normal temperature for standby.
EXAMPLE 1A Spirulina protein hydrolysate (SPPH)
Is prepared by the following steps:
s1, wall breaking: preparing 10% suspension of blunt-topped spirulina powder by Tris-HCl buffer solution, adjusting pH to 7.4, soaking for 1 day, freezing at-20 deg.C for 12h, thawing at 4 deg.C, repeatedly freezing and thawing for 3 times, and centrifuging at 5000rpm for 30min to obtain supernatant;
s2, protein isolate: taking the supernatant obtained in the step S1, and slowly adding NH4SO4To saturation thereofKeeping the temperature at 50%, continuously stirring, standing at 4 deg.C overnight, centrifuging at 5000rpm for 30min, re-dissolving the precipitate in first-stage water, placing in 3.5KDa dialysis belt, dialyzing with flowing water for 3d, dialyzing with deionized water for 3 times, changing deionized water every 12 hr, and lyophilizing the dialyzed protein solution to obtain Spirulina protein extract;
s3, enzymolysis: dissolving the spirulina protein extract obtained in the step S2 in primary water to prepare a protein solution with the weight volume percentage of 20%, adjusting the pH to 7.0 by using 0.5mol/L NaOH solution, adding alkaline protease according to 4300U/g, adjusting the temperature to 55 ℃, stirring for enzymolysis for 160min, cooling to room temperature, adjusting the pH to 6.5 by using 1mol/L HCl, adding papain according to 4.5% of the weight percentage of the enzyme and the substrate, adjusting the temperature to 60 ℃, stirring for hydrolysis for 210min, placing in a constant-temperature water bath kettle at 99 ℃ for 10min, cooling and freeze-drying to obtain the spirulina protein extract.
Example 2A Spirulina protein zymolyte (SPPH)
Is prepared by the following steps:
s1, wall breaking: preparing 12% suspension of blunt-topped spirulina powder by Tris-HCl buffer solution, adjusting pH to 7.5, soaking for 2 days, freezing at-20 deg.C for 24h, thawing at 4 deg.C, repeatedly freezing and thawing for 4 times, and centrifuging at 6000rpm for 10min to obtain supernatant;
s2, protein isolate: taking the supernatant obtained in the step S1, and slowly adding NH4SO4Continuously stirring until the saturation degree is 50%, standing at 4 ℃ overnight, centrifuging at 6000rpm for 10min, taking the precipitate to redissolve in primary water, placing in a dialysis belt of 3.5KDa, dialyzing with flowing water for 3d, dialyzing with deionized water for 4 times, changing the deionized water every 12h, taking the dialyzed protein solution, and freeze-drying to obtain the spirulina protein extract;
s3, enzymolysis: dissolving the spirulina protein extract obtained in the step S2 in primary water to prepare a protein solution with the weight volume percentage of 25%, adjusting the pH to 7.5 by using 0.5mol/L NaOH solution, adding alkaline protease according to 4500U/g, adjusting the temperature to 55 ℃, stirring for enzymolysis for 180min, cooling to room temperature, adjusting the pH to 6.5 by using 1mol/L HCl, adding papain according to the weight percentage of 4.8% of enzyme and substrate, adjusting the temperature to 63 ℃, stirring for hydrolysis for 200min, placing in a constant-temperature water bath kettle at 100 ℃ for 10min, cooling and freeze-drying to obtain the spirulina protein extract.
EXAMPLE 3A Spirulina protein hydrolysate (SPPH)
Is prepared by the following steps:
s1, wall breaking: preparing suspension with weight volume percentage of 8% from Spirulina platensis powder with Tris-HCl buffer solution, adjusting pH to 7.3, soaking for 2 days, freezing at-20 deg.C for 18h, thawing at 25 deg.C, repeatedly freezing and thawing for 5 times, and centrifuging at 8000rpm for 10min to obtain supernatant;
s2, protein isolate: taking the supernatant obtained in the step S1, and slowly adding NH4SO4Continuously stirring until the saturation degree is 50%, standing at 4 deg.C overnight, centrifuging at 8000rpm for 10min, re-dissolving the precipitate in first-stage water, placing in 3.5KDa dialysis belt, dialyzing with flowing water for 3d, dialyzing with deionized water for 5 times, changing deionized water every 12 hr, and lyophilizing the dialyzed protein solution to obtain Spirulina protein extract;
s3, enzymolysis: dissolving the spirulina protein extract obtained in the step S2 in primary water to prepare a protein solution with the weight volume percentage of 20%, adjusting the pH to 7.5 by using 0.5mol/L NaOH solution, adding alkaline protease according to 4000U/g, adjusting the temperature to 52 ℃, stirring for enzymolysis for 170min, cooling to room temperature, adjusting the pH to 6.5 by using 1mol/L HCl, adding papain according to the weight percentage of 4.2% of the enzyme and the substrate, adjusting the temperature to 62 ℃, stirring for hydrolysis for 240min, placing in a constant-temperature water bath kettle at 98 ℃ for 15min, cooling and freeze-drying to obtain the spirulina protein extract.
Test example 1 Effect of Spirulina protein zymolyte on the Life of caenorhabditis elegans under Natural conditions
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: wild type C.elegans (N2).
3. The test method comprises the following steps:
transferring the caenorhabditis elegans in the L4 stage into a 1.5ml sterile EP tube, washing for 3 times by using S Medium, removing eggs and nematode fragments which do not hatch, adjusting the density to 20 strips/10 muL by using S Medium solution, shaking uniformly, adding 10 muL caenorhabditis elegans solution into each hole, adding spirulina protein zymolyte dissolved in the S Medium solution to enable the final concentration to be 2mg/ml (adding equal volume of S Medium solution into a control group), then adding ampicillin with the final concentration of 100 mug/ml and 5-fluorouracil deoxynucleoside (5-FUDR) with the final concentration of 75 mug/ml, finally complementing the S Medium containing NA22 bacteria liquid to 100 muL, placing the solution in a shaking table for culturing at 20 ℃, and counting the survival ratio of the caenorhabditis elegans every 12 hours until all the caenorhabditis elegans are dead.
4. And (3) test results:
the survival rate of C.elegans is shown as a survival curve and the results are analyzed by the Kaplan-Meier method, as shown in FIG. 1. As can be seen from FIG. 1, the spirulina protein zymolyte can prolong the life of caenorhabditis elegans under natural conditions, which shows that the spirulina protein zymolyte provided by the invention has the function of delaying senility.
Test example 2 Effect of Spirulina protein hydrolysate on survival Rate of caenorhabditis elegans under Heat shock conditions
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: wild type C.elegans (N2).
3. The test method comprises the following steps:
transferring the caenorhabditis elegans in the L4 stage into a 1.5ml sterile EP tube, washing for 3 times by using S Medium, removing eggs and nematode fragments which do not hatch, adjusting the density to 20 strips/10 muL by using S Medium solution, adding 10 muL of caenorhabditis elegans solution into each hole after shaking uniformly, adding spirulina protein zymolyte dissolved in the S Medium solution to enable the final concentration to be 2mg/ml (adding equal-volume S Medium solution into a control group), then adding ampicillin with the final concentration of 100 mug/ml and 5-fluorouracil deoxynucleoside (5-FUDR) with the final concentration of 75 mug/ml, finally complementing the S Medium with NA22 to 100 muL, placing the plate into a 20 ℃ shaking table for culturing for 48h, placing the plate into a 35 ℃ heat shock for 6h, placing the plate into a 20 ℃ for incubation for 15h, and counting the survival rate of the caenorhabditis elegans.
4. And (3) test results:
as can be seen from FIG. 2, compared with the control group, the spirulina protein zymolyte can significantly improve the survival rate of the caenorhabditis elegans under heat stress, which shows that the spirulina protein zymolyte prepared by the invention can significantly enhance the heat stress resistance of the caenorhabditis elegans.
Test example 3 Effect of Spirulina protein hydrolysate on survival Rate of caenorhabditis elegans under oxidative stress
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: wild type C.elegans (N2).
3. The test method comprises the following steps:
transferring the caenorhabditis elegans in L4 stage into a 1.5ml sterile EP tube, washing for 3 times by using S Medium, removing eggs and nematode fragments which do not hatch, adjusting the density to 20 strips/10 muL by using S Medium solution, shaking uniformly, adding 10 muL caenorhabditis elegans solution into each hole, adding spirulina protein zymolyte dissolved in the S Medium solution to enable the final concentration to be 2mg/ml (adding equal-volume S Medium solution into a control group), adding ampicillin with the final concentration of 100 mug/ml and 5-fluorouracil deoxynucleoside (5-FUDR) with the final concentration of 75 mug/ml, finally complementing the S Medium containing NA22 to 100 muL, placing in a shaking table at 20 ℃ for 24h, adding white hay with the final concentration of 50mmol/L to perform oxidative stress molding, and counting the survival ratio of the caenorhabditis elegans every 12h, until all of them die.
4. And (3) test results:
the survival rate of C.elegans was expressed as a survival curve, and the results were analyzed by the Kaplan-Meier method. As can be seen from FIG. 3, the spirulina protein zymolyte can significantly improve the survival rate of caenorhabditis elegans under oxidative stress, which shows that the spirulina protein zymolyte prepared by the invention can enhance the oxidation resistance of the caenorhabditis elegans and relieve the damage caused by oxidative stress.
Test example 4 Effect of Spirulina protein hydrolysate on the aggregation of lipofuscin in C.elegans
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: wild type C.elegans (N2).
3. The test method comprises the following steps:
transferring the C.elegans of L4 stage into a 1.5ml sterile EP tube, washing with S Medium for 3 times to remove eggs and nematode debris that do not hatch, adjusting the density to 20 strips/10 μ L with S Medium solution, shaking, adding 10 μ L of C.elegans solution into each well, adding Spirulina protein zymolyte dissolved in S Medium solution to make the final concentration to be 2mg/ml (adding equal volume of S Medium solution into control group), then adding ampicillin with a final concentration of 100. mu.g/ml and 5-fluorouracil deoxynucleoside (5-FUDR) with a final concentration of 75. mu.g/ml, finally complementing S Medium containing NA22 bacterial solution to 100. mu.l, placing the mixture in a shaking table for 10d at 20 ℃, collecting caenorhabditis elegans, washing the caenorhabditis elegans with M9 buffer solution for 3 times, transferring the washed solution to a 2% agarose gel sheet, and dripping 10. mu.l NaN with a concentration of 100 mmol/L.3And (3) anaesthetizing the caenorhabditis elegans, setting the excitation wavelength to be 355nm and the emission wavelength to be 460nm, photographing by using a fluorescence microscope, analyzing pictures by using Image J software, and calculating the fluorescence density of each group.
4. And (3) test results:
as can be seen from FIG. 4, the fluorescence intensity in the caenorhabditis elegans added with the spirulina protein zymolyte is obviously lower than that of the contrast group, which shows that the spirulina protein zymolyte prepared by the invention can reduce the aggregation of caenorhabditis elegans lipofuscin, reduce the formation of color spots and delay senility.
Test example 5 Effect of Spirulina protein hydrolysate on aggregation of polyQ protein of Huntington's disease model (AM141)
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: the transgenic nematode strain AM141 can express polyQ40 YFP fusion protein, and the polyQ protein is continuously aggregated from the hatching to the adult growth stage, namely, the yellow-green fluorescence spots are continuously increased along with the aging of the nematode, so that the transgenic nematode strain AM141 can be applied to the research of related pathways of polyQ toxicity, and is a common disease model for researching HD.
3. The test method comprises the following steps:
taking the synchronized L1 stage transgenic nematodeAdding strain AM141 into a 24-well plate, adding about 500 strains of spirulina protein zymolyte and NA22 bacterial liquid into each well, shaking, placing at 20 ℃ for shake culture, collecting transgenic nematodes in an EP tube every 24h, washing for 3 times by using M9 buffer solution, dripping the collected transgenic nematodes on a 2% agarose pad, and dripping 10 mu L of NaN with the concentration of 100mmol/L3Anaesthetizing the transgenic nematodes, setting an excitation wavelength of 485nm and an emission wavelength of 538nm, observing by a fluorescence microscope, and counting yellow-green fluorescence accumulation points of each transgenic nematode in each group (about 20 transgenic nematodes).
4. And (3) test results:
as can be seen from FIG. 5, the fluorescence aggregation amount of the transgenic nematodes in the spirulina protein zymolyte group is less than that in the control group within 3 days, wherein the fluorescence point aggregation amount of the transgenic nematodes treated by the spirulina protein zymolyte on the 3 rd day is obviously reduced compared with that in the control group, and the result shows that the spirulina protein zymolyte prepared by the invention can obviously inhibit the aggregation of polyQ protein and has certain relieving and treating effects on polyglutamine diseases.
Test example 6 Effect of Spirulina protein zymolyte on aggregation of α -synuclein in the Parkinson model (NL5901)
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: the transgenic nematode strain NL5901 is a transgenic nematode strain expressing human alpha-synuclein marked by green fluorescent protein in body wall muscle cells, and along with the aging, the more alpha-synuclein is expressed, the stronger green fluorescence is, and the transgenic nematode strain is a nematode model commonly used for researching PD.
3. The test method comprises the following steps:
adding synchronized L1-stage transgenic nematode NL5901 into 24-well plate, adding about 500 strains per well, adding spirulina protein zymolyte and NA22 bacterial liquid, shaking, culturing at 20 deg.C for 72h, collecting transgenic nematodes in EP tube, washing with M9 buffer solution for 3 times, dropping the transgenic nematodes on 2% agarose pad, and dropping 10 μ L NaN with concentration of 100mmol/L3Anaesthetizing caenorhabditis elegans, setting excitation wavelength to be 485nm and emission wavelength to be 538nAnd m, observing through a fluorescence microscope, and analyzing and counting the fluorescence density of each nematode by using Image J software.
4. And (3) test results:
as can be seen from FIG. 6, the fluorescence intensity of the fluorescent protein expressed by the transgenic nematodes treated with the spirulina protein zymolyte is significantly lower than that of the control group, which indicates that the spirulina protein zymolyte prepared by the invention can significantly inhibit the aggregation of alpha-synuclein and has certain relieving and treating effects on alpha-synucleinopathy.
Test example 7 Effect of Spirulina protein hydrolysate on chemotactic ability of Alzheimer's disease model (CL2355)
1. Test materials: the spirulina protein hydrolysate (SPPH) prepared in example 1 of the present invention.
2. Test subjects: the transgenic nematode strain CL2355 can induce and express Abeta protein at high temperature, is an important substance causing Alzheimer disease, can cause nerve damage, has reduced sensitivity to chemical substances (cANP, benzaldehyde, diacetyl and the like), and can judge whether a sample can relieve neurotoxicity generated by Abeta aggregation or not by detecting chemotactic capacity of the heat-shocked transgenic nematodes.
3. The test method comprises the following steps:
taking synchronized eggs of a transgenic nematode strain CL2355, performing shake cultivation at 15 ℃ for 23 hours, adjusting the density of the nematode, equivalently adding the eggs into a 24-hole plate, wherein each hole is about 500, then adding spirulina protein zymolyte and NA22 bacterial liquid, shaking, then placing at 15 ℃ for cultivation for 36 hours, then raising the temperature to 23 ℃ for continuous cultivation for 36 hours, collecting the transgenic nematodes in an EP (Ephedra) tube, washing 3 times by using M9 buffer solution, transferring the transgenic nematodes to the center of a 9cm chemotaxis flat plate, respectively dripping attractant benzaldehyde (A) and absolute ethyl alcohol (B) at positions 1cm away from the edge of the two sides of the flat plate, performing cultivation for 1 hour at 23 ℃, taking out, heating in a 50 ℃ oven for 15min for killing, and counting the condition of the transgenic nematodes induced by the benzaldehyde under a microscope.
The chemotaxis index CI ═ number (A) -number (B))/(number (A) + number (B)).
4. And (3) test results:
as can be seen from fig. 7, the chemotactic index of the transgenic nematodes after the treatment of the spirulina protease prepared in example 1 of the present invention is significantly higher than that of the control group, which indicates that the spirulina protease prepared in the present invention can effectively alleviate neurotoxicity caused by aggregation of a β protein, and has certain effects of alleviating and treating alzheimer's disease.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (2)
1. Use of a spirulina protein hydrolysate for the preparation of a medicament for inhibiting aggregation of neurotoxic proteins or alleviating neurotoxicity;
the neurotoxic protein includes polyQ protein and alpha-synuclein;
the neurotoxicity is caused by aggregation of A beta protein;
the preparation method of the spirulina protein zymolyte comprises the following steps:
s1, wall breaking: preparing suspension with the weight volume percentage of 8-12% by using Tris-HCl buffer solution, adjusting the pH to 7.0-7.5, soaking for 1-3 days, freezing at-20-40 ℃ for 12-24 h, then unfreezing at 4-25 ℃, repeatedly freezing and thawing for 3-5 times, and centrifuging at 5000-8000 rpm for 10-30 min to obtain supernatant;
s2, protein isolate: taking the supernatant obtained in the step S1, and slowly adding NH4SO4Continuously stirring until the saturation degree is 50%, standing overnight at 4 ℃, centrifuging at 5000-8000 rpm for 10-30 min, re-dissolving the precipitate in primary water, placing the primary water in a dialysis belt of 3.5KDa, dialyzing with flowing water for 2-3 d, dialyzing with deionized water for 3-5 times, changing the deionized water every 12h, and freeze-drying the dialyzed protein solution to obtain a spirulina protein extract;
s3, enzymolysis: dissolving the spirulina protein extract obtained in the step S2 in primary water to prepare a protein solution with the weight volume percentage of 15-30%, adjusting the pH to 7.0-8.0 by using 0.5mol/L NaOH solution, adding alkaline protease according to 4000-4500U/g, adjusting the temperature to 45-60 ℃, stirring and performing enzymolysis for 120-180 min, cooling to room temperature, adjusting the pH to 6.0-7.0 by using 1mol/L HCl, adding papain according to the weight percentage of 3.5-5.0% of enzyme and substrate, adjusting the temperature to 55-65 ℃, stirring and hydrolyzing for 180-250 min, placing in a constant-temperature water bath kettle at 95-100 ℃ for 10-15 min, cooling and freeze-drying to obtain the spirulina protein extract.
2. The use of claim 1, wherein said medicament mitigates toxicity associated with aggregation of a β protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810897805.9A CN108939039B (en) | 2018-08-08 | 2018-08-08 | Application of spirulina protein zymolyte and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810897805.9A CN108939039B (en) | 2018-08-08 | 2018-08-08 | Application of spirulina protein zymolyte and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108939039A CN108939039A (en) | 2018-12-07 |
| CN108939039B true CN108939039B (en) | 2022-04-15 |
Family
ID=64467778
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810897805.9A Active CN108939039B (en) | 2018-08-08 | 2018-08-08 | Application of spirulina protein zymolyte and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108939039B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114306204A (en) * | 2022-01-04 | 2022-04-12 | 内蒙古科技大学 | Pure natural spirulina polypeptide mask and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899102A (en) * | 2010-07-12 | 2010-12-01 | 华南理工大学 | Method for separating high purity phycocyanin from spirulina |
| CN101928743A (en) * | 2009-09-29 | 2010-12-29 | 香港中文大学 | Enzymolysis product of spirulina phycocyanin and application thereof |
| CN107674905A (en) * | 2017-11-02 | 2018-02-09 | 林峰 | Spirulina bioactive peptide, composition and preparation method |
-
2018
- 2018-08-08 CN CN201810897805.9A patent/CN108939039B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928743A (en) * | 2009-09-29 | 2010-12-29 | 香港中文大学 | Enzymolysis product of spirulina phycocyanin and application thereof |
| CN101899102A (en) * | 2010-07-12 | 2010-12-01 | 华南理工大学 | Method for separating high purity phycocyanin from spirulina |
| CN107674905A (en) * | 2017-11-02 | 2018-02-09 | 林峰 | Spirulina bioactive peptide, composition and preparation method |
Non-Patent Citations (12)
| Title |
|---|
| A Spirulina-Enhanced Diet Provides Neuroprotection in an a-Synuclein Model of Parkinson’s Disease;Pabon et al.;《PLOS ONE》;20120930;第7卷(第9期);第e45265-e45256页 * |
| Neuroprotective Activities of Spirulina platensis in the 6-OHDA Model of Parkinson’s Disease Are Related to Its Anti-Inflammatory Effects;Lima et al.;《Neurochem Res》;20170831;第3390-3400 * |
| Phycocyanin moderates aging and proteotoxicity in Caenorhabditis elegans;Singh et al.;《J Appl Phycol》;20151216;第28卷;第2414页左栏第1段及图5c，d * |
| Phycocyanin protects against Alpha-Synuclein toxicity in yeast;Macedo et al.;《Journal of Functional Foods》;20171004;第38卷;摘要、第554页左栏第2段和第556页左栏第2段以及图1c，d * |
| Simple In Vitro Assays to Identify Amyloid-β Aggregation Blockers for Alzheimer’s Disease Therapy;Guo et al.;《Journal of Alzheimer’s Disease》;20101231;摘要、第1360页右栏第2段和第1366页左栏第2段及表1 * |
| The antioxidant, immunomodulatory, and anti‑inflammatory activities of Spirulina: an overview;Wu et al.;《Arch Toxicol》;20160603;第90卷;第1817-1840页 * |
| Therapeutic Potentials of Microalgae in the Treatment of Alzheimer’s Disease;Olasehinde et al.;《Molecules》;20171231;第1-18页 * |
| 藻蓝蛋白及其酶解产物的抗肿瘤效应研究进展;邓伟等;《海洋通报》;20100630;第29卷(第3期);第359页左栏倒数第2段及右栏第2段 * |
| 螺旋藻提取物在化妆品中的应用;朱晓龙等;《云南民族学院学报(自然科学版)》;19981031;第7卷(第2期);第17页2.2.1、第18页第3段及第18-19页3.2-3.3部分、第22页第3段 * |
| 螺旋藻水解肽的制备及其抗肿瘤活性研究;王竹君等;《现代食品科技》;20151231;第31卷(第11期);第26页右栏第3段、第27页左栏第1段以及表1-2 * |
| 螺旋藻酸奶的研制;蒋秋燕等;《食品工业》;20011231(第1期);第29-30和20页 * |
| 钝顶螺旋藻蛋白提取物的延缓衰老功效研究;余程明等;《食品科技》;20180420;第43卷(第4期);第229页右栏第4段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108939039A (en) | 2018-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2017209066C1 (en) | Walnut oligopeptide powder, preparation method and application thereof | |
| Kumar et al. | In vitro anti-cancer, anti-diabetic, anti-inflammation and wound healing properties of collagen peptides derived from unicorn leatherjacket (Aluterus monoceros) at different hydrolysis | |
| CN102046777A (en) | Novel lactic acid bacterium having anti-allergic activity, anti-allergic agent, food and pharmaceutical composition each comprising the lactic acid bacterium, and process for production of the anti-allergic agent | |
| CN100402548C (en) | Method for preparing physiological active polypeptide of deer placenta | |
| CN108939039B (en) | Application of spirulina protein zymolyte and preparation method thereof | |
| US20220110984A1 (en) | Application of Dubosiella Newyorkensis in Prolonging Lifespan and Anti-Aging | |
| JP4132635B2 (en) | Uninactivated enzyme-enhanced composition | |
| CN102134287B (en) | Preparation method for pipelined extraction of sodium hyaluronate through combs | |
| CN108402472B (en) | Method for extracting biological protein from female silkworm chrysalis | |
| CN114272356A (en) | Formula and preparation method of anti-aging pharmaceutical composition | |
| JP4052534B2 (en) | Seafood products and feed | |
| JP3428356B2 (en) | Physiologically active substance and method for producing the same | |
| Snieszko | Natural resistance and susceptibility to infections | |
| US20210393501A1 (en) | Preparation method and application of recombinant mutant collagenase | |
| CN110464756A (en) | It is a kind of for adjusting the gegen qinlian decoction water extract of fat body | |
| CN109568335A (en) | Laver amylose is intervening the application in Caenorhabditis elegans aging | |
| CN1170587C (en) | I (Robinia Pseudoacacia) lectin and its uses | |
| KR100557467B1 (en) | Process for feeding fish and livestock a liquid extract of mushroom mycelial culture and product thereof | |
| CN107712914A (en) | A kind of compound product with lipid-reducing function and preparation method thereof | |
| CN114041603A (en) | Application of peptide-zinc chelate in antiallergic product | |
| CN103509102A (en) | Wild type human growth hormone mutant | |
| JP2001069921A (en) | Health food for pet animal and livestock | |
| CN105358164A (en) | Strain for the treatment and/or prevention of chronic inflammatory diseases | |
| CN101528771B (en) | Method for purification of DNAK | |
| CN1144597C (en) | Method for preparing genetic antibiotic peptide medicine for animals from transgenic antibiotic peptide muscidian vermicule and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 510006 room 405-2, building 1, Department of Guangdong University of pharmacy, No. 280, Waihuan East Road, Xiaoguwei street, Panyu District, Guangzhou, Guangdong Province Applicant after: Tangshidai (Guangzhou) Biomedical Technology Co.,Ltd. Address before: 510006 room 405-2, building 1, Department of Guangdong University of pharmacy, No. 280, Waihuan East Road, Xiaoguwei street, Panyu District, Guangzhou, Guangdong Province Applicant before: GUANGZHOU ABBS BIOTECHNOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220317 Address after: 410012 station 4-7, room 103-5, basement 1, building B, entrepreneurship building, University Science Park, No. 283, middle section of Xiaoxiang Avenue, Juzizhou street, Yuelushan University Science and Technology City, Changsha City, Hunan Province Applicant after: Hunan tangshidai Biotechnology Co.,Ltd. Address before: 510006 room 405-2, building 1, Department of Guangdong University of pharmacy, No. 280, Waihuan East Road, Xiaoguwei street, Panyu District, Guangzhou, Guangdong Province Applicant before: Tangshidai (Guangzhou) Biomedical Technology Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230920 Address after: No. 66, Chaosan Group, Pengfang Village, Wentang Town, Yuanzhou District, Yichun City, Jiangxi Province, 336000 (self committed) Patentee after: Jiangxi Zaoquan Biotechnology Co.,Ltd. Address before: 410012 station 4-7, room 103-5, basement 1, building B, entrepreneurship building, University Science Park, No. 283, middle section of Xiaoxiang Avenue, Juzizhou street, Yuelushan University Science and Technology City, Changsha City, Hunan Province Patentee before: Hunan tangshidai Biotechnology Co.,Ltd. |