CN106632668A - Preparation method and application of sleeve-fish collagen polypeptide and iron complex thereof - Google Patents
Preparation method and application of sleeve-fish collagen polypeptide and iron complex thereof Download PDFInfo
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- CN106632668A CN106632668A CN201710026172.XA CN201710026172A CN106632668A CN 106632668 A CN106632668 A CN 106632668A CN 201710026172 A CN201710026172 A CN 201710026172A CN 106632668 A CN106632668 A CN 106632668A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 63
- 102000008186 Collagen Human genes 0.000 title claims abstract description 57
- 108010035532 Collagen Proteins 0.000 title claims abstract description 57
- 229920001436 collagen Polymers 0.000 title claims abstract description 57
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 57
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 53
- 150000004698 iron complex Chemical class 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 15
- 206010021143 Hypoxia Diseases 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 241000238366 Cephalopoda Species 0.000 claims description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 52
- 235000019441 ethanol Nutrition 0.000 claims description 16
- 239000012141 concentrate Substances 0.000 claims description 12
- 239000000706 filtrate Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 230000007062 hydrolysis Effects 0.000 claims description 6
- 238000006460 hydrolysis reaction Methods 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 238000004821 distillation Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 4
- 239000011790 ferrous sulphate Substances 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000004064 recycling Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000000967 suction filtration Methods 0.000 claims description 4
- 206010002660 Anoxia Diseases 0.000 claims description 3
- 241000976983 Anoxia Species 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 230000007953 anoxia Effects 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000009413 insulation Methods 0.000 claims description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 229910052742 iron Inorganic materials 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 20
- 210000004369 blood Anatomy 0.000 abstract description 11
- 239000008280 blood Substances 0.000 abstract description 11
- 239000004310 lactic acid Substances 0.000 abstract description 10
- 235000014655 lactic acid Nutrition 0.000 abstract description 10
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 101710088194 Dehydrogenase Proteins 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 235000013376 functional food Nutrition 0.000 abstract description 3
- 238000005903 acid hydrolysis reaction Methods 0.000 abstract description 2
- 239000003344 environmental pollutant Substances 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 231100000719 pollutant Toxicity 0.000 abstract description 2
- 230000007954 hypoxia Effects 0.000 abstract 2
- 238000001727 in vivo Methods 0.000 abstract 2
- 239000006227 byproduct Substances 0.000 abstract 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 9
- 239000001301 oxygen Substances 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 241000251468 Actinopterygii Species 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229960001722 verapamil Drugs 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102100026189 Beta-galactosidase Human genes 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010059881 Lactase Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 2
- 229940116108 lactase Drugs 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 235000014102 seafood Nutrition 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 240000006909 Tilia x europaea Species 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 241001311778 Uroteuthis chinensis Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- -1 water 580L Chemical compound 0.000 description 1
Classifications
-
- 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
-
- 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
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Polymers & Plastics (AREA)
- Marine Sciences & Fisheries (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method and application of sleeve-fish collagen polypeptide and an iron complex thereof. A high-temperature acetic acid hydrolysis method is utilized to prepare the sleeve-fish collagen polypeptide and the iron complex thereof from sleeve-fish skin which is a byproduct of sleeve-fish processing, and the preparation method is stable in reaction condition, easy to control and free of pollutant emission and meets requirements on clean production. Animal experiments show that the sleeve-fish collagen polypeptide and the iron complex thereof can remarkably increase hypoxia tolerance survival time, improve activity of in-vivo lactic dehydrogenase and reduce content of in-vivo blood lactic acid and blood urea nitrogen. Results show that hypoxia tolerance activity can be remarkably improved by adopting either the sleeve-fish collagen polypeptide or the iron complex, and the sleeve-fish collagen polypeptide and the iron complex can be used for preparing hypoxia-tolerant drug and functional food and are of important significance in developing and utilizing marine medicinal biological resources in China.
Description
Technical field
The invention belongs to pharmaceutical technology field, more particularly, it is related to squid collagen polypeptide and its iron complex
Preparation method and application.
Background technology
Squid (Loligo chinensis) belongs to marine organisms for Mollusca squid.It is distributed widely in the south of China
Sea and Southeast Asia marine site.Its is nutritious, and protein content is high, can be processed as various ticbits.In squid process, remove
Crust is removed, squid skin is often as discarded object.Research both at home and abroad finds, discarded object accounts for the 30% left of total amount in fish process
The right side, wherein containing rich in protein resource, based on collagen, its hydrolysate contains various active peptide.Active peptide has
Physiology and the pharmacologically actives such as antitumor, antiulcer, hypotensive, promoting bone growing, promotion skin metabolism and enhancing immunity.In doctor
Medicine, health care and beauty and make-up aspect has a good application prospect.Research to fish scrap collagen is carried out at home
Seldom, often as offal treatment, living resources are caused to waste and environmental pollution.China's collagen is generally from slaughtered animals
Waste material in produce, the utilization of the collagen peptide in aquatic livestock is always blank.Collagen is extracted using modern biotechnology
Polypeptide, improves biologically active, and further the raw material of exploitation medicine, health care and cosmetics, there are no at present both at home and abroad
Cross report.Seafood fish leftover bits and pieces is extracted into collagen and new active collagen polypeptide is prepared, structural modification, Ji Nengman is carried out
Demand of the sufficient every profession and trade to collagen polypeptide, can lift the added value of aquatic products and recycle discarded object again, environmental protection,
Aquatic products industry is developed wide prospect is provided.
The content of the invention
First purpose of the present invention is the preparation method for providing squid collagen polypeptide.
Second object of the present invention is the preparation method for providing squid collagen polypeptide iron complex.
Third object of the present invention is to provide that squid collagen polypeptide is good and squid collagen polypeptide iron complex.
Fourth object of the present invention is to provide the application of squid collagen polypeptide and its iron complex.
To realize first purpose of the invention, the present invention discloses technical scheme below：The preparation method of squid collagen polypeptide,
Characterized in that, the preparation method comprises the steps：
(1) take fresh squid skin rubbing to put in reaction under high pressure pot, add the aqueous acetic acid that mass percent is 2-10%,
Stirring while adding, steam heating after stirring, temperature control stops immediately heating at 110-130 DEG C, after hydrolysis 1-3 hours,
It is allowed to be naturally cooling to less than 105 DEG C；
(2) vent valve is slowly opened, charge door is opened and is added activated carbon, 90～100 DEG C of insulation 20-60 minutes, Ran Houli
The heart, centrifugate is concentrated into 1/3-1/5 weight and obtains concentrate 1；
(3) concentrate 1 that step (2) is obtained is cooled to after room temperature, adds the ethanol of 3-5 times of weight, stirring while adding, is allowed
Its precipitates overnight, precipitated liquid centrifugation, filtrate press filtration again, filtrate recycling ethanol is simultaneously concentrated into 1/9-1/11 weight and obtains concentrate
2, concentrate 2 is spray-dried, obtain orange-yellow porous solid and be squid collagen polypeptide (SP-1).Jing mass spectral analyses, contain 20
Remaining kind of peptide constituents.
Used as a preferred version, step (1) is hydrolyzed 1 hour.
To realize second purpose of the invention, the present invention discloses technical scheme below：Squid collagen polypeptide iron complex
Preparation method, it is characterised in that the preparation method comprises the steps：
(1) above-mentioned squid collagen polypeptide is dissolved in distilled water, percentage by weight 2.8% is slowly added under magnetic agitation
Ferrous sulfate aqueous solution, room temperature reaction 3h-4h；
(2) after reacting liquid filtering, the ethanol of 3-5 times of weight is added to be precipitated in filtrate, suction filtration after 2h, absolute ethyl alcohol
Washing, filter residue is dried in room temperature in vacuo and removes ethanol, and dried object multiplexing distillation water dissolves, freeze-drying obtains brown color scale loose
Thing is squid collagen polypeptide iron complex (SP-F).Yield is 47.2%.
To realize the 3rd purpose of the invention, the present invention discloses technical scheme below：Using above-mentioned squid collagen polypeptide
The squid collagen polypeptide that preparation method is prepared, and, using the preparation method system of above-mentioned squid collagen polypeptide iron complex
The standby squid collagen polypeptide iron complex for obtaining.
To realize the 4th purpose of the invention, the present invention discloses technical scheme below：Squid collagen polypeptide prepare it is resistance to
Application in anoxic medicine or anoxia-resistant food, and, squid collagen polypeptide iron complex is preparing anti-hypoxia medicament or resistance to scarce
Application in oxygen food.
It is an advantage of the current invention that：This method than traditional sulfuric acid and Hydrochloric Acid Hydrolysis Method, with stable reaction conditions, easily
In control, non-pollutant discharge, meet cleanly production.The present invention is carried from the seafood fish leftover bits and pieces such as squid leftover bits and pieces fish-skin
Take collagen and prepare new active collagen polypeptide, carry out structural modification, every profession and trade can be met to collagen polypeptide
Demand, can lift the added value of aquatic products and recycle discarded object again, environmental protection, to aquatic products industry development provide it is wide before
Scape.Zoopery shows that squid collagen polypeptide (SP-1) and its iron complex (SP-F) are remarkably improved the resist oxygen lack time-to-live；
Increase internal lactic acid dehydrogenase activity；Reduce the content of internal blood lactase acid and blood urea nitrogen.SP-1 and SP-F prepared by the present invention
Oxygen-deficient endurance is remarkably improved, can be used to prepare anti-hypoxia medicament and functional food, the Ocean Medicinal to developing China
Living resources are significant.
Description of the drawings
Fig. 1 is the resist oxygen lack time-to-live.
Fig. 2 is lactic acid dehydrogenase activity.
Fig. 3 is Serum lactic acid content.
Fig. 4 is blood urea nitrogen detection.
Fig. 5 mass spectroscopy results.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.Experimental technique used in following embodiments for example without
Specified otherwise, is conventional method.Material used, reagent etc. in following embodiments, if no special instructions, can be from business way
Footpath obtains.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
The preparation of the squid skin collagen polypeptide of embodiment 1.
Fresh squid skin 200Kg, is rubbed with meat grinder.The squid skin of rubbing is put in reaction under high pressure pot.Add 1200L's
3% acetic acid water (i.e. the glacial acetic acid of water 1160L, glacial acetic acid 36Kg or 72 bottles of 500ml is mixed), stirring while adding, Zhi Daojia
Till complete.After acetic acid water is added and stirred, begin to warm up, temperature control at 121 DEG C, after hydrolysis 1 hour, stop immediately plus
Heat, allows it to be naturally cooling to～105 DEG C, then slowly opens vent valve, opens charge door, adds 2.5Kg activated carbons, 90～
100 DEG C are incubated 20 minutes.It is then centrifuged for, centrifugate is concentrated into 300L or so.Concentrate is cooled to after room temperature, adds 3 times of amount (V/
V) 95% ethanol, side edged is stirred, and allows its precipitates overnight.Precipitated liquid is centrifuged, filtrate press filtration again.Filtrate recycling ethanol is simultaneously concentrated into
200L or so, concentrate is spray-dried again, obtains 4.5 kilograms of orange-yellow porous solid (squid collagen polypeptide).
Mass spectroscopy result：Using direct-injection technique, the total polypeptide Jing mass spectrums anion spectrum of collagen and cation spectrum are determined, glue
Former total polypeptide plants oligopeptides constituents in the range of molecular weight 170-1334.5 containing more than 20.Fig. 5 is shown in mass spectral analysis.
The preparation of the squid skin collagen polypeptide of embodiment 2.
Fresh squid skin 100Kg, is rubbed with meat grinder.The squid skin of rubbing is put in reaction under high pressure pot.Add the 8% of 600L
Acetic acid water (i.e. water 580L, glacial acetic acid 18Kg are mixed), it is stirring while adding, till adding.Acetic acid water is added and stirred
After uniform, begin to warm up, temperature control after hydrolyzing 1 hour, stops immediately heating at 110 DEG C, allows it to be naturally cooling to～105
DEG C, vent valve is then slowly opened, charge door is opened, 1Kg activated carbons are added, 90～100 DEG C are incubated 20 minutes.It is then centrifuged for,
Centrifugate is concentrated into 150L or so.Concentrate is cooled to after room temperature, adds 3 times of amount (V/V) 95% ethanol, side edged to stir, and allows it
Precipitates overnight.Precipitated liquid is centrifuged, filtrate press filtration again.Filtrate recycling ethanol is simultaneously concentrated into 200L or so, and concentrate is spray-dried again,
Obtain orange-yellow 2.3 kilograms of porous solid (squid collagen polypeptide).
The preparation of the squid collagen polypeptide iron complex of embodiment 3.
Squid skin range of hydrolysed peptides 30.00g is dissolved in the distilled water of 120mL, adds 2.8% ferrous sulfate to be dissolved in 30mL.
Under magnetic agitation, copperas solution is slowly added into squid skin hydrolysis peptide solution, room temperature reaction 3h.Reactant liquor degreasing
After cotton is filtered, 95% ethanol of 3 times of amounts is added in filtrate, precipitated.Suction filtration, twice, filter residue is in room for absolute ethanol washing
Temperature vacuum drying (removes ethanol).Multiplexing 420mL distillation water dissolves, freeze-drying obtains squid skin range of hydrolysed peptides ferrous salt (brown color squama
Piece loose matter).Yield is 47.8%.
The preparation of the squid collagen polypeptide iron complex of embodiment 4.
Squid skin range of hydrolysed peptides 100.00g is dissolved in the distilled water of 400mL, 2.8% ferrous sulfate 100mL is added.In magnetic
Under power stirring, copperas solution is slowly added into squid skin hydrolysis peptide solution, room temperature reaction 3h.Reactant liquor absorbent cotton
After filtration, 95% ethanol of 3 times of amounts is added in filtrate, precipitated.Suction filtration, twice, filter residue is in room temperature for absolute ethanol washing
Vacuum drying (removes ethanol).Multiplexing 1400mL distillation water dissolves, freeze-drying obtains squid skin range of hydrolysed peptides ferrous salt (brown color squama
Piece loose matter).Yield is 48.1%.
The squid polypeptide resist oxygen lack animal experiment of embodiment 5.
1. research material
1.1 animals used as test：Kunming mice, male, 6-8 week old, 18-22g, 8 per group, high concentration group 9.
1.2 reagents and medicine：Blood lactase acid kit, lactic dehydrogenase enzyme reagent kit and blood urea nitrogen kit (are built by Nanjing
Cheng companies provide)；Verapamil (is provided) by Tianjin Central Pharmaceutical Co., Ltd, given the test agent SP-1 (squid collagen polypeptide)
It is standby by The 2nd Army Medical College Yi Yanghua professor's project team systems with SP-F (squid collagen polypeptide iron complex).
1.3 laboratory apparatus：ELIASA, centrifuge.
2. experimental technique
Mouse is randomly divided into 8 groups, control group：Two given the test agent SP-F and SP-1, set up separately：50mg/kg, 100mg/kg,
200mg/kg3 dosage group, resist oxygen lack experiment positive drug (Verapamil) and saline control group.Daily gastric infusion,
Verapamil drug administration by injection, totally 21 days.After last dose 1h, mouse is by being only put into the 125ml wide-mouth bottles that fill 20g soda limes
In, the closed bottleneck of paraffin records the mouse survival time.
Heart extracting blood immediately after dead mouse, illustrates according to kit, carries out blood urea nitrogen, Serum lactic acid content and lactic acid and takes off
Hydrogenase activity is detected.
Experimental data adopts the software processings of SPSS 13.0
3. experimental result
The each group animal survival condition of table 3.1
Resist oxygen lack time-to-live (s) of table 3.2
The lactic acid dehydrogenase activity of table 3.3
The Serum lactic acid content of table 3.4
The blood urea nitrogen of table 3.5 is detected
The organ index of table 3.6 (mg/g)
The Avoirdupois monitoring of table 3.7
Above test result indicate that, in experimentation, the survival rate of mouse, body weight and each organ index are showed no and substantially change
Become；The resist oxygen lack time-to-live：50mg/kg the and 100mg/kg dosage groups of SP-1 and the 100mg/kg dosage groups of SP-F compare physiology
Salt solution group significantly extends；Lactic acid dehydrogenase activity：The 100mg/kg dosage groups of SP-1 are dramatically increased；Serum lactic acid content：SP-1's
50mg/kg the and 100mg/kg dosage groups of 50mg/kg and 100mg/kg dosage groups and SP-F are substantially reduced than physiological saline group；
Blood urea nitrogen content:The 50mg/kg dosage of SP-1 and the 200mg/kg dosage groups of SP-F are substantially reduced than physiological saline group.Experiment
As a result show that SP-1 and SP-F have obvious Oxygen-deficient endurance, can be used to prepare anti-hypoxia medicament and functional food.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise without departing from the principles of the invention, can also make some improvements and modifications, and these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (7)
1. the preparation method of squid collagen polypeptide, it is characterised in that the preparation method comprises the steps：
(1) take fresh squid skin rubbing to put in reaction under high pressure pot, add mass percent for the aqueous acetic acid of 2-10%, Bian Jia
Side is stirred, steam heating after stirring, and temperature control stops immediately heating at 110-130 DEG C, after hydrolysis 1-3 hours, allows it
It is naturally cooling to less than 105 DEG C；
(2) vent valve is slowly opened, charge door is opened and is added activated carbon, 90～100 DEG C of insulation 20-60 minutes, be then centrifuged for, from
Heart liquid is concentrated into 1/3-1/5 weight and obtains concentrate 1；
(3) concentrate 1 that step (2) is obtained is cooled to after room temperature, adds the ethanol of 3-5 times of weight, stirring while adding, allows it to sink
Form sediment overnight, precipitated liquid centrifugation, filtrate press filtration again, filtrate recycling ethanol is simultaneously concentrated into 1/9-1/11 weight and obtains concentrate 2, will
Concentrate 2 is spray-dried, and obtains orange-yellow porous solid and is squid collagen polypeptide.
2. the preparation method of the squid collagen polypeptide according to right and described in requiring 1, it is characterised in that step (1) hydrolysis 1 is little
When.
3. the squid collagen polypeptide for being prepared using the preparation method of squid collagen polypeptide described in claim 1.
4. the preparation method of squid collagen polypeptide iron complex, it is characterised in that the preparation method comprises the steps：
(1) the squid collagen polypeptide described in claim 3 is dissolved in distilled water, under magnetic agitation weight percent is slowly added into
Than 2.8% ferrous sulfate aqueous solution, room temperature reaction 3h-4h；
(2) after reacting liquid filtering, the ethanol of 3-5 times of weight is added to be precipitated in filtrate, suction filtration after 2h, absolute ethyl alcohol is washed
Wash, filter residue is dried in room temperature in vacuo and removes ethanol, dried object multiplexing distillation water dissolves, freeze-drying obtains brown color scale loose matter
As squid collagen polypeptide iron complex.
5. the squid collagen polypeptide iron for being prepared using the preparation method of squid collagen polypeptide iron complex described in claim 4
Complex compound.
6. the squid collagen polypeptide described in claim 3 is preparing anti-hypoxia medicament or the application in anoxia-resistant food.
7. the squid collagen polypeptide iron complex described in claim 5 in anti-hypoxia medicament or anoxia-resistant food is prepared should
With.
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