CN105693840A - Preparation method of fly larva polypeptides - Google Patents
Preparation method of fly larva polypeptides Download PDFInfo
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- CN105693840A CN105693840A CN201610184180.2A CN201610184180A CN105693840A CN 105693840 A CN105693840 A CN 105693840A CN 201610184180 A CN201610184180 A CN 201610184180A CN 105693840 A CN105693840 A CN 105693840A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 42
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 41
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 16
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 238000004537 pulping Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 abstract description 7
- 230000003078 antioxidant effect Effects 0.000 abstract description 6
- 238000000502 dialysis Methods 0.000 abstract description 5
- 238000007254 oxidation reaction Methods 0.000 abstract description 5
- 239000003963 antioxidant agent Substances 0.000 abstract description 4
- 230000003647 oxidation Effects 0.000 abstract description 4
- 238000001914 filtration Methods 0.000 abstract description 2
- 238000000227 grinding Methods 0.000 abstract 1
- 239000012153 distilled water Substances 0.000 description 19
- 239000000523 sample Substances 0.000 description 19
- 239000000243 solution Substances 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000003292 glue Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000007865 diluting Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 150000003254 radicals Chemical class 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 239000004141 Sodium laurylsulphate Substances 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 230000003064 anti-oxidating effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 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 2
- 230000009970 fire resistant effect Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229960002477 riboflavin Drugs 0.000 description 2
- 235000019192 riboflavin Nutrition 0.000 description 2
- 239000002151 riboflavin Substances 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000000881 depressing effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- DIHKMUNUGQVFES-UHFFFAOYSA-N n,n,n',n'-tetraethylethane-1,2-diamine Chemical compound CCN(CC)CCN(CC)CC DIHKMUNUGQVFES-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000007540 photo-reduction reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000033764 rhythmic process Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Insects & Arthropods (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a preparation method of fly larva polypeptides. The method comprises the following steps: grinding fly larvae into pulp, adding an appropriate amount of water to dilute the pulp, heating for 10 min to 20 min under the condition of 80 to 120 DEG C, filtering, lowering the temperature to 4 DEG C, centrifuging, collecting supernatant, dialyzing by adopting a dialysis bag, and removing impurities with the molecular weight of 3000Da or less, thereby obtaining the fly larva polypeptides. The fly larva polypeptides obtained by virtue of the method still have relatively high oxidation resistance at the high temperature of 80 to 100 DEG C and are suitable for being used as a high-temperature-resistant antioxidant.
Description
Invention field
The present invention relates to the polypeptide from animal, be specifically related to fly larvae polypeptide。
Background technology
Free radical O2 -There is bio-toxicity, lipid peroxidation, damaging cells film can be made, cause multiple disease, promote body aging, involutional generation in advance。Daily diet is unhealthy, Exposure to Sunlight, pressure, environmental pollution can allow the free radical in organism spread unchecked。The oxidation resistance of body is more strong, turns out body vitality more strong, and the life-span is more long
Antioxidation is exactly any material that there is the oxidation reaction that just can effectively suppress free radical with low concentration, and its mechanism of action can be act directly on free radical, or indirectly consumes the material easily generating free radical, it is prevented that reaction further occurs。
Quickening along with people's rhythm of life, requirement for antioxidant is more and more higher in daily life, a lot of commercial production are owing to can not find suitable resistant to elevated temperatures powerful antioxidant and cost increases severely, and also do not occurred the biological anti-oxidant of a kind of high temperature resistance with high-quality inside and outside China。
Summary of the invention
The preparation method that the technical problem to be solved is to provide a kind of fly larvae polypeptide, the obtained fly larvae polypeptide of the method has the advantage that non-oxidizability is strong。
This invention address that the technical scheme of the problems referred to above is:
A kind of preparation method of fly larvae polypeptide, the method comprises the following steps:
Fly larvae being ground pulping, adds appropriate water dilution, heat 10min～20min when 80～120 DEG C, filter, be down to 4 DEG C and be centrifuged, take supernatant, adopt bag filter dialysis, removing molecular weight is the impurity of below 3000Da, obtains fly larvae polypeptide。
The condition of the centrifugation step described in such scheme is: rotating speed is 12000rpm, and centrifugation time is 5～25min。
The molecular weight of the fly larvae polypeptide obtained described in said method is 20000Da, and this polypeptide still has stronger non-oxidizability under the hot conditions of 80～100 DEG C, is suitable for as fire-resistant oxidation resistant agent。
The present invention relatively prior art has the advantage that
1, utilize the fly larvae difference albumen tolerance to temperature, adopt the temperature conditions of 80～120 DEG C by lethal for non-targeted albumen precipitation, and then extraction process becomes very simple。
2, obtained fly larvae polypeptide is resistant to the high temperature of 80～100 DEG C, can be used for biology, medicine and other fields, wide market。
Accompanying drawing explanation
Fig. 1 is the relation curve of Extracting temperature and obtained fly larvae polypeptide non-oxidizability。
Fig. 2 is the relation curve extracting heat time heating time with obtained fly larvae polypeptide non-oxidizability。
Fig. 3 is the PAGE-SDS Gel electrophoresis results figure of fly larvae polypeptide obtained under different Extracting temperature；In figure, 1～5 hurdle is the electrophoretic effects of sample 1, the electrophoretic effects of 6～10 hurdles respectively sample 2～6
Fig. 4 is the different PAGE-SDS Gel electrophoresis results figure extracting obtained fly larvae polypeptide heat time heating time；In figure, 1,2 hurdles are the electrophoretic effects of sample 1, and 3～6 hurdles are the electrophoretic effects of sample 2～5, and 7 hurdles are the electrophoretic effects of marker albumen
Detailed description of the invention
Embodiment 1
Fly larvae is ground pulping, takes serosity 50g and add 50g distilled water diluting, heat 20min when 80 DEG C, filter, be down to 4 DEG C with the centrifugal 20min of 12000rpm, take supernatant, adopt the dialysis of 3000Da bag filter to remove impurity, obtain fly larvae polypeptide；
Fly larvae polypeptide lyophilization will be obtained, save backup at-20 DEG C。
Embodiment 2
Fly larvae is ground pulping, takes serosity 50g and add 50g distilled water diluting, heat 15min when 90 DEG C, filter, be down to 4 DEG C with the centrifugal 20min of 12000rpm, take supernatant, adopt the dialysis of 3000Da bag filter to remove impurity, obtain fly larvae polypeptide；
Fly larvae polypeptide lyophilization will be obtained, save backup at-20 DEG C。
Embodiment 3
Fly larvae is ground pulping, takes serosity 50g and add 50g distilled water diluting, heat 10min when 100 DEG C, filter, be down to 4 DEG C with the centrifugal 20min of 12000rpm, take supernatant, adopt the dialysis of 3000Da bag filter to remove impurity, obtain fly larvae polypeptide；
Fly larvae polypeptide lyophilization will be obtained, save backup at-20 DEG C。
Embodiment 4 (mensuration of the different temperatures impact on fly larvae Purity and molecular weight thereof)
The configuration of 4.1 reagent:
4.1.1. sample buffer
Weigh trishydroxymethylaminomethane 0.303g, bromophenol blue 2mg, sodium lauryl sulphate 0.8g, measure hydrochloric acid 0.189ml, glycerol 4ml, beta-mercaptoethanol 2ml, be dissolved in water after mixing and be diluted to 10ml。
4.1.2.30%Acr-Bis
Weigh 30g acrylamide, 0.8g methylene diacrylamide, add pure water and be dissolved to 100ml, with filter paper filtering after it is completely dissolved, keep in Dark Place。Put in brown bottle, the several months can be deposited at 4 DEG C。
4.1.3.pH8.8Tris-HCl
Weigh trishydroxymethylaminomethane 18.15g, add suitable quantity of water and dissolve, adjust pH value to 8.8 with hydrochloric acid, be diluted with water to 100ml。
4.1.4.pH6.7 concentration glue buffer
Weigh trishydroxymethylaminomethane 6.05g, add suitable quantity of water and dissolve, adjust pH value to 6.8 with hydrochloric acid, be diluted with water to 100ml。
4.1.5.TEMED (tetraethylethylenediamine) stock solution
4.1.6.10%AP
Weigh 100mg Ammonium persulfate., be dissolved in water to 1ml。
4.1.710g/LSDS
Weigh 1g sodium lauryl sulphate, be dissolved in water to 100ml, room temperature preservation。
4.1.8.pH8.3Tris-glycine electrode buffer
Weigh trishydroxymethylaminomethane 3g, glycine 14.4g, sodium lauryl sulphate 1g, add suitable quantity of water and dissolve, adjust pH value to 8.3 with hydrochloric acid, be diluted with water to 1000ml。
4.1.9. coomassie brilliant blue R250 dyeing liquor
Weigh coomassie brilliant blue R250 1g, add methanol 200ml, glacial acetic acid 50ml, water 250ml mixing。
4.1.10. destaining solution
Take methanol 400ml, glacial acetic acid 100ml and water 500ml mixing。
4.2. experimental implementation
4.2.1. sample preparation
Prepare fly larvae polypeptide by method described in embodiment 1, institute the difference is that after fly larvae serosity is added distilled water diluting room temperature preserve 20min and filter；Obtained fly larvae polypeptide 0.1g, adds 10mL distilled water rare as test sample 1；
Prepare fly larvae polypeptide by method described in embodiment 1, the difference is that fly larvae serosity being added the heating-up temperature respectively 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C after distilled water diluting；Taking heating-up temperature respectively is fly larvae polypeptide 0.1g obtained under 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C of conditions, and it is rare to add 10mL distilled water, and successively as test sample 2～6。
Each polypeptide sample is mixed in an eppendorf pipe with sample buffer (10uL+10uL)。Put into 100 DEG C of heating 10min, take supernatant point sample。
4.2.2. the preparation of separation gel and concentration glue
Glass plate, sample comb, Spacer detergent are cleaned, rinses for several times with ddH2O, then with ethanol, dry；
Spacer will be added between glass plate two pieces clean, install glass plate according to the prompting of Bio-RadMini II/III description；
12% separation gel 20.0ml is prepared, mixing by such as lower volume:
DdH2O3.3ml；1.5mol/LTris-HClpH=8.82.5ml；30%Acr-Bis4.0ml；10%SDS200ul；10%AP100ul；TEMED10ul。
To between glass plate, record separation gel, cover one layer of redistilled water immediately, glue and polymerizable after about 20min；
6% concentration glue 3.0ml is prepared, mixing by such as lower volume:
DdH2O5.3mL；1.5mol/LTris-HClpH=6.82.5mL；30%Acr-Bis2.0mL；10%SDS200ul；10%AP100ul；TEMED10ul。
Being inclined by upper strata redistilled water, filter paper blots, and records concentration glue, inserts sample comb；
4.2.3. electrophoresis and result
Install electrophoresis system, add electrode buffer, loading；
Voltage stabilizing 80V, when bromophenol blue has just run out of concentration glue, changes voltage 120V, stops electrophoresis, when bromophenol blue just runs out of separation gel, about need 45min～1hr。
Unload offset plate, peel off glue and put in dyeing liquor, room temperature dyeing 1～2hr；Being placed in destaining solution, put into by glue and decolour on 80rpm decolorization swinging table, every 20min changes a destaining solution to purifying completely。
Electrophoresis result is as shown in Figure 3。More than 80 DEG C only remaining wall scroll colored zones, illustrate that its impurity protein heats in 80 DEG C of environment above and all remove。
Embodiment 5 (different temperatures impact on fly larvae polypeptide antioxidant activity)
In order to be better understood from the present invention, adopt NBT photoreduction met hod that the antioxidation of this fire-resistant oxidation resistant peptide is detected by the present invention below。Concrete grammar is as described below。
5.1. the configuration of reagent:
5.1.1.1.44×10-2Mol/L methionine solution liquid
Accurately weigh METHIONINE 215.6mg in 100mL small beaker, after dissolving with a small amount of distilled water, move in 100mL volumetric flask and distill the water capacity to scale with 0, fully mixing (matching while using)。4 DEG C of refrigerators preserve available 1～2d。
5.1.2.2.25×10-4Mol/LNBT solution
Accurately weigh NBT (C4OH3OCl2N10O6, MW=817.7) 92mg in 50mL small beaker, after dissolving with a small amount of distilled water, move in 50mL volumetric flask and be settled to scale with distilled water, fully mixing (now with the current)。4 DEG C of refrigerators preserve available 2～3d。
5.1.3.6×10-4Mol/LEDTA solution
Accurately weigh EDTA (MW=292) 87.86mg in 50mL small beaker, after dissolving with a small amount of distilled water, move in 50mL volumetric flask and be settled to scale with distilled water, fully mix, (now with the current)。4 DEG C of refrigerators preserve available 8～10d。
5.1.4.6×10-6Mol/L riboflavin solution
Accurately weigh riboflavin (MW=376.36) 0.00113mg in 50mL small beaker, after dissolving with a small amount of distilled water, move in 50mL volumetric flask and be settled to scale with distilled water, fully mix, (now with the current)。4 DEG C of refrigerators preserve available 8～10d。This solution should keep in Dark Place, and namely will be equipped with the brown bottle of this liquid with black paper and wraps。
5.2. the preparation of test sample
(1) prepare fly larvae polypeptide by method described in embodiment 1, the difference is that fly larvae serosity is added the heating-up temperature respectively room temperature after distilled water diluting, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C。
(2) taking heating-up temperature respectively is fly larvae polypeptide 0.01g obtained under room temperature, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C of conditions, and it is rare to add 10mL distilled water, and successively as test sample 1～6。
5.3.NBT method measures fly larvae polypeptide non-oxidizability
5.3.1 the configuration of reaction system
Taking 8,10ml test tube according to the form below respectively and add following reagent, added sample number into spectrum is identical with test tube numbering。
Table 1NBT method reagent adds table
5.3.2. the process of reaction system
1-7 system reaction sample is joined porous plate is placed in 4000ux illumination 20min, No. 8 system reactant liquor dark processing 20min, click and enter porous plate。1-6 reaction system compares group, and as a control group, No. 8 reaction system dark processing are as zeroing group for No. 7 reaction systems。
5.3.3. the mensuration of absorbance
Utilize microplate reader measure its wavelength for 560nm when absorbance。
Fly larvae extracting solution NBT method OD value is heated under table 2 different temperatures
Oxidizing and depressing rate (%)=(control wells OD value-experimental port OD value)/(control wells OD value-blank well OD) × 100% calculates。
Obtain above-mentioned each statistical average and draw temperature absorbance curve, as shown in Figure 1
Embodiment 6 (impact on fly larvae extracting solution antioxidant activity and purity of the different heating time)
6.1. the preparation of test sample
(1) prepare fly larvae polypeptide by method described in embodiment 2, institute the difference is that after fly larvae serosity is added distilled water diluting room temperature preserve 20min and filter；Obtained fly larvae polypeptide 0.1g, adds 10mL distilled water rare as test sample 1；
(2) prepare fly larvae polypeptide by method described in embodiment 2, the difference is that heat time heating time respectively 5min, 10min, 15min, the 20min when 90 DEG C；Fly larvae polypeptide 0.1g obtained when heat time heating time respectively 5min, 10min, 15min, 20min when taking 90 DEG C respectively, it is rare to add 10mL distilled water, and successively as test sample 2～5。
The mensuration of 6.2 molecular weight and purity
As described in Example 4, result is shown in Fig. 4 to assay method, and after purification, sample (10min～20min) shows single strap；Show that polypeptide molecular weight is 20000Da
The mensuration of 6.3 non-oxidizabilitys
By diluted sample 10 times before using, as described in Example 5, result is shown in Fig. 2 to assay method。
Claims (2)
1. the preparation method of a fly larvae polypeptide, the method comprises the following steps: fly larvae is ground pulping, add appropriate water dilution, heat 10min～20min when 80～120 DEG C, filter, be down to 4 DEG C and be centrifuged, take supernatant, employing bag filter is dialysed, and removing molecular weight is the impurity of below 3000Da, obtains fly larvae polypeptide。
2. the preparation method of a kind of fly larvae polypeptide according to claim 1, the condition of described centrifugation step is: rotating speed is 12000rpm, and centrifugation time is 5～25min。
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|---|---|---|---|---|
| CN101292998A (en) * | 2008-05-27 | 2008-10-29 | 沈荣法 | Use of fly-maggot powder in anti-fatigue, anti-senescence and anti-adiposity health care function aspects |
| CN103397068A (en) * | 2013-08-15 | 2013-11-20 | 河北大学 | Preparation method of fly maggot peptides |
| CN105255829A (en) * | 2015-11-10 | 2016-01-20 | 广州赛莱拉干细胞科技股份有限公司 | Method for separating PBMC (peripheral blood mononuclear cell) |
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2016
- 2016-03-28 CN CN201610184180.2A patent/CN105693840A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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