CN106417194A - N15 stable isotope labeling method for insect protein quantification and tracing - Google Patents
N15 stable isotope labeling method for insect protein quantification and tracing Download PDFInfo
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- CN106417194A CN106417194A CN201611126023.2A CN201611126023A CN106417194A CN 106417194 A CN106417194 A CN 106417194A CN 201611126023 A CN201611126023 A CN 201611126023A CN 106417194 A CN106417194 A CN 106417194A
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- 108010060231 Insect Proteins Proteins 0.000 title claims abstract description 15
- 238000011002 quantification Methods 0.000 title abstract description 4
- 238000003141 isotope labeling method Methods 0.000 title abstract 2
- 239000000843 powder Substances 0.000 claims abstract description 37
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 13
- 241000238631 Hexapoda Species 0.000 claims abstract description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 26
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 19
- 238000002372 labelling Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- 239000002917 insecticide Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 230000012010 growth Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000006151 minimal media Substances 0.000 claims description 2
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 11
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 238000001948 isotopic labelling Methods 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract description 2
- 108010026552 Proteome Proteins 0.000 abstract 1
- 230000001131 transforming effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 16
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- 238000003756 stirring Methods 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 210000000582 semen Anatomy 0.000 description 4
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 235000014103 egg white Nutrition 0.000 description 3
- 210000000969 egg white Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
- 241000255967 Helicoverpa zea Species 0.000 description 2
- 241001477931 Mythimna unipuncta Species 0.000 description 2
- 241000256247 Spodoptera exigua Species 0.000 description 2
- 229910000004 White lead Inorganic materials 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- DUWWHGPELOTTOE-UHFFFAOYSA-N n-(5-chloro-2,4-dimethoxyphenyl)-3-oxobutanamide Chemical compound COC1=CC(OC)=C(NC(=O)CC(C)=O)C=C1Cl DUWWHGPELOTTOE-UHFFFAOYSA-N 0.000 description 2
- 235000019260 propionic acid Nutrition 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000021405 artificial diet Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000001466 metabolic labeling Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229910052573 porcelain Inorganic materials 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000029983 protein stabilization Effects 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicinal Chemistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a N15 stable isotope labeling method for insect protein quantification and tracing. The method comprises the following steps: transforming N15 inorganic salt into biologic protein by use of a yeast, wherein if the number of yeast culture generations exceeds 6, over 98% of the extracted protein is N15 protein; grinding the yeast to obtain N15-containing mycoprotein powder; and replacing conventional N14 protein with the N15-containing mycoprotein powder to culture multiple kinds of insects and provide specific N15 isotope labeling for insect protein quantification and tracing. The method disclosed by the invention can be applied to the quantitative analysis of insect protein and has the advantages of convenience in operation, low test cost and short test period and facilitates efficient and low-cost proteome analysis of a large number of samples.
Description
Technical field
The present invention relates to the technical field of protein stabilization isotope marks quantitation, refers in particular to one kind for insect protein
The N of the quantitative and spike of matter15Cold labeling method.
Background technology
The quantitative analyses of insecticide vivo protein for research protein function and seek disease protein label and drug targets
Bring new mode of thinking and research direction.Insecticide vivo protein quantitative technique mainly has Isotope metabolism labelling method at present
SILAC technology.
SILAC (Stable isotope labeling with amino acids in cell culture, SILAC)
Mammalian cell stable isotope metabolic labeling approaches, its ultimate principle be using containing light, in or heavy external source stable
Isotope aminoacid (mainly having Lys and Arg) adds culture medium culturing cell, and cell has been fitted together to same in the protein of new synthesis
The plain aminoacid in position, (2 after cultivating 5-6 generation6), the protein of all proteins basic more than 98% of cell is by isotope mark
Note.Processed by different condition, protein mixing under the conditions of equivalent different disposal is taken, is separated and mass spectrum through SDS-PAGE afterwards
Analysis, carries out relative quantification by comparing the size of first mass spectrometric in figure isotope peak type, while two grades of spectrograms identify egg
White matter.
SILAC has obvious limitation, and its situation is as follows：
1st, complex operation.SILAC quantitation flow process includes：Cell culture → protein extraction → immunoprecipitation → electrophoretic separation →
Digestions → Mass Spectrometer Method → detection by quantitative, complex operation, primitive cell culture labeling effciency is low, it has not been convenient to implement.
2nd, experimentation cost height.SILAC reagent is very expensive.
3rd, the test period is long.As SILAC needs to add labelling during being cultivated, so needing long
Time could compare thoroughly labelling.Although the speed that the time of whole experiment depends primarily on cell growth is each with adopted
Sample handling procedure is planted, but in general, SILAC is tested from start to end, including data analysiss, takes around 20 to 25
My god.
Content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, provide a kind of for the quantitative and spike of insect protein
N15Cold labeling method, the method can be applicable to the Protein quantitative analysis of insecticide, and easy to operate, experimentation cost is low,
It is widely used, beneficial to people are efficient, inexpensive, Proteomic analysis is carried out to a large amount of samples.
For achieving the above object, technical scheme provided by the present invention is：One kind is for insect protein quantitation and spike
N15Cold labeling method, first, using yeast by N15Inorganic salt changes into bioprotein, when Yeast Cultivation algebraically
Reach 6 more than generation, it is all N which extracts albumen more than 98%15Albumen, then yeast is ground, protein contained by release thalline, warp
Extraction purification is made containing N15Tropina powder, using contain N15Tropina powder replace conventional N14Albumen, for training
Foster various insects, are that the quantitative and spike of insect protein labelling provides specific N15Isotope marks.
The basic nutrition demand of insecticide includes saccharide, protein, lipid, vitamin, inorganic salt and moisture etc..In great majority
In the man-made feeds of insecticide, Fructus Hordei Germinatus, Semen sojae atricolor powder and yeast powder are main protein sources.Aminoacid in Fructus Hordei Germinatus and Semen sojae atricolor powder
Content is simultaneously unbalanced, and the aminoacid purity in Semen sojae atricolor powder is not high, it is impossible to meet demand of the insecticide in growth and development process.And
Yeast powder contains complete aminoacid group, purity and utilization rate height, is a kind of protein source of high-quality.In addition, yeast powder
Multiple vitamin B group can be provided, the development of insecticide normal growth be ensured, maintains multiple metabolic processes.Therefore, in the present invention containing only
N15The yeast powder of protein can provide N as protein sources and nitrogen source15Isotope marks, coordinate saccharide, lipid and other battalion
Foster material makes insect artificial diet, for cultivating various insects.
Protocol step provided by the present invention is as follows：
1) prepare containing only N according to formula as below15(without N14) inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
2) use high-pressure sterilizing pot, by step 1) in medium sterilization；
3) after step 2) in culture medium be cooled to room temperature after, picking single bacterium colony yeast inoculation trained in the medium
Support；
4) treat that yeast culture algebraically reaches 6 more than generation, culture medium is taken out, is stood in room temperature to yeast thalline and sink completely
Bottom, pours out fluid medium and gives up, and obtains yeast thalline；
5) with sterilized normal saline or buffer solution yeast thalline, and buchner funnel sucking filtration is used；
6) after the thalline that collects is weighed, with buffer with 1:1～1.5 ratio is mixed into new bacterium solution, is slowly added to
To in liquid nitrogen so that bacterium solution is entered in liquid nitrogen and forms little particle, thalline liquid nitrogen granule is added in high speed disintegrator and is crushed, obtain
The thalline of powder；
7) thalli powder is poured out, after being melted with water-bath, is re-injected in liquid nitrogen, repeat step 6)；
8) by step 7) in thalli powder melt after, be put in high speed centrifuge be centrifuged, after centrifugation, supernatant contains ferment
Female mycoprotein matter, collects supernatant, gives up precipitation；
9) liquid containing protein loads in bag filter, is dialysed using circulating water, eluting inorganic salt therein；
10) the liquid elder generation pre-freeze after dialysing, after condensing into solid, places in freezer dryer and dries, obtain thalline egg
White lead end；
11)N15The preparation of insect feedstuff：Using step 10) in the tropina powder made as feedstuff basic albumen
Source, replaces conventional N14Albumen is used for cultivating, and adds the saccharide without N element and other inorganic salt materials, makes final
N15Insect feedstuff.
In step 2) in, yeast is by the N in culture medium15Inorganic salt changes into bioprotein, when Yeast Cultivation algebraically reaches 6
More than generation, it is all N which extracts albumen more than 98%15Albumen, and can be according to the final insect feedstuff for preparing in practical operation
Select different yeast and containing only N15(without N14) minimal media based formulas.
In step 11) in, N can be prepared according to situation15Fruit bat feedstuff, N15Bollworm feedstuff, N15Beet armyworm feedstuff etc.
Insect feedstuff.In the final feedstuff for preparing, N element is all from step 4) to step 10) in extracted tropina after purification
The N of powder15, using N15Protein powder replaces conventional N14Albumen is used for cultivating insecticide, is insect protein labelling quantitatively and shows
Track provides specific N15Isotope marks, and the concrete manufacturing conditions of inorganic salt and feedstuff will be according to the growth of caste spy
Depending on levying.
The present invention compared with prior art, has the advantage that and beneficial effect：
1st, simple to operate.The operation being related in the present invention all relatively easy and easy left-hand seats, operator is through simple training just
Whole flow process can be completed.
2nd, experimentation cost is low.Agents useful for same of the present invention and material are general reagent and material mostly, low cost；Institute of the present invention
The equipment for using is simple and easy to get, operates without the need for special messenger, saves high cost of equipment and cost of labor.
3rd, it is widely used.Scheme proposed by the invention, it is adaptable to the raising and research of various insects.
Specific embodiment
With reference to multiple specific embodiments, the invention will be further described.
1 (N of embodiment15The preparation of fruit bat feedstuff)
The N for the quantitative and spike of insect protein described in the present embodiment15Cold labeling method, specifically
N15The preparation method of cold labeling fruit bat feedstuff, comprises the following steps：
1) prepare containing only N according to formula as below15(without N14) inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
2) use high-pressure sterilizing pot, by step 1) in medium sterilization；
3) after step 2) in culture medium be cooled to room temperature after, picking single bacterium colony yeast inoculation trained in the medium
Support；
4) treat that yeast culture algebraically reaches 6 more than generation, culture medium is taken out, is stood in room temperature to yeast thalline and sink completely
Bottom, pours out fluid medium and gives up, and obtains yeast thalline；
5) with sterilized normal saline or buffer solution yeast thalline, and buchner funnel sucking filtration is used；
6) after the thalline that collects is weighed, with buffer with 1:1～1.5 ratio is mixed into new bacterium solution, is slowly added to
To in liquid nitrogen so that bacterium solution is entered in liquid nitrogen and forms little particle, thalline liquid nitrogen granule is added in high speed disintegrator and is crushed, obtain
The thalline of powder；
7) thalli powder is poured out, after being melted with water-bath, is re-injected in liquid nitrogen, repeat step 6)；
8) by step 7) in thalli powder melt after, be put in high speed centrifuge be centrifuged, after centrifugation, supernatant contains ferment
Female mycoprotein matter, collects supernatant, gives up precipitation；
9) liquid containing protein loads in bag filter, is dialysed using circulating water, eluting inorganic salt therein；
10) the liquid elder generation pre-freeze after dialysing, after condensing into solid, places in freezer dryer and dries, obtain thalline egg
White lead end；
N15The preparation of fruit bat feedstuff：
Explanation：According to formula weigh needed for article, first with 2/3 water heating dissolve agar, add sugar or banana pulp with
And tropina powder；Remaining 1/3 water is mixed with Semen Maydis powder or rice flour, wheat bran, is slowly poured in agar sugar liquid, Bian Jia after stirring evenly
Hot side stirring, can stop heating after several minutes are boiled.The feedstuff of flowing is poured in feeder, the feedstuff in each feeder
Thickness is about 2cm, immediately seals, upright standing.Should be noted that during operation does not make feedstuff be attached on feeder side wall.Treat
After feedstuff condenses, the globule on feeder medial wall and feedstuff are wiped clean, instill propanoic acid and feeder is rotated, make propanoic acid uniform
It is distributed in feed surface.
2 (N of embodiment15The preparation of bollworm feedstuff)
The present embodiment makes culture N as different from Example 115Bollworm feedstuff, prepares containing only N according to formula as below15
Inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
According to technique productions same as Example 1, tropina powder is obtained thereafter.
N15The preparation of bollworm feedstuff：
Explanation：Starch and tropina powder are weighed according to formula, mixed well with 500～550ml water after mixing, separately agar is put
Enter in container, 400～450ml water added, is heated to agar and dissolves, remaining 50ml water is first put in high temperature sterilize pot and sterilizes,
The compositions such as ascorbic acid is dissolved after cooling.The agar for dissolving is poured into the pastel that corn starch and yeast powder mixing are tuned into
In, mix homogeneously, sterilized 15～20 minutes with 120 degrees Celsius in high-pressure sterilizing pot, add preservative after taking-up immediately, not
Disconnected stirring, when temperature is reduced to 55～60 DEG C or so, adds cholesterol and with aseptic water-soluble ascorbic acid solution, fully
After stirring, poured in the container for containing feedstuff rapidly, room temperature is cooled to, is then stored in standby in about 4 DEG C of refrigerator.
3 (N of embodiment15The preparation of beet armyworm feedstuff)
The present embodiment makes culture N as different from Example 115Beet armyworm feedstuff, according to formula as below prepare containing only
N15Inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
According to technique productions same as Example 1, tropina powder is obtained thereafter.
N15The preparation of beet armyworm feedstuff：
| Feed ingredient | Consumption (g) |
| Starch | 100～150 |
| Tropina powder | 40～55 |
| Vitamin C | 4～6 |
| P-hydroxybenzoic acid | 2～4 |
| Sorbic acid | 1.5～2.5 |
| 10% formaldehyde | 12～15 (ml) |
| Agar | 14～18 |
| Water | 950～1050 (ml) |
Explanation：First agar being boiled in water and dissolve, is subsequently adding and wet starch and tropina powder is mixed with a small amount of water, stir
Mix uniform and boil again.Vitamin and preservative are added when temperature drop is to 60～70 DEG C afterwards, then change into about 2cm's
Thickness is placed in cooled and solidified in porcelain dish, is covered with preservative film stand-by in 4 DEG C of refrigerators of placement after cooling.
Embodiment described above is only the preferred embodiments of the invention, not to limit the practical range of the present invention with this, therefore
The change made by all shapes according to the present invention, principle, all should cover within the scope of the present invention.
Claims (3)
1. a kind of for the quantitative N with spike of insect protein15Cold labeling method, it is characterised in that：First, utilize
Yeast is by N15Inorganic salt changes into bioprotein, and when Yeast Cultivation algebraically reaches 6 more than generation, which extracts albumen more than 98% is all
N15Albumen, then yeast is ground, makes containing N15Tropina powder, using contain N15Tropina powder replace conventional
N14Albumen, for cultivating various insects, is that the quantitative and spike of insect protein labelling provides specific N15Isotope mark
Note；Which comprises the following steps：
1) prepare containing only N according to formula as below15Inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
2) use high-pressure sterilizing pot, by step 1) in medium sterilization；
3) after step 2) in culture medium be cooled to room temperature after, picking single bacterium colony yeast inoculation cultivated in the medium；
4) treat that yeast culture algebraically reaches 6 more than generation, culture medium is taken out, is stood in room temperature to yeast thalline and sink to the bottom completely,
Go out fluid medium and give up, obtain yeast thalline；
5) with sterilized normal saline or buffer solution yeast thalline, and buchner funnel sucking filtration is used；
6) after the thalline that collects is weighed, with buffer with 1:1～1.5 ratio is mixed into new bacterium solution, is slowly added into liquid
In nitrogen so that bacterium solution is entered in liquid nitrogen and forms little particle, thalline liquid nitrogen granule is added in high speed disintegrator and is crushed, obtain powder
The thalline of shape；
7) thalli powder is poured out, after being melted with water-bath, is re-injected in liquid nitrogen, repeat step 6)；
8) by step 7) in thalli powder melt after, be put in high speed centrifuge be centrifuged, after centrifugation, supernatant contains yeast
Protein, collects supernatant, gives up precipitation；
9) liquid containing protein loads in bag filter, is dialysed using circulating water, eluting inorganic salt therein；
10) the liquid elder generation pre-freeze after dialysing, after condensing into solid, places in freezer dryer and dries, obtain tropina powder
End；
11)N15The preparation of insect feedstuff：Using step 10) in the tropina powder made as the basic protein sources of feedstuff, take
The N of generation routine14Albumen is used for cultivating, and adds the saccharide without N element and other inorganic salt materials, makes final N15Elder brother
Worm feedstuff.
2. according to claim 1 a kind of for the quantitative N with spike of insect protein15Cold labeling method,
It is characterized in that：In step 2) in, yeast is by the N in culture medium15Inorganic salt changes into bioprotein, when Yeast Cultivation algebraically reaches
To 6 more than generation, it is all N which extracts albumen more than 98%15Albumen, and can be raised according to the final insecticide for preparing in practical operation
Material selects different yeast and containing only N15Minimal media based formulas.
3. according to claim 1 a kind of for the quantitative N with spike of insect protein15Cold labeling method,
It is characterized in that：In step 11) in, in the final feedstuff for preparing, N element is all from step 4) to step 10) in extracted
The N of tropina powder after purification15, using N15Protein powder replaces conventional N14Albumen is used for cultivating insecticide, is insect protein
The quantitative and spike of matter labelling provides specific N15Isotope marks, and the concrete manufacturing conditions of inorganic salt and feedstuff will be according to elder brother
Depending on the growth characteristics of worm species.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110862928A (en) * | 2019-12-03 | 2020-03-06 | 华东理工大学 | A kind of15Preparation method and application of N-labeled mycoprotein |
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| CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
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| CN102313713A (en) * | 2011-07-14 | 2012-01-11 | 浙江大学 | Rapid detection method of abundance of tracer isotope <15>N in plant based on midinfrared spectrum |
| US20150253332A1 (en) * | 2014-03-05 | 2015-09-10 | GlycoScientific LLC | Isotopically Labeled Glycans and Methods for Producing the Same |
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