CN107236775A - A kind of deer SPP1 polypeptide and application - Google Patents
A kind of deer SPP1 polypeptide and application Download PDFInfo
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- CN107236775A CN107236775A CN201710412957.0A CN201710412957A CN107236775A CN 107236775 A CN107236775 A CN 107236775A CN 201710412957 A CN201710412957 A CN 201710412957A CN 107236775 A CN107236775 A CN 107236775A
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 53
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 53
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 241000282994 Cervidae Species 0.000 title claims abstract description 45
- 102100030684 Sphingosine-1-phosphate phosphatase 1 Human genes 0.000 title claims abstract description 35
- 101710168942 Sphingosine-1-phosphate phosphatase 1 Proteins 0.000 title claims abstract description 35
- 239000003513 alkali Substances 0.000 claims abstract description 24
- 238000005516 engineering process Methods 0.000 claims abstract description 20
- 230000005855 radiation Effects 0.000 claims abstract description 19
- 238000005238 degreasing Methods 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 229940036811 bone meal Drugs 0.000 claims description 27
- 239000002374 bone meal Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 24
- 210000000988 bone and bone Anatomy 0.000 claims description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 11
- 108091005804 Peptidases Proteins 0.000 claims description 8
- 239000004365 Protease Substances 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000005237 degreasing agent Methods 0.000 claims description 7
- 239000013527 degreasing agent Substances 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 230000002779 inactivation Effects 0.000 claims description 6
- 238000007670 refining Methods 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 235000019419 proteases Nutrition 0.000 claims description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 2
- -1 chloromethanes Alkane Chemical class 0.000 claims 1
- 102000015636 Oligopeptides Human genes 0.000 abstract description 8
- 108010038807 Oligopeptides Proteins 0.000 abstract description 8
- 235000014101 wine Nutrition 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 4
- 102000004142 Trypsin Human genes 0.000 abstract description 2
- 108090000631 Trypsin Proteins 0.000 abstract description 2
- 230000002929 anti-fatigue Effects 0.000 abstract description 2
- 230000029087 digestion Effects 0.000 abstract description 2
- 239000012588 trypsin Substances 0.000 abstract description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241001550206 Colla Species 0.000 description 3
- 102000005158 Subtilisins Human genes 0.000 description 3
- 108010056079 Subtilisins Proteins 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- GLEVLJDDWXEYCO-UHFFFAOYSA-N Trolox Chemical compound O1C(C)(C(O)=O)CCC2=C1C(C)=C(C)C(O)=C2C GLEVLJDDWXEYCO-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000006193 liquid solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282985 Cervus Species 0.000 description 1
- 241000283007 Cervus nippon Species 0.000 description 1
- 208000008967 Enuresis Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 241000270708 Testudinidae Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 206010003549 asthenia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- JTEVUCQIVRGWHT-UHFFFAOYSA-N phosphoric acid;phthalic acid Chemical compound OP(O)(O)=O.OC(=O)C1=CC=CC=C1C(O)=O JTEVUCQIVRGWHT-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 235000020097 white wine Nutrition 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/04—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
-
- 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
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a kind of deer SPP1 polypeptide, the preparation method of the deer SPP1 polypeptide includes successively：Degreasing, decalcification step, microwave radiation technology alkali carries, microwave radiation technology alkali enzymolysis, microwave radiation technology neutrality enzymolysis and trypsin digestion and concentration step.The deer SPP1 polypeptide has higher oligopeptides ratio, with higher inoxidizability and antifatigue effect of refreshing oneself.The deer SPP1 polypeptide can be used for preparing on health products, its health preserving wine prepared, with higher stability.
Description
Technical field
The present invention relates to health treatment technical field, more particularly to a kind of deer SPP1 polypeptide and application.
Background technology
Animal Bone active component is mainly polypeptide and some protein such as ostosis albumen and bone growth factor etc..Deer
Bone is generally the bone of Cervidae sika deer and red deer animal.Traditional Chinese medical science information, with qi-restoratives win, strengthening the bones and muscles, blood-enrich, wind-dispelling
The function of dehumidifying, for diseases such as rheumatalgia pain, impotence, the enuresis, oedema.Colla 0ssis Cervi is to eipathia asthenia, marrow deficiency, anaemia, wind
The diseases such as wet, limbs pain have preferable clinical efficacy.
Because Colla 0ssis Cervi has huge molecular weight, the colloidal stability formed in alcohol water blend is poor, chance temperature,
The change stability of oxidation-reduction potential etc. is easily destroyed, and produces loss of gloss, muddiness, deposited phenomenon, and this is in food and drink wine (dew
Wine) in be flagrant.On the other hand, may in velvet deerhorn and tortoise plastron liquor process for preparation because Colla 0ssis Cervi has huge molecular weight
The loss caused is as follows:1) precipitated with the tannin in Chinese medicine and polyphenols formation；2) formed under certain alcohol concentration
Precipitation；3) precipitation is formed in traditional aging process；4) loss of gloss, turbidity and precipitation are formed when temperature declines suddenly, vinosity is influenceed.
Collagen polypeptide is that collagen or gelatin handle obtained product by proteasome degradation, is had compared with gelatin stronger
Water solubility, is easily absorbed by the body.With progress of research, it has been found that many skins of collagen have more physiological function, for example, resist
Hypertension, prevention and treatment osteoarthritis and osteoporosis, treatment gastric ulcer etc. disease, anti-aging and anti-oxidant etc..
Modern study finds that the anti-inflammatory active ingredient in deer bone is polypeptide.
The content of the invention
In order to overcome the deficiencies in the prior art, an object of the present invention is to provide a kind of deer SPP1 polypeptide.
The second object of the present invention is the application for providing the deer SPP1 polypeptide.
An object of the present invention adopts the following technical scheme that realization：
A kind of deer SPP1 polypeptide, it is characterised in that its preparation method includes successively：
Defatting step：Deer aggregate is crushed to 60-80 mesh, loads filter screen, after being rinsed repeatedly with distilled water, uses degreasing agent
Degreasing is carried out, dries, obtains bone tankage；
Decalcification step：Bone tankage is washed with decalcifying agent, decalcification bone meal is obtained；
Extraction step：Dilute NaOH solution is added into decalcification bone meal to pH=12-13,50-60 DEG C of microwave radiation technology is carried
Take, obtain first extract；
Alkali enzymolysis step：Watery hydrochloric acid is added into first extract to pH=9-10, alkali protease, 55-65 DEG C of microwave is added
Assistance enzymolysis, obtains primary enzymolysis liquid；
Enzymolysis step again：Neutral proteinase and trypsase, 45-55 DEG C of microwave-assisted enzyme are added into primary enzymolysis liquid
Solution, is heated to 80-90 DEG C of inactivation, stands, and filtering obtains polypeptide liquid；
Concentration step：Refining liquid is concentrated under reduced pressure into Brix for 45-55 °, the body of paste of deer-bone collagenous polypeptide is produced.
Further, in defatting step, degreasing agent is chloroform：Ethanol：Water=(1-2):(1-2):1.
Further, in decalcification step, decalcifying agent is 0.5mol/L EDTA.
Further, in decalcification step, decalcifying agent is 0.5mol/L watery hydrochloric acid.
Further, extraction step and/or alkali enzymolysis step and/or again in enzymolysis step, microwave frequency is 1500-
3000MHz, power is 5kW.
Further, in alkali enzymolysis step, the addition of alkali protease is 2000-3000U/g decalcification bone meal.
Further, again in enzymolysis step, the addition of neutral proteinase is 2000-3000U/g decalcification bone meal.
Further, again in enzymolysis step, the addition of trypsase is 3000-4000U/g decalcification bone meal.
Further, using micro porous filtration.
The second object of the present invention adopts the following technical scheme that realization：
The application of the deer SPP1 polypeptide on health products are prepared.
Compared with prior art, the beneficial effects of the present invention are：
(1) molecular structure for the deer SPP1 polypeptide that the present invention is provided is more uniform, and the accounting of oligopeptides is high, it is easy to absorb；Should
Deer SPP1 polypeptide has preferable antifatigue and antioxidation activity, with preferable effect；
(2) the deer SPP1 polypeptide that provides of the present invention has higher stability after being used to preparing health preserving wine, is difficult to sink
Form sediment, good quality.
Embodiment
Below, with reference to embodiment, the present invention is described further, it is necessary to which explanation is, what is do not collided
Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.
In detailed description below, such as non-specified otherwise, the reagent or material used can be by commercially available or conventional
The mode of research technique is obtained.
The present invention provides a kind of deer SPP1 polypeptide, and its preparation method includes successively：
Defatting step：Deer aggregate is crushed to 60-80 mesh, loads filter screen, after being rinsed repeatedly with distilled water, uses degreasing agent
Degreasing is carried out, dries, obtains bone tankage；
Decalcification step：Bone tankage is washed with decalcifying agent, decalcification bone meal is obtained；
In the degreasing and decalcification step, it can effectively remove liposoluble constituent in deer aggregate and calcareous, preparation can be reduced
Process, the loss of collagen improves the yield of polypeptide.Degreasing agent can selected from including but not limited to chloroform and
The mixed aqueous solution of ethanol, such as chloroform：Ethanol：Water=(1-2):(1-2):1, it can be lost using this kind of degreasing agent
On the basis of SPP1, the liposoluble constituent in aggregate is effectively removed.Decalcifying agent can be selected from EDTA or watery hydrochloric acid, the former with
Chelation is precipitated with calcium formation, and the latter's dissolving is calcareous, under the 0.5mol/L concentration ranges, to the damageability of deer SPP1
It is small；
Extraction step：Dilute NaOH solution is added into decalcification bone meal to pH=12-13,50-60 DEG C of microwave radiation technology is carried
Take, obtain first extract；
In this step, by the way of sig water is extracted, it can be efficiently reduced while extraction efficiency is ensured to deer bone
The loss of albumen；And excessive concentration, it is possible to albuminous degeneration is caused, concentration is too low, extraction efficiency is not high；
Alkali enzymolysis step：Watery hydrochloric acid is added into first extract to pH=9-10, alkali protease, 55-65 DEG C of microwave is added
Assistance enzymolysis, obtains primary enzymolysis liquid；
Enzymolysis step again：Neutral proteinase and trypsase, 45-55 DEG C of microwave-assisted enzyme are added into primary enzymolysis liquid
Solution, is heated to 80-90 DEG C of inactivation, stands, and filtering obtains polypeptide liquid；
Alkali enzymolysis step and again enzymolysis step are combined, the trend that on the one hand effectively can be changed using system pH, more thorough
Digest to bottom, to obtain the polypeptide that strand is shorter, be more easy to absorption, that is, add after alkali enzyme enzymolysis, under system pH slightly has
Drop, adds neutral proteinase and trypsin digestion, two kinds of enzymes enzymatic activity under the pH value of system reaches higher level；This
In step, filter type can using conventional multilayer gauze natural filtration, in order to a step high protein polypeptide liquid homogeneity, can
Using micro porous filtration, optional 22 μm of the aperture of micro porous filtration；
Concentration step：Refining liquid is concentrated under reduced pressure into Brix for 45-55 °, the body of paste of deer-bone collagenous polypeptide is produced,
Body of paste made from this step has preferable stability, not mutability.
Embodiment 1：
A kind of deer SPP1 polypeptide, its preparation method includes successively：
Defatting step：Deer aggregate is crushed to 60-80 mesh, loads the filter being formed by stacking by three layers of aperture for the gauze of 22 mesh
Net, after being rinsed repeatedly with distilled water, uses chloroform：Ethanol：Water=1.5:1.5:1 carries out degreasing, dries, obtains degreasing bone
Powder；
Decalcification step：Bone tankage is washed with 0.5mol/L EDTA, decalcification bone meal is obtained；
Extraction step：Dilute NaOH solution is added into decalcification bone meal to pH=12-13,55 DEG C of microwave radiation technologies are extracted,
Obtain first extract；Wherein microwave frequency is 1500-3000MHz, and power is 5kW；
Alkali enzymolysis step：Watery hydrochloric acid is added into first extract to pH=9-10, is added with the feed molar ratio of 2500U/g decalcification bone meal
Enter Alcalase alkali proteases, 60 DEG C of microwave radiation technology enzymolysis obtain primary enzymolysis liquid；Wherein microwave frequency is 2000MHz, work(
Rate is 5kW；
Enzymolysis step again：Neutral proteinase and trypsase are added into primary enzymolysis liquid, 50 DEG C of microwave radiation technologies are digested,
85 DEG C of inactivations are heated to, stands, using 22 μm of micro-pore-film filtrations, obtains polypeptide liquid；The wherein feed molar ratio of neutral proteinase
For 2500U/g decalcification bone meal, the feed molar ratio of trypsase is 3500U/g decalcification bone meal；
Concentration step：Refining liquid is concentrated under reduced pressure into Brix for 50 °, the body of paste of deer-bone collagenous polypeptide is produced, receives
Rate is 86.6%, and oligopeptides ratio is 77.3%.
Embodiment 2：
A kind of deer SPP1 polypeptide, its preparation method includes successively：
Defatting step：Deer aggregate is crushed to 60-80 mesh, loads the filter being formed by stacking by three layers of aperture for the gauze of 22 mesh
Net, after being rinsed repeatedly with distilled water, uses chloroform：Ethanol：Water=1:2:1 carries out degreasing, dries, obtains bone tankage；
Decalcification step：Bone tankage is washed with 0.5mol/LHCl, decalcification bone meal is obtained；
Extraction step：Dilute NaOH solution is added into decalcification bone meal to pH=12-13,60 DEG C of microwave radiation technologies are extracted,
Obtain first extract；Wherein microwave frequency is 1500-3000MHz, and power is 5kW；
Alkali enzymolysis step：Watery hydrochloric acid is added into first extract to pH=9-10, is added with the feed molar ratio of 2000U/g decalcification bone meal
Enter Alcalase alkali proteases, 65 DEG C of microwave radiation technology enzymolysis obtain primary enzymolysis liquid；Wherein microwave frequency is 2000MHz, work(
Rate is 5kW；
Enzymolysis step again：45 DEG C of microwave radiation technology enzymolysis of neutral proteinase and trypsase are added into primary enzymolysis liquid,
80 DEG C of inactivations are heated to, stands, using 22 μm of micro-pore-film filtrations, obtains polypeptide liquid；The wherein feed molar ratio of neutral proteinase
For 3000U/g decalcification bone meal, the feed molar ratio of trypsase is 4000U/g decalcification bone meal；
Concentration step：Refining liquid is concentrated under reduced pressure into Brix for 45 °, the body of paste of deer-bone collagenous polypeptide is produced, receives
Rate is 85.1%, and oligopeptides ratio is 79.0%.
Embodiment 3：
A kind of deer SPP1 polypeptide, its preparation method includes successively：
Defatting step：Deer aggregate is crushed to 60-80 mesh, loads the filter being formed by stacking by three layers of aperture for the gauze of 22 mesh
Net, after being rinsed repeatedly with distilled water, uses chloroform：Ethanol：Water=2:1:1 carries out degreasing, dries, obtains bone tankage；
Decalcification step：Bone tankage is washed with 0.5mol/L EDTA, decalcification bone meal is obtained；
Extraction step：Dilute NaOH solution is added into decalcification bone meal to pH=12-13,60 DEG C of microwave radiation technologies are extracted,
Obtain first extract；Wherein microwave frequency is 1500-3000MHz, and power is 5kW；
Alkali enzymolysis step：Watery hydrochloric acid is added into first extract to pH=9-10, is added with the feed molar ratio of 2000U/g decalcification bone meal
Enter Alcalase alkali proteases, 55 DEG C of microwave radiation technology enzymolysis obtain primary enzymolysis liquid；Wherein microwave frequency is 2000MHz, work(
Rate is 5kW；
Enzymolysis step again：45 DEG C of microwave radiation technology enzymolysis of neutral proteinase and trypsase are added into primary enzymolysis liquid,
80 DEG C of inactivations are heated to, stands, using 22 μm of micro-pore-film filtrations, obtains polypeptide liquid；The wherein feed molar ratio of neutral proteinase
For 2000U/g decalcification bone meal, the feed molar ratio of trypsase is 3000U/g decalcification bone meal；
Concentration step：Refining liquid is concentrated under reduced pressure into Brix for 55 °, the body of paste of deer-bone collagenous polypeptide is produced, receives
Rate is 84.7%, and oligopeptides ratio is 75.4%.
Comparative example 1：
As different from Example 1, extraction step does not use microwave radiation technology to comparative example 1, and yield is 54.1%, oligopeptides ratio
For 80.4%.
Comparative example 2：
As different from Example 1, alkali enzymolysis step and again enzymolysis step do not use microwave radiation technology to comparative example 2, receive
Rate is 76.4%, and oligopeptides ratio is 61.7%.
Comparative example 3：
Comparative example 3 as different from Example 1, extraction step, alkali enzymolysis step and again enzymolysis step using micro-
Ripple is aided in, and yield is 56.4%, and oligopeptides ratio is 64.9%.
Performance detection and effective evaluation
1st, determination oxidative
By 10 μm of ol/L Trolox storing solutions phosphorate phthalate buffer be diluted to concentration for 0.05,0.10,0.20,
0.40th, 0.80,1.6 μm of ol/L standard liquid, using phosphate buffer as negative control, using fluorescence half-life as vertical seat
Mark, using Trolox concentration as abscissa, draws standard curve.
0.01g embodiment 1-3 deer SPP1 polypeptide is taken, using distilled water as blank control, plus 100mL phosphate delays
Fliud flushing is diluted, then be respectively adopted phosphate buffer dilution by 10 times, 100 times, 1000 times, 10000 times, 100000 times it is dilute
Release, dilution is measured, its result is as shown in the table：
The oxidation resistance test result of table 1
2nd, mouse, which bears a heavy burden, tests
Using SPF male mices, body weight is 18-22g, per test group 10, totally 40, and swimming with a load attached to the body survey is carried out respectively
Fixed, environment temperature is 22-24 DEG C, humidity 50-54% during experiment.The deer bone egg polypeptide obtained using embodiment 1, setting dosage
For 0.1g/kg.bw, 0.2g/kg.bw, 0.5g/kg.bw, using aqua sterilisa as control group, mix food and feed 30 days, carry out swimming with a load attached to the body
Experiment.
The Loaned swimming test of table 2
After gavage is fed 30 days, each dosage group mouse, the walking weight load relative to control group mice is obviously prolonged.Say
The deer SPP1 polypeptide that bright the application is provided, which has, improves body fatigue resistance effect.
3rd, stability test
By embodiment 1-3 and comparative example 1-3 deer SPP1 polypeptide with 1:10 proportioning is added in 50 ° of white wine, concussion
Dissolving, obtains the health preserving wine of deer SPP1 polypeptide, takes 1L health preserving wines with 3000rpm centrifugation 5min, observation health preserving wine
Outward appearance and whether there is precipitation, measure precipitation volume, it is as a result as shown in the table：
The stability test result of table 3
As shown above, the deer SPP1 polypeptide that this application is obtained has higher dynamic stability, the polypeptide
Prepare and be unlikely to deteriorate after health preserving wine, be not likely to produce precipitation, quality is higher.
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to limit the scope of protection of the invention with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (10)
1. a kind of deer SPP1 polypeptide, it is characterised in that its preparation method includes successively：
Defatting step：Deer aggregate is crushed to 60-80 mesh, loads filter screen, after being rinsed repeatedly with distilled water, is carried out using degreasing agent
Degreasing, dries, obtains bone tankage；
Decalcification step：Bone tankage is washed with decalcifying agent, decalcification bone meal is obtained；
Extraction step：Dilute NaOH solution is added into decalcification bone meal to pH=12-13,50-60 DEG C of microwave radiation technology is extracted, obtained
To first extract；
Alkali enzymolysis step：Watery hydrochloric acid is added into first extract to pH=9-10, alkali protease, 55-65 DEG C of microwave radiation technology is added
Enzymolysis, obtains primary enzymolysis liquid；
Enzymolysis step again：Neutral proteinase and trypsase are added into primary enzymolysis liquid, 45-55 DEG C of microwave radiation technology is digested,
80-90 DEG C of inactivation is heated to, is stood, filtering obtains polypeptide liquid；
Concentration step：Refining liquid is concentrated under reduced pressure into Brix for 45-55 °, the body of paste of deer-bone collagenous polypeptide is produced.
2. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in defatting step, the degreasing agent is three chloromethanes
Alkane：Ethanol：Water=(1-2):(1-2):1.
3. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in decalcification step, the decalcifying agent is 0.5mol/
L EDTA.
4. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in decalcification step, the decalcifying agent is 0.5mol/
L watery hydrochloric acid.
5. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that extraction step and/or alkali enzymolysis step and/or again
In secondary enzymolysis step, microwave frequency is 1500-3000MHz, and power is 5kW.
6. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in alkali enzymolysis step, the addition of alkali protease
Measure as 2000-3000U/g decalcification bone meal.
7. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that again in enzymolysis step, the neutral proteinase
Addition be 2000-3000U/g decalcification bone meal.
8. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that again in enzymolysis step, the trypsase
Addition is 3000-4000U/g decalcification bone meal.
9. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that again in enzymolysis step, using micro porous filtration.
10. application of the deer SPP1 polypeptide on health products are prepared as described in claim any one of 1-9.
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| CN107897943A (en) * | 2017-11-28 | 2018-04-13 | 苏洁 | A kind of method that deer bone native peptides are extracted in the bone from deer |
| CN108396052A (en) * | 2018-03-28 | 2018-08-14 | 通化百泉保健食品有限公司 | A kind of industrialized producing technology of deer's sinew albumen oligopeptide |
| CN108623653A (en) * | 2018-04-27 | 2018-10-09 | 罗乌支 | A method of it extracting antler native peptides from antler and enzymolysis prepares polypeptide |
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| CN108623653A (en) * | 2018-04-27 | 2018-10-09 | 罗乌支 | A method of it extracting antler native peptides from antler and enzymolysis prepares polypeptide |
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