CN116421708B - Extraction of ovalbumin from pigeon egg white and application of ovalbumin in wound repair - Google Patents
Extraction of ovalbumin from pigeon egg white and application of ovalbumin in wound repair Download PDFInfo
- Publication number
- CN116421708B CN116421708B CN202310334539.XA CN202310334539A CN116421708B CN 116421708 B CN116421708 B CN 116421708B CN 202310334539 A CN202310334539 A CN 202310334539A CN 116421708 B CN116421708 B CN 116421708B
- Authority
- CN
- China
- Prior art keywords
- ovalbumin
- egg white
- polyethylene glycol
- protein
- supernatant
- 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
- 108010058846 Ovalbumin Proteins 0.000 title claims abstract description 152
- 229940092253 ovalbumin Drugs 0.000 title claims abstract description 106
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 99
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 99
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 title claims abstract description 93
- 235000014103 egg white Nutrition 0.000 title claims abstract description 93
- 210000000969 egg white Anatomy 0.000 title claims abstract description 93
- 241000272201 Columbiformes Species 0.000 title claims abstract description 84
- 230000037314 wound repair Effects 0.000 title claims abstract description 9
- 238000000605 extraction Methods 0.000 title abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 64
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 43
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 230000001737 promoting effect Effects 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims description 34
- 238000003756 stirring Methods 0.000 claims description 26
- 239000007787 solid Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- 235000013601 eggs Nutrition 0.000 claims description 16
- 239000012071 phase Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 13
- 230000001105 regulatory effect Effects 0.000 claims description 11
- 101710173438 Late L2 mu core protein Proteins 0.000 claims description 10
- 101710188315 Protein X Proteins 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 101710188306 Protein Y Proteins 0.000 claims description 5
- 239000007791 liquid phase Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 210000002969 egg yolk Anatomy 0.000 claims description 4
- 235000013345 egg yolk Nutrition 0.000 claims description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims 3
- 230000000694 effects Effects 0.000 abstract description 12
- 230000004071 biological effect Effects 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 4
- 238000005185 salting out Methods 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 3
- 239000002932 luster Substances 0.000 abstract description 2
- 238000001556 precipitation Methods 0.000 abstract description 2
- 235000015598 salt intake Nutrition 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 61
- 239000000243 solution Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 19
- 101710086987 X protein Proteins 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 230000029663 wound healing Effects 0.000 description 9
- 229940057838 polyethylene glycol 4000 Drugs 0.000 description 8
- 238000000502 dialysis Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 239000012266 salt solution Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 101710144217 Ovalbumin-related protein Y Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 101150086731 ges-1 gene Proteins 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 229940068918 polyethylene glycol 400 Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011112 process operation Methods 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 210000001626 skin fibroblast Anatomy 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010026206 Conalbumin Proteins 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010064983 Ovomucin Proteins 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 231100000991 Reactive Oxygen Species (ROS) Photosafety Assay Toxicity 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003316 Vitamin D Natural products 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 235000001465 calcium Nutrition 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229960003284 iron Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 229940046008 vitamin d Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
-
- 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
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Birds (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses extraction of ovalbumin in pigeon egg white and application of ovalbumin in wound repair, and belongs to the field of biology and medicine. The method adopts a technical route of polyethylene glycol fractional precipitation preseparation and double water phase system purification to realize an egg albumin extraction method aiming at pigeon egg white, and has the advantages of high biocompatibility, mild condition, obviously lower salt consumption than a salting-out method, simple operation and the like; the obtained ovalbumin has high purity, good color and luster and rehydration solubility, greatly retains the structure and biological activity of the protein, is easy to amplify, has low equipment cost, is suitable for large-scale industrial production, and has the effect of promoting wound repair.
Description
Technical Field
The invention relates to extraction of ovalbumin in pigeon egg white and application of ovalbumin in wound repair, and belongs to the field of biology and medicine.
Background
The pigeon eggs are called as 'animal ginseng', contain a large amount of high-quality protein and a small amount of fat, and contain a small amount of total sugar and phospholipid, and microelements and nutritional ingredients such as iron, calcium, vitamin A, vitamin B, vitamin D and the like, and are easy to digest and absorb by human bodies. With the acceptance of wide consumers of pigeon egg products, meat pigeon breeding is generally promoted nationwide, but the problems of thin and fragile eggshells, short preservation time and the like limit the circulation of the pigeon egg products. Meanwhile, the pigeon egg deep processing technology is relatively backward, and the potential of the pigeon egg deep processing technology serving as a high-quality protein source is not excavated, so that the extraction method and the application of the novel pigeon egg white protein are provided, and the method has important significance for pigeon egg product deep processing.
Ovalbumin is a major component of egg white proteins and has a wide range of applications in the food industry due to its foaming and gelling properties. In addition, the research discovers that the ovalbumin has biological and physiological functions of resisting oxidation, reducing blood pressure, regulating immunity, inhibiting blood vessel tension and the like, can be used as a biochemical reagent, a nutritional supplement, a medicine and the like, and has high application value in the fields of biology and medicine. Ovalbumin has two paralogues, namely ovalbumin-associated Y protein (molecular weight about 44 kDa) and ovalbumin-associated X protein (molecular weight about 43 kDa), and the difference in amino acid composition and molecular weight of the two component proteins necessarily results in a difference in physiological functions of the proteins. Egg albumin accompanies Y protein but is very little in eggs, but is the most main protein in pigeon egg white, and the content is more than 50%. Meanwhile, egg albumin in the pigeon egg white and the egg white is combined with sugar chains through covalent bonds, and exists in a glycoprotein structure, so that the overall molecular weight of the egg white is larger than that of single protein; it has been reported (Isolation and Characterization of Major Glycoproteins of Pigeon Egg White) that the molecular weights of glycoproteins after binding sugar chains of ovalbumin-associated Y protein and ovalbumin-associated X protein are 44-65 kDa and 43-55 kDa, respectively. Some studies have reported that ovalbumin accompanies the structural parameters of the Y protein, but its physiological function is not yet clear. As a protein with high content in pigeon egg white, the research on the biological activity of the protein is very important.
Egg is used as egg with high yield and wide application, egg albumin is used as one of the earliest proteins separated from egg white, and at present, most of egg albumin is extracted by a salting-out method, and a large amount of ammonium sulfate can cause protein denaturation, so that the physiological activity of the egg albumin is damaged to a certain extent. Other common extraction methods such as ion exchange chromatography, gel chromatography, ultrafiltration, adsorption and the like are mostly complicated in operation, and equipment cost is high. The aqueous two-phase system is a liquid-liquid extraction technology, and mainly comprises water, polymer and salt, two phases are formed due to incompatibility between the polymer and the salt, and separation of samples is realized by means of different partition coefficients of proteins in the two phases. Matheus M. Pereira et al succeeded in extracting ovalbumin from egg white using a two aqueous phase system.
The early-stage research of the invention shows that the separation process of the egg white in the egg white is not suitable for the pigeon eggs because the composition and the structure of the egg white in the egg white are different from those of the egg white in the pigeon eggs, including a method for extracting the egg white from the egg white by Matheus M. Therefore, it is necessary to develop a separation process for ovalbumin in pigeon eggs.
In addition, if more functions of the egg albumin in the pigeon egg white can be found, related application products are developed, the added value of the egg albumin in the pigeon egg white is improved, and the method has great significance for deep processing of the pigeon egg products.
Disclosure of Invention
In order to solve at least one problem, the invention discovers that the ovalbumin with the molecular weight of 44-65 kDa and 43-55 kDa is extracted from the pigeon egg white by adopting an improved aqueous two-phase system extraction method, and the method has the advantages of simple and quick process operation, no need of complex equipment, suitability for industrial production, high product purity and good biological activity. The invention further discovers that the egg albumin from the pigeon egg white accompanies the Y protein (the accession number on NCBI is PKK33949.1, ovalbumin-related protein Y or referred to as an egg albumin associated protein Y gene), has the effect of promoting wound repair, and is beneficial to improving the added value of the egg albumin in the pigeon egg white.
A first object of the present method is to provide the use of ovalbumin with Y protein for the preparation of a product for promoting wound repair.
In one embodiment, the ovalbumin is an ovalbumin under accession number PKK33949.1 on NCBI, designated ovalbumin companion Y protein (ovalbumin-related protein Y).
In one embodiment, the ovalbumin is present in the glycoprotein structure along with the Y protein also bound to the sugar chain by covalent bonds.
In one embodiment, the ovalbumin can be obtained by adopting a synthetic mode, and can also be extracted from pigeon egg white.
In one embodiment, the molecular weight of the ovalbumin is 44-65 kDa. Preferably, the egg albumin derived from the pigeon egg white is prepared by the improved aqueous two-phase system extraction method.
In one embodiment, the product is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient.
In one embodiment, the product is a dressing.
A second object of the present invention is to provide a wound repair promoting product comprising egg albumin derived from pigeon egg white as a main active ingredient.
In one embodiment, the molecular weight of the ovalbumin is 44-65 kDa. Preferably, the egg albumin derived from the pigeon egg white is prepared by the improved aqueous two-phase system extraction method.
In one embodiment, the product is a dressing.
In one embodiment, the product is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient.
A third object of the present method is to provide a method for extracting ovalbumin from pigeon egg white, comprising the steps of:
(1) Separating egg white and egg yolk, diluting the pigeon egg white with 1-3 times of water, fully stirring, centrifuging, and regulating the pH of the supernatant to 6-8;
(2) Adding polyethylene glycol with molecular weight of 4000-6000 into the treated egg white liquid to make the mass percentage of polyethylene glycol in the egg white liquid be 5-9%, fully stirring and centrifuging to obtain precipitate and supernatant A; continuously adding polyethylene glycol with the molecular weight of 4000-6000 into the supernatant A to ensure that the mass percentage of the polyethylene glycol in the supernatant A is 15-20%, and fully stirring;
(3) Adding a salt solution with pH of 3-8, and further extracting ovalbumin by utilizing a double water phase system; after fully reacting for 12-24 hours, centrifugally separating two liquid phases, and collecting polyethylene glycol solution B.
(4) And (3) removing polyethylene glycol from the solution B by dialysis or ultrafiltration technology, and drying to obtain a solid A, namely the ovalbumin mixture.
(5) And (3) redissolving the obtained solid A in deionized water, regulating the pH value of the solution to 3.5-6.5, and generating a precipitate C, namely the ovalbumin accompanied with the Y protein, and freeze-drying the supernatant C to obtain the ovalbumin X protein.
Wherein, the pigeon egg white in the step (1) is pigeon egg white or pigeon egg white powder is rehydrated and dissolved.
In one embodiment, the supernatant in step (1) has a pH of 6.5 to 7.5.
In one embodiment, the average molecular weight of polyethylene glycol in the step (2) is 4000-6000, so that the mass percentage of polyethylene glycol in egg white liquid is 5-9%, polyethylene glycol is slowly added while stirring, and then the mixture is fully stirred for 2-4 hours and centrifuged to obtain precipitate and supernatant A; continuously adding polyethylene glycol with the molecular weight of 4000-6000 into the supernatant A to make the mass percentage of the polyethylene glycol in the supernatant A be 15-20%, adding the polyethylene glycol, stirring at the same time, and fully stirring for 2-4 hours.
In one embodiment, the step (3) is performed at room temperature.
In one embodiment, the salt solution in step (3) has a pH of 3 to 8 and a reaction time of 12 to 18 hours at room temperature.
In one embodiment, the salt solution in the step (3) is a phosphate solution or a citrate solution, and the concentration is 5-8 mol/L.
In one embodiment, the polyethylene glycol removing method in the step (4) can adopt a dialysis or ultrafiltration technology, the dialysis method adopts a dialysis bag with a molecular weight cut-off of 1000-20000, the ultrafiltration method adopts an ultrafiltration membrane with a molecular weight cut-off of 1000-20000 for desalination, and the drying method can adopt freeze drying or spray drying.
The pigeon egg white ovalbumin prepared by the method comprises two components, wherein the molecular weight of the two components is 44-65 kDa and 43-55 kDa, the two components are special protein components in pigeon eggs, the 44-65 kDa part is identified as ovalbumin accompanying Y protein, and the 43-55 kDa part is identified as ovalbumin accompanying X protein.
The method adopts a technical route of polyethylene glycol fractional precipitation preseparation and double water phase system purification. Polyethylene glycol is a nonionic linear macromolecular polymer with very strong hydrophilicity, so that a hydration layer can be destroyed to dehydrate protein molecules, and simultaneously, the protein molecules are extruded through space rejection and are entangled by means of chain length to precipitate. Because of its good hydrophilicity and relatively small molecular weight, ovalbumin can be dissolved in the supernatant in polyethylene glycol solution to be roughly separated from other precipitated proteins such as ovotransferrin, ovomucin, etc. In a double water phase system formed by polyethylene glycol and salt solution, residual hybrid protein is separated out due to salting-out effect, meanwhile, the residual hybrid protein cannot be dissolved in the polyethylene glycol solution, pigment in egg white is concentrated in the salt solution phase, so that the purification effect is achieved, and the polyethylene glycol solution contains high-purity ovalbumin.
The invention has the advantages and effects that:
(1) The invention discovers that the 44-65 kDa ovalbumin accompanied with the Y protein has the functions of promoting wound healing and the like.
(2) The invention develops a method for extracting and obtaining 44-65 kDa ovalbumin concomitant with Y protein from pigeon egg white, and the method has the advantages of high biocompatibility, mild condition, obviously lower salt consumption than that of a salting-out method, simplicity in operation and the like, is easy to amplify, has lower equipment cost and is suitable for large-scale industrial production.
(3) The pigeon egg white ovalbumin prepared by the method has pure white color, high water solubility, high purity, good color and luster and rehydration solubility, and the structure and biological activity of the protein are reserved to a great extent.
Drawings
FIG. 1 is a schematic operation flow chart of a process for extracting ovalbumin from pigeon egg white;
FIG. 2 is a schematic diagram of a two-aqueous phase system.
FIG. 3 shows detection of pigeon egg white, solid B obtained in comparative example 2, solid A obtained in example 1, ovalbumin-associated Y protein and ovalbumin-associated X protein, and ovalbumin-associated Y protein and ovalbumin-associated X protein obtained in example 2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Wherein, lane 1: protein molecular weight standard; lane 2: pigeon egg white; lane 3: egg white; lane 4: comparative example 2 was a pigeon egg white extract solid B obtained by an egg white ovalbumin extraction method; lane 5: solid a obtained in example 1; lane 6: the ovalbumin obtained in example 1 was accompanied by a Y protein; lane 7: the ovalbumin obtained in example 1 was accompanied by protein X; lane 8: the ovalbumin obtained in example 2 was accompanied by a Y protein; lane 9: the ovalbumin obtained in example 2 was accompanied by protein X.
FIG. 4 is a UV spectrum of ovalbumin obtained in example 2 along with Y protein.
FIG. 5 shows the finished product of ovalbumin-associated Y protein prepared by the method and an aqueous solution thereof.
FIG. 6 shows the results of cell scratch experiments.
FIG. 7 is an effect of pigeon egg albumin and egg albumin on ROS production by cells under UV irradiation.
FIG. 8 shows the results of cytotoxicity experiments.
FIG. 9 shows the results of partial identification of ovalbumin along with Y protein.
FIG. 10 shows the results of partial identification of ovalbumin along with protein X.
Detailed Description
A flow chart of the process operation of the present invention is shown in fig. 1.
The invention is further described below with reference to the accompanying drawings and specific examples, which are not intended to limit the invention in any way.
The purity and protein composition of ovalbumin in the following examples were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the recovery rate of ovalbumin was calculated by the following method: the protein content of the pigeon egg white was determined by coomassie brilliant blue method, and the egg albumin content in the pigeon egg white was calculated according to the ratio, and the recovery rate = (the amount of protein obtained after drying/the protein content in the pigeon egg white) ×100%.
Example 1: preparation of ovalbumin
(1) Taking 100g of fresh pigeon egg white, adding water with equal mass for dilution, fully stirring for 2 hours, centrifuging at 6000rpm for 10 minutes at 4 ℃, and adjusting the pH of the supernatant to 7.5 by using 1 mol/L hydrochloric acid;
(2) Adding polyethylene glycol 4000 into the processed egg white liquid while stirring to make the mass percentage of the polyethylene glycol 4000 in the egg white liquid be 9%, fully stirring for 2 hours, centrifuging, wherein the centrifuging condition is 4 ℃ and centrifuging at 6000rpm for 20 minutes. The precipitate and supernatant A were obtained. Continuously adding polyethylene glycol 4000 into the supernatant A while stirring to make the mass percentage of the polyethylene glycol 4000 in the supernatant be 18%, and fully stirring for 2 hours;
(3) Sodium phosphate solution with pH of 5.3 is added to form a double water phase system, after the reaction is fully carried out for 18 hours at room temperature, the mixture is centrifuged at 9000rpm for ten minutes, two phases are separated as shown in figure 2, and polyethylene glycol solution B is collected.
(4) Dialyzing the solution B with 10000 Da dialysis bag for 24 hr, and lyophilizing to obtain solid A which is ovalbumin mixture.
(5) And re-dissolving the solid A in water, regulating the pH value to 4.5, and obtaining precipitate C which is the protein of the ovalbumin with the protein Y, and obtaining supernatant B which is the protein of the ovalbumin with the protein X after freeze drying.
The detection of the obtained ovalbumin-associated Y protein and ovalbumin-associated X protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the purities are 99.8% and 99.6% respectively, and the recovery rates are 72.3% and 74.1% respectively.
Example 2: preparation of ovalbumin
(1) 1000g of fresh pigeon egg white is taken, water with equal mass is added for dilution, after being fully stirred for 2 hours, the mixture is centrifuged for 10 minutes at the temperature of 4 ℃ and the rpm of 6000rpm, and the pH value of the supernatant is regulated to 7.5 by 1 mol/L hydrochloric acid;
(2) Adding polyethylene glycol 4000 into the processed egg white liquid while stirring to make the mass percentage of the polyethylene glycol 4000 in the egg white liquid be 9%, and centrifuging after stirring for 2 hours, wherein the centrifuging condition is 4 ℃ and 600 rpm for 20 minutes. The precipitate and supernatant A were obtained. Continuously adding polyethylene glycol 4000 into the supernatant A while stirring to make the mass percentage of the polyethylene glycol 4000 in the supernatant be 18%, and fully stirring for 2 hours;
(3) Adding sodium citrate solution with pH of 6.5, and further extracting ovalbumin by using a double water phase system. After the reaction was completed at room temperature for 18 hours, the mixture was centrifuged at 9000rpm for ten minutes, and the two liquid phases were separated to collect polyethylene glycol solution B.
(4) Dialyzing the solution B with 10000 Da dialysis bag for 24 hr, and lyophilizing to obtain solid A which is ovalbumin mixture.
(5) And re-dissolving the solid A in water, regulating the pH value to 4.5, and obtaining precipitate C which is the protein of the ovalbumin with the protein Y, and obtaining supernatant B which is the protein of the ovalbumin with the protein X after freeze drying.
The detection of the obtained ovalbumin-associated Y protein and ovalbumin-associated X protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the purities are 96.4% and 95.7% respectively, and the recovery rates are 75.6% and 72.8% respectively.
The difference between example 1 and example 2 is that the pigeon egg white used has a different mass than the salt solution used in step (3).
Comparative example 1: influence of different extraction processes on the extraction effect of ovalbumin in pigeon egg white.
The inventors varied the ratio of polyethylene glycol first added in step (2) to analyze its effect on ovalbumin concomitant Y protein extraction, other steps and methods remained consistent with example 1. The results are shown in Table 1.
TABLE 1
The inventors also compared the effect of the salt solution used in step (3) on ovalbumin concomitant Y protein extraction, other steps and methods being consistent with example 1. The results are shown in Table 2:
TABLE 2
As shown in tables 1 and 2, the key factors in the extraction process of the egg white ovalbumin of the pigeon egg white are compared by the inventor, so that the extraction method of the ovalbumin is applicable to the pigeon egg white, and the obtained ovalbumin has higher purity and yield along with the Y protein.
Comparative example 2: extraction method of egg white ovalbumin from pigeon egg white
The inventor also uses an extraction method of egg white ovalbumin to extract the ovalbumin in the pigeon egg white, and the specific method is as follows:
(1) Taking fresh pigeon eggs, separating pigeon egg white and egg yolk to obtain 100g pigeon egg white, adding deionized water with equal mass for dilution, fully stirring for 2 hours, centrifuging at 4 ℃ and 6000rpm for 10 minutes, and regulating the pH of the supernatant to 7.5 by using 1 mol/L hydrochloric acid;
(2) Adding 90% (w/v) polyethylene glycol 400 solution into the treated egg white liquid while stirring to make the mass ratio of the polyethylene glycol 400 solution to the egg white liquid be 1:2, after thorough stirring, add sodium citrate solution at ph=7. The reaction was completed at room temperature for 12 hours.
(3) Centrifugal for ten minutes at 9000rpm, separate the two liquid phases and collect the polyethylene glycol solution.
(4) Dialyzing polyethylene glycol solution with 10000 Da dialysis bag for 24 hr, and drying to obtain solid B.
The solid B component is detected by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis, and the electrophoresis result shows that: three proteins in the pigeon egg white are not separated, and the ovalbumin is not successfully extracted. It is explained that the egg white ovalbumin extraction method is not suitable for pigeon egg white.
Example 3: structure identification and quality analysis of pigeon egg white ovalbumin
The pigeon egg white, egg white and egg albumin obtained in example 1, example 2 and comparative example were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results are shown in FIG. 3. Wherein, lane 1: protein molecular weight standard; lane 2: pigeon egg white; lane 3: egg white; lane 4: comparative example 2 was a pigeon egg white extract solid B obtained by an egg white ovalbumin extraction method; lane 5: solid a obtained in example 1; lane 6: the ovalbumin obtained in example 1 was accompanied by a Y protein; lane 7: the ovalbumin obtained in example 1 was accompanied by protein X; lane 8: the ovalbumin obtained in example 2 was accompanied by a Y protein; lane 9: the ovalbumin obtained in example 2 was accompanied by protein X.
The electrophoresis result shows that the egg white and the pigeon egg white have different protein compositions, and two types of egg albumin exist in the pigeon egg white, namely, the egg albumin (ova 1) with larger molecular weight and the egg albumin (ova 2) with smaller molecular weight.
The electrophoretic bands of the two ovalbumin proteins obtained in example 1 were mass-identified. FIG. 9 is a partial identification of ova1, showing that ova1 is ovalbumin-associated Y protein (ovalbumin-related protein Y) and has an accession number PKK33949.1 at NCBI; FIG. 10 shows partial identification of ova2, showing that ova2 is ovalbumin with protein X having an accession number PKK33879.1 on NCBI. The ultraviolet spectrum result (figure 4) is combined, so that the structure of the ovalbumin accompanied by the Y protein in the pigeon egg white can be completely obtained, meanwhile, the ovalbumin accompanied by the Y protein and the accompanied by the X protein in the pigeon egg white both contain sugar chain parts, and the band molecular weight of the electrophoresis pattern is in the range of 50-65 kDa.
The pigeon egg white ovalbumin prepared in example 1 was pure white with the Y protein powder, dissolved in water at 5% concentration, and the solution was clear and transparent, see fig. 5.
The extraction method of the pigeon egg white ovalbumin in the embodiments 1-2 shows that the purity of the pigeon egg white ovalbumin obtained under the extraction conditions is above 95%, and the recovery rate is above 72%, and the method has excellent purity and recovery rate for the pigeon egg white ovalbumin, can still keep high purity and recovery rate when the ovalbumin is prepared on a large scale, and is suitable for industrial production.
Example 4: application of ovalbumin in promoting wound healing
The effect of ovalbumin on human skin fibroblast (NHDF) migration ability was determined by cell streaking experiments from example 1, step (5), to obtain ovalbumin-associated Y protein (OVAY) and-associated X protein (OVAX), and egg white ovalbumin.
The method specifically comprises the following steps: NHDF cells with good growth state are prepared according to 1×10 5 Inoculating each of the above materials to 6-well plate, streaking at bottom of the plate with pipette tip when fusion degree is about 90%, adding DMEM culture solution into control group, adding pigeon egg white ovalbumin solution (1 mg/ml) into sample group, placing at 37deg.C, and 5% CO 2 After culturing in the incubator for 0, 24, 48, 72 hours, the cell scratch area was observed, and the wound healing rate= (1-scratch area/initial scratch area) ×100%, and the initial scratch area was a scratch area of 0 hour.
As a result, as shown in fig. 6, it was found that the cell wound healing rate of each sample group was significantly higher than that of the control group (the wound healing rate of 72 hours was about 28%), with the highest healing rate of pigeon egg white OVAY (the wound healing rate of 72 hours was about 45%), followed by pigeon egg white OVAX (the wound healing rate of 72 hours was about 32%) and egg white-derived ovalbumin (the wound healing rate of 72 hours was about 30%). The pigeon egg white ovalbumin prepared by the invention has the function of promoting wound healing along with the Y protein.
Example 5: application of ovalbumin in resisting skin oxidative damage
The skin fibroblasts were treated at 2.5X10 4 Inoculating/hole in 96-well plate, adhering 24-h, changing liquid, adding DMEM culture liquid into control group, adding DMEM culture liquid into sample group, and adding DMEM culture medium containing pigeon egg white Ovalbumin (OVAY) and associated X protein (OVAX) prepared in example 1, and egg white ovalbumin (1 mg/ml)The cell viability and ROS assay were determined by CCK after incubation of 48h and 0.02 mJ UVB irradiation, 24h, except for the sample set. Cell relative ROS content = experimental group absorbance/control group absorbance x 100/cell relative survival x 100%.
As a result, as shown in fig. 7, the pigeon egg white ovalbumin and ovalbumin-associated X proteins reduced ROS content in cells by 24.5%, 12.8%, respectively, while the egg white ovalbumin reduced ROS content in cells by 8.21%, respectively, as compared to UV-irradiated groups. The egg albumin from pigeon egg white has obvious effect of resisting oxidative damage caused by UV radiation along with the Y protein.
Example 6: cytotoxicity experiments show that ovalbumin is nontoxic
The effect of ovalbumin on gastric mucosal epithelial cell (GES-1) viability was determined by cell experiments, using the ovalbumin-associated Y protein (OVAY) and associated X protein (OVAX) obtained in step (5) of example 1.
The method specifically comprises the following steps: GES-1 cells with good growth state were cultured according to a scale of 5X 10 4 Each sample was inoculated into 96-well plates at a volume of 10. Mu.g/mL after culturing 12 h cells for adherent growth, and each sample was subjected to stimulation treatment 12, 24, 48h, respectively. After the incubation time, 10. Mu.LCCK-8 solution was added to each well to add the corresponding amount of cell culture medium as a blank. After further incubation in the cell incubator for 4h, absorbance at 450 nm was measured with a microplate reader and data recorded and analyzed.
As a result, as shown in fig. 8, the cell viability of each sample group was higher than 70% compared to the control group. Wherein, the survival rate of the cells treated by the pigeon egg white ovalbumin along with the Y protein is highest (the survival rate of the cells in 48 hours is about 93 percent), and the survival rate of the cells in 48 hours is next to the egg white ovalbumin along with the X protein (the survival rate of the cells in 48 hours is about 82 percent), and the survival rate of the cells in 48 hours is about 72 percent. The egg white-derived ovalbumin of the pigeon egg white is lower in cytotoxicity along with the Y protein.
It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design alternative embodiments without departing from the scope of the appended claims.
Claims (6)
1. Use of ovalbumin for the preparation of a pharmaceutical composition for promoting wound repair, characterized in that said ovalbumin is a protein with accession number PKK33949.1 on NCBI.
2. The use according to claim 1, wherein the molecular weight of the ovalbumin is 44-65 kDa.
3. Use according to claim 1, wherein the ovalbumin is derived from pigeon egg white.
4. The use according to claim 1, characterized in that the method for preparing ovalbumin comprises the following steps:
(1) Separating egg white and egg yolk of pigeon eggs, diluting the egg white with 1-3 times of water, fully stirring and centrifuging, and regulating the pH of the supernatant to 6-8;
(2) Adding polyethylene glycol with the molecular weight of 2000-6000 into the treated egg white liquid to enable the mass percentage of the polyethylene glycol to be 5-9% of the egg white liquid, fully stirring and centrifuging to obtain sediment and supernatant A; continuously adding polyethylene glycol with the molecular weight of 2000-6000 into the supernatant A to ensure that the mass percentage of the polyethylene glycol in the supernatant A is 15-20%, and fully stirring;
(3) Adding phosphate solution or citrate solution with pH of 3-8, and further extracting ovalbumin by using a double water phase system; fully reacting for 12-24 hours at room temperature, standing or centrifugally separating two liquid phases, and collecting polyethylene glycol solution B;
(4) Removing polyethylene glycol from the solution B, and drying to obtain a solid A, namely an ovalbumin mixture;
(5) And (3) redissolving the obtained solid A in deionized water, regulating the pH value of the solution to 3.5-6.5, and generating a precipitate C, namely the protein Y associated with the ovalbumin with the accession number PKK33949.1 on NCBI, and freeze-drying the supernatant C to obtain the protein X associated with the ovalbumin with the accession number PKK33879.1 on NCBI.
5. The use according to claim 4, wherein the average molecular weight of the polyethylene glycol in the step (2) is 4000-6000, so that the polyethylene glycol accounts for 5-9% of the mass of the egg white liquid, and the polyethylene glycol is centrifuged after being fully stirred for 2-4 hours to obtain sediment and supernatant A; continuously adding polyethylene glycol with the molecular weight of 4000-6000 into the supernatant A to ensure that the mass percentage of the polyethylene glycol in the supernatant A is 15-20%, slowly adding the polyethylene glycol while stirring, and fully stirring for 2-4 hours.
6. The preparation method of the egg albumin with Y protein from pigeon egg white is characterized by comprising the following steps:
(1) Separating egg white and egg yolk, diluting the egg white with 1-3 times of water, fully stirring and centrifuging, and regulating the pH of the supernatant to 6-8;
(2) Adding polyethylene glycol with the molecular weight of 2000-6000 into the treated egg white liquid to enable the mass percentage of the polyethylene glycol to be 5-9% of the egg white liquid, fully stirring and centrifuging to obtain sediment and supernatant A; continuously adding polyethylene glycol with the molecular weight of 2000-6000 into the supernatant A to ensure that the mass percentage of the polyethylene glycol in the supernatant A is 15-20%, and fully stirring;
(3) Adding phosphate solution or citrate solution with pH of 3-8, and further extracting ovalbumin by using a double water phase system; fully reacting for 12-24 hours at room temperature, standing or centrifugally separating two liquid phases, and collecting polyethylene glycol solution B;
(4) Removing polyethylene glycol from the solution B, and drying to obtain a solid A, namely an ovalbumin mixture;
(5) And (3) redissolving the obtained solid A in deionized water, regulating the pH value of the solution to 3.5-6.5, and generating a precipitate C, namely the protein Y associated with the ovalbumin with the accession number PKK33949.1 on NCBI, and freeze-drying the supernatant C to obtain the protein X associated with the ovalbumin with the accession number PKK33879.1 on NCBI.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310334539.XA CN116421708B (en) | 2023-03-31 | 2023-03-31 | Extraction of ovalbumin from pigeon egg white and application of ovalbumin in wound repair |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310334539.XA CN116421708B (en) | 2023-03-31 | 2023-03-31 | Extraction of ovalbumin from pigeon egg white and application of ovalbumin in wound repair |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116421708A CN116421708A (en) | 2023-07-14 |
| CN116421708B true CN116421708B (en) | 2024-02-09 |
Family
ID=87084840
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310334539.XA Active CN116421708B (en) | 2023-03-31 | 2023-03-31 | Extraction of ovalbumin from pigeon egg white and application of ovalbumin in wound repair |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116421708B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001089540A1 (en) * | 2000-05-23 | 2001-11-29 | N.V. Nutricia | Use of egg white and egg white fractions for tissue regeneration |
| CN103524612A (en) * | 2013-09-30 | 2014-01-22 | 暨南大学 | Method for separating milk-derived whey proteins in PEG/phosphate aqueous two-phase system |
| CN104961823A (en) * | 2015-06-19 | 2015-10-07 | 江苏大学 | Purification method applicable to manufacturing food-grade ovalbumin on large scale |
| CN110003323A (en) * | 2019-04-02 | 2019-07-12 | 武汉大学 | A kind of method that double-aqueous phase system isolates and purifies protein |
| CN112028990A (en) * | 2020-09-08 | 2020-12-04 | 兴宁市金鸽养殖专业合作社 | Extraction method of pigeon egg white ovalbumin |
-
2023
- 2023-03-31 CN CN202310334539.XA patent/CN116421708B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001089540A1 (en) * | 2000-05-23 | 2001-11-29 | N.V. Nutricia | Use of egg white and egg white fractions for tissue regeneration |
| CN103524612A (en) * | 2013-09-30 | 2014-01-22 | 暨南大学 | Method for separating milk-derived whey proteins in PEG/phosphate aqueous two-phase system |
| CN104961823A (en) * | 2015-06-19 | 2015-10-07 | 江苏大学 | Purification method applicable to manufacturing food-grade ovalbumin on large scale |
| CN110003323A (en) * | 2019-04-02 | 2019-07-12 | 武汉大学 | A kind of method that double-aqueous phase system isolates and purifies protein |
| CN112028990A (en) * | 2020-09-08 | 2020-12-04 | 兴宁市金鸽养殖专业合作社 | Extraction method of pigeon egg white ovalbumin |
Non-Patent Citations (3)
| Title |
|---|
| Junko Hirose et al..Ovalbumin-related gene Y protein bears carbohydrate chains of the ovomucoid type.Biosci Biotechnol Biochem.2006,第70卷(第1期),144-151. * |
| Valerie Lechevalier, et al..Bioactive Egg Compounds .Springer,2007,51-60. * |
| 徐小钦 等.白羽王鸽卵清蛋白关联蛋白Y基因(OVALY)克隆与生物信息学分析.浙江农业学报.2019,第31卷(第05期),704-707. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116421708A (en) | 2023-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Herrera et al. | Recovery of c-phycocyanin from the cyanobacterium Spirulina maxima | |
| WO1991010369A1 (en) | Method for preparing an enzymatic hydrolyzate | |
| CN103882083B (en) | A kind of preparation method of antioxidant collagen peptide | |
| CA3057780A1 (en) | Purification of phycobiliproteins | |
| CN104131055B (en) | Preparation method for phycoerythrin ACE inhibitory peptide | |
| KR100280030B1 (en) | Hydroxyfurin-rich protein and pharmaceutical and cosmetic preparations containing it | |
| CN103740797B (en) | Method for preparing high-hydrolysis degree functional oligopeptide by use of high-temperature peanut meal | |
| JP7022453B2 (en) | Phycocyanin purification method | |
| CN116421708B (en) | Extraction of ovalbumin from pigeon egg white and application of ovalbumin in wound repair | |
| CN116963614A (en) | Preparation method of fermented cornu Cervi Pantotrichum extract processed by three steps | |
| EP3849995B1 (en) | Methods for separation of chlorophyll and soluble proteins | |
| CN107529554A (en) | A kind of production technology of pig blood ball peptide | |
| RU2320195C1 (en) | Method for producing of protein preparation from cyanobacteria | |
| CN117965668A (en) | Bovine bone collagen peptide with antioxidant and anti-aging functions and preparation method and application thereof | |
| CN117551186A (en) | Method for preparing collagen peptide by countercurrent immobilized enzyme column enzymolysis | |
| KR101437259B1 (en) | Sequential Separation of Lysozyme and Ovalbumin from Chicken Egg White | |
| CN114480542B (en) | Method for extracting bitter peptide from wheat gluten enzymolysis product | |
| Melanie et al. | Separation and concentration of phycocyanin from Spirulina sp. using microfiltration membrane for natural pigment | |
| CN108504709B (en) | Corn oligopeptide and industrial production method thereof | |
| RU2204262C2 (en) | Method for preparing biologically active protein concentrate enriched with pancreatic ribonuclease a, angiogenin and lysozyme from dairy ultrafiltrate | |
| CN112048534B (en) | Spanish mackerel protein hydrolysate with resveratrol efficacy improving activity and preparation method thereof | |
| CN102093469B (en) | Milk-derived antioxidant active peptide and application thereof | |
| CN114836505B (en) | Small molecule anti-alcohol peptide and preparation method and application thereof | |
| RU2781831C2 (en) | Phycobiliprotein purification | |
| CN116508848B (en) | Compound capable of reducing inflammation and rich in sialoglycan and preparation method thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |