CN109444314A - The method and application of -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group are quickly analyzed using GC-MS method - Google Patents
The method and application of -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group are quickly analyzed using GC-MS method Download PDFInfo
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- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 67
- 244000068988 Glycine max Species 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 45
- RSEBUVRVKCANEP-UHFFFAOYSA-N 2-pyrroline Chemical compound C1CC=CN1 RSEBUVRVKCANEP-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract 11
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 230000001488 breeding effect Effects 0.000 claims abstract description 8
- 238000009395 breeding Methods 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 5
- MOMFXATYAINJML-UHFFFAOYSA-N 2-Acetylthiazole Chemical group CC(=O)C1=NC=CS1 MOMFXATYAINJML-UHFFFAOYSA-N 0.000 claims description 29
- 150000002500 ions Chemical class 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 17
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 15
- 239000012086 standard solution Substances 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 12
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 238000002137 ultrasound extraction Methods 0.000 claims description 8
- 238000005374 membrane filtration Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 238000004949 mass spectrometry Methods 0.000 claims description 6
- 239000012159 carrier gas Substances 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 239000001307 helium Substances 0.000 claims description 5
- 229910052734 helium Inorganic materials 0.000 claims description 5
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 238000009400 out breeding Methods 0.000 claims description 3
- ZVJHJDDKYZXRJI-UHFFFAOYSA-N 1-Pyrroline Chemical compound C1CC=NC1 ZVJHJDDKYZXRJI-UHFFFAOYSA-N 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 9
- 239000003205 fragrance Substances 0.000 abstract description 7
- 150000001875 compounds Chemical class 0.000 abstract description 4
- 239000000796 flavoring agent Substances 0.000 abstract description 4
- 235000019634 flavors Nutrition 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 3
- 238000001819 mass spectrum Methods 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 230000002068 genetic effect Effects 0.000 abstract 1
- 150000002894 organic compounds Chemical class 0.000 abstract 1
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 12
- 229960004756 ethanol Drugs 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 150000003233 pyrroles Chemical class 0.000 description 3
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000003988 headspace gas chromatography Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- SZQCPPRPWDXLMM-UHFFFAOYSA-N 1,4-dimethylpyrazole Chemical compound CC=1C=NN(C)C=1 SZQCPPRPWDXLMM-UHFFFAOYSA-N 0.000 description 1
- 241001270131 Agaricus moelleri Species 0.000 description 1
- 241000272814 Anser sp. Species 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 235000001497 healthy food Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
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Abstract
The present invention provides the methods for quickly analyzing -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group using GC-MS method, belong to field of organic compound detection.The present invention quickly measures odor characteristic compound 2-AP in soybean with gas chromatography-mass spectrum technology, have the characteristics that simple detection method, easy to operate, sample and reagent consumption is few and result is accurate and reliable, it is particularly suitable for carrying out the high-volume screening varieties before soybean breeder by measurement 2-AP content, to the accuracy and reliability of fragrance appraise result when improving soybean breeding, technical foundation is provided from " language description subjective type " to " numerical value metered dose " transformation for soy flavor appraise result, is had great importance to the deep fragrance resource for excavating soybean.And it is applied in external kind, domestic place and improved variety and genetic group material using this method.
Description
Technical field
The present invention relates to organic matter detection technique field and method applications more particularly to a kind of utilization GC-MS method quickly to divide
Analyse the method and application of -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group.
Background technique
Soybean originates from China, is important vegetable protein and oil source, has very high nutritive value and medicinal valence
Value, is described as " healthy food of 21 century " by people.The soybean of China's production is mainly used for eating, and only payes attention to yield for a long time
With general quality, fragrance quality is had ignored.However fragrance is important one of the index of quality of soybean, it can not only improve people's
Appetite, it may have important commodity value, therefore, research soy flavor is not only to cultivation high quality soybean, improvement bean product flavor
With important theory significance, have important practical significance to the happiness for improving people.The study found that the fragrance of soybean is equal
Derived from 2- acetyl group -1- pyrrolin (2AP), there is puffed rice taste.But due to the unstable chemcial property of 2-AP in soybean and contain
Measure it is extremely low so that the difficulty of 2AP quantitative detection increases.
Currently, the method for detecting 2AP in soybean in the prior art has: with soybean podding duration seed (Section four of stem top
When blade is sufficiently spread out) it is material, pass through internal standard substance 2,4- dimethyl pyrazole using automatic headspace gas chromatography (HS-GC)
Pyridine (DMP) measures soybean 2AP concentration；2AP concentration is measured using Isotopic Internal Standard object method.These methods are cumbersome, instrument used
Device is expensive, and analysis must all add internal standard compound or external standard every time, and use cost is relatively high and complicated for operation.And related general
Measure 2AP in breeding using less.
Summary of the invention
In consideration of it, quickly analyzing soybean odor characteristic compound using GC-MS method the purpose of the present invention is to provide a kind of
The method and application of -1 pyrrolin content of 2- acetyl group.The present invention quickly measures 2-AP in soybean using GC-MS method, has detection
Method is simple, easy to operate, sample and reagent consumption is few and result is accurate and reliable feature.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
A method of -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group quickly being analyzed using GC-MS method,
The following steps are included:
(1) ultrasonic extraction is carried out after mixing soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate, gained is extracted
It is centrifuged after taking liquid to mix with NaCl, by 0.22 μm of membrane filtration of gained supernatant, solution to be measured is obtained, to described
Solution to be measured carries out GC-MS detection, obtains GC-MS testing result, the GC-MS testing result includes 2- acetyl group -1- pyrroles
The appearance time and peak area of quinoline；
(2) peak area and scheduled standard curve obtained according to the step (1), obtains 2- acetyl in solution to be measured
The concentration of base -1- pyrrolin；The standard curve using the concentration of 2- acetyl group -1- pyrrolin standard solution as abscissa, with 2-
The peak area that acetyl group -1- pyrrolin standard solution carries out GC-MS detection is ordinate；
GC-MS testing conditions independently include: in the step (1) and (2)
Chromatographic condition: chromatographic column is An Jielong DB-WAX capillary column: 30m × 0.25mm × 0.25 μm, column temperature heating journey
Sequence is that 60 DEG C of holdings 2min, 10 DEG C/min rise to 100 DEG C, then rises to 230 DEG C with 30 DEG C/min, keeps 5min；Constant linear velocity
41.2cm/s, injector temperature are 250 DEG C；Carrier gas is high-purity helium；Splitless injecting samples, sample volume are 1 μ L；
Mass Spectrometry Conditions: electron impact ion source, ion source temperature are 200 DEG C；Ionization energy is 70eV；Interface temperature is
220℃；Full scan mode, scanning range are m/z 35~500.
Preferably, the amount ranges of soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate are in the step (1)
(0.4~0.8g): (1.5~3mL): (0.1~0.2g).
Preferably, the time of ultrasonic extraction is 20~30min in the step (1).
Preferably, the mass ratio of soybean bean powder or florescence blade and NaCl is (0.4~0.8) in the step (1):
(0.1~0.2).
Preferably, the concentration of 2- acetyl group -1- pyrrolin standard solution is 4~0.25ppm in the step (2).
Preferably, soybean bloom blade process shreds processing in the step (1).
Preferably, the appearance time of the 2- acetyl group -1- pyrrolin of GC-MS detection is in the step (1) and (2)
7.016min。
It preferably, with fragments characteristic ion m/z 83 is object ion when GC-MS detection in the step (1) and (2), m/
Z 69 and m/z 111 is with reference to ion.
- 1 pyrroles of soybean odor characteristic compound 2- acetyl group is quickly analyzed using GC-MS method the present invention also provides a kind of
Application of the method for quinoline content in soybean breeding, detects soybean using detection method described in above-mentioned technical proposal,
Screening obtains the high soybean of -1 pyrrolin content of 2- acetyl group and carries out breeding.
- 1 pyrrolin of soybean odor characteristic compound 2- acetyl group is quickly analyzed using GC-MS method the present invention provides a kind of
The method of content, the present invention quickly measure odor characteristic compound 2-AP in soybean with gas chromatography-mass spectrum technology (GC-MS),
Have the characteristics that simple detection method, easy to operate, sample and reagent consumption is few and result is accurate and reliable, is particularly suitable for passing through
It measures 2-AP content and carries out the high-volume screening varieties before soybean breeder, fragrance appraise result is accurate when to raising soybean breeding
Property and reliability, for soy flavor appraise result from " language description subjective type " to " numerical value metered dose " transformation provide technology base
Plinth has great importance to the deep fragrance resource for excavating soybean.Embodiment statistics indicate that, detection side provided by the invention
Method can accurately obtain the characteristic peak of -1 pyrrolin of 2- acetyl group, and the coefficient R of standard curve2=0.9998, to same
The relative standard deviation that sample is detected is 1.5%, and detection method precision is high, and at home and abroad soybean high-content 2AP educates
It will be significant in kind.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is that 1ppm standard items scan scans total ion chromatogram；
Fig. 2 is the mass spectrogram that Nist composes library 2-AP；
Fig. 3 is the curve of normal equation.
Specific embodiment
- 1 pyrrolin of soybean odor characteristic compound 2- acetyl group is quickly analyzed using GC-MS method the present invention provides a kind of
The method of content, comprising the following steps:
(1) ultrasonic extraction is carried out after mixing soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate, gained is extracted
It is centrifuged after taking liquid to mix with NaCl, by 0.22 μm of membrane filtration of gained supernatant, solution to be measured is obtained, to described
Solution to be measured carries out GC-MS detection, obtains GC-MS testing result, the GC-MS testing result includes 2- acetyl group -1- pyrroles
The appearance time and peak area of quinoline；
(2) peak area and scheduled standard curve obtained according to the step (1), obtains 2- acetyl in solution to be measured
The concentration of base -1- pyrrolin；The standard curve using the concentration of 2- acetyl group -1- pyrrolin standard solution as abscissa, with 2-
The peak area that acetyl group -1- pyrrolin standard solution carries out GC-MS detection is ordinate；
GC-MS testing conditions independently include: in the step (1) and (2)
Chromatographic condition: chromatographic column is An Jielong DB-WAX capillary column: 30m × 0.25mm × 0.25 μm, column temperature heating journey
Sequence is that 60 DEG C of holdings 2min, 10 DEG C/min rise to 100 DEG C, then rises to 230 DEG C with 30 DEG C/min, keeps 5min；Constant linear velocity
41.2cm/s, injector temperature are 250 DEG C；Carrier gas is high-purity helium；Splitless injecting samples, sample volume are 1 μ L；
Mass Spectrometry Conditions: electron impact ion source, ion source temperature are 200 DEG C；Ionization energy is 70eV；Interface temperature is
220℃；Full scan mode, scanning range are m/z 35~500.
The present invention carries out ultrasonic extraction after mixing soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate, by institute
It obtains after extract liquor is mixed with NaCl and is centrifuged, by 0.22 μm of membrane filtration of gained supernatant, obtain solution to be measured, it is right
The solution to be measured carries out GC-MS detection, obtains GC-MS testing result, the GC-MS testing result includes 2- acetyl group -1-
The appearance time and peak area of pyrrolin.
In the present invention, the amount ranges of the soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate are preferably
(0.4~0.8g): (1.5~3mL): (0.1~0.2g), more preferably 0.8g:3mL:0.2g.
In the present invention, the soybean bloom bean powder or florescence blade are preferably through shredding processing.In the present invention,
It is described shred processing preferably shred into the shape that diameter is 2cm size with scissors.
In the present invention, the soybean bloom blade is preferably top vane, and the soybean bean powder is preferably Tube-
Mill-100 tube mill (German IKA company production) milling.
In the present invention, the time of the ultrasonic extraction is preferably 20~30min.
In the present invention, the mixing of the soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate is preferably in whirlpool
Concussion carries out in whirlpool mixer (model: XH-C).
In the present invention, the mass ratio of the soybean bean powder or florescence blade and NaCl is preferably (0.4~0.8):
(0.1~0.2), more preferably 4:1.In the present invention, the effect of NaCl is to increase the solubility of organic matter in a solvent, and reduction has
Machine object water phase concentration, to improve 2- acetyl group -1- pyrrolin extraction yield.
After mixing with NaCl, the present invention is centrifuged again after mix products preferably to be stood to 2h at room temperature.At this
In invention, the centrifuge separation is preferably centrifuged 10min at 4 DEG C of refrigerated centrifuge, 12000rpm.
After obtaining solution to be measured, the solution to be measured is preferably placed in makings bottle (32mm × 11mm) by the present invention,
It is stand-by to screw lid sealing.
In the present invention, the GC-MS testing conditions include:
Chromatographic condition: chromatographic column is An Jielong DB-WAX capillary column: 30m × 0.25mm × 0.25 μm, column temperature heating journey
Sequence is that 60 DEG C of holdings 2min, 10 DEG C/min rise to 100 DEG C, then rises to 230 DEG C with 30 DEG C/min, keeps 5min；Constant linear velocity
41.2cm/s, injector temperature are 250 DEG C；Carrier gas is high-purity helium；Splitless injecting samples, sample volume are 1 μ L；
Mass Spectrometry Conditions: electron impact ion source, ion source temperature are 200 DEG C；Ionization energy is 70eV；Interface temperature is
220℃；Full scan mode, scanning range are m/z 35~500.
In the present invention, the appearance time of the 2- acetyl group -1- pyrrolin of the GC-MS detection is preferably 7.016min.
In the present invention, the appearance time of 2- acetyl group -1- pyrrolin with the library NIST by comparing to obtain.
It in the present invention, is preferably object ion with fragments characteristic ion m/z 83 when the GC-MS is detected, 69 and of m/z
M/z 111 is with reference to ion.
According to obtained peak area and scheduled standard curve, 2- acetyl group -1- pyrrolin in solution to be measured is obtained
Concentration；The standard curve using the concentration of 2- acetyl group -1- pyrrolin standard solution as abscissa, with 2- acetyl group -1- pyrroles
The peak area that quinoline standard solution carries out GC-MS detection is ordinate.
In the present invention, the concentration of the 2- acetyl group -1- pyrrolin standard solution is preferably 4~0.25ppm, more preferably
For 4ppm, 2ppm, 1ppm, 0.5ppm or 0.25ppm.
In the present invention, the 2- acetyl group -1- pyrrolin standard solution is preferably prepared by following methods: by 2-AP
Standard items 10mg is dissolved in 100mL dehydrated alcohol, is configured to the standard reserving solution of 1000ppm, be placed in 4 DEG C of refrigerators save it is standby
With；Standard reserving solution is configured to the mark of 4ppm, 2ppm, 1ppm, 0.5ppm, 0.25ppm respectively using diluted method step by step
Quasi- solution draws 1mL with disposable syringe, and (32mm × 11mm) is numbered in makings bottle with 0.22 μm of membrane filtration,
It is placed in sample bottle and numbers, it is stand-by to screw lid sealing.
In the present invention, the step of GC-MS is detected is consistent with above scheme, and details are not described herein.
In the present invention, the standard curve is y=115373x+1483.1, coefficient R2=0.9998.
- 1 pyrroles of soybean odor characteristic compound 2- acetyl group is quickly analyzed using GC-MS method the present invention also provides a kind of
Application of the method for quinoline content in soybean breeding, detects soybean using detection method described in above-mentioned technical proposal,
Screening obtains the high soybean of -1 pyrrolin content of 2- acetyl group and carries out breeding.
Below with reference to embodiment to the detection method and its application of -1 pyrrolin of 2- acetyl group in soybean provided by the invention
It is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1. the preparation of standard reserving solution: 2-AP standard items 10mg being dissolved in 100mL dehydrated alcohol, is configured to
The stock solution of 1000ppm is placed in 4 DEG C of refrigerators and saves backup.
2. the preparation of standard solution: standard reserving solution is configured to respectively using diluted method step by step 4ppm, 2ppm,
The standard solution of 1ppm, 0.5ppm, 0.25ppm draw 1mL with disposable syringe, and with 0.22 μm of membrane filtration in makings
(32mm × 11mm) is numbered in bottle, is placed in sample bottle and is numbered, and it is stand-by to screw lid sealing.
3. the preparation of sample: taking soybean bean powder or florescence top vane 2, accurately weigh 0.8g, shredded into scissors
Diameter is the shape of 2cm size, is placed in the 10mL centrifuge tube for filling 3mL alcohol and 0.2g anhydrous sodium sulfate, is mixed with whirlpool
In refrigerated centrifuge 4 after device (model: XH-C) concussion several minutes of rear ultrasonic extraction 20min of mixing, addition 0.2gNaCl, standing 2h
DEG C 12000rpm is centrifuged 10min, takes 1mL supernatant and with 0.22 μm of membrane filtration in makings bottle with disposable syringe
It is stand-by to screw lid sealing for (32mm × 11mm) number.
2010 chromatographic mass spectrometry condition of 4.GC-MS:
Chromatographic condition: chromatographic column is An Jielong DB-WAX capillary column: 30m × 0.25mm × 0.25 μm, column temperature heating journey
Sequence is that 60 DEG C of holdings 2min, 10 DEG C/min rise to 100 DEG C, then rises to 230 DEG C with 30 DEG C/min, keeps 5min；Constant linear velocity
41.2cm/s, injector temperature are 250 DEG C；Carrier gas is high-purity helium (purity > 99.999%)；Splitless injecting samples, sample volume are
1μL；
Mass Spectrometry Conditions: electron impact ion source (EI), ion source temperature are 200 DEG C；Ionization energy is 70eV；Interface temperature
Degree is 220 DEG C；Full scan mode, scanning range are m/z 35~500.
5. qualitative analysis and quantitative analysis method:
The qualitative analysis of 5.1 2-AP uses NIST library searching.
The determination of 5.2 2-AP appearance times and the drafting of standard curve: by taking 1ppm as an example, according to above-mentioned 4th part GC-
MS condition carries out SCAN scanning, obtains GC-MS total ion chromatogram (see Fig. 1), integrates to resulting peak and is composed by NIST
Library searching 2-AP standard mass spectrogram (see Fig. 2) determines that the appearance time of 2-AP (peak 1) is 7.016min, similar to spectrum library 2-AP
Degree is 91%, establishes sim method also according to the 4th part GC-MS condition, setting fragments characteristic ion m/z 83 be target from
Son, m/z 69 and m/z 111 be with reference to ion, with 7.016 for standard analysis standard items, by standard solution according to concentration from as low as
Height arrangement, is put in sample disc, each 3 needle of concentration sample introduction, and the average value by its gained peak area is ordinate, corresponding standard liquid
Concentration is abscissa fit standard curve, and calculating regression equation is y=115373x+1483.1 and coefficient R2=
0.9998 (see Fig. 3) obtains various concentration gradient 2-AP fragment ion flow graph.
The precision of 5.2 methods:
5.2.1 the precision of instrument:
Taking concentration is that 1.0ppm standard solution is repeated sample introduction 6 times by selected chromatographic condition, obtains the peak area of 2AP, counts
Calculating its relative standard deviation RSD is respectively 0.8%, illustrates that instrument repeatability is met the requirements.
5.2.2 the precision of method
The soybean sample to be tested containing 2AP is chosen, 4 parallel determinations are carried out, experimental result is shown in Table 1, relative standard deviation
1.5%, show method precision height.
1 method precision experimental result of table
Application of the 6.2AP in China's incubation and local varieties breeding
Using the method for the above measurement soybean 2AP is bred as China, local varieties are screened wherein 10 parts of materials for totally 240 parts
Expect the 2AP containing higher concentration, is respectively as follows: specific material, 2AP content and geographical distribution and is shown in Table 2, as shown in Table 2, soybean 2AP
Content it is lower, the order of magnitude is hundred a ten thousandths, and 2AP content contained by different cultivars is different, range of variation 0.003ppm-
0.215ppm, wherein Ningling day goose egg is higher, and 2AP content is 0.215ppm, and secondly fragrant for Tonghua flat-top, 2AP content is
0.203ppm。
The 2AP content of 2 10 parts of table domestic materials
Application of the 7.2AP in external breed breeding
External kind is screened for 50 parts totally using the method for the above measurement soybean 2AP, wherein 10 parts of materials contain compared with
The 2AP of high concentration, specific material, 2AP content and geographical distribution are shown in Table 3, as shown in Table 3, contained by the soybean varieties of country variant
2AP content it is also different, range of variation 0.003ppm-0.331ppm, wherein the saikai20 higher of Japan, 2AP content is
0.331ppm, is secondly the Essex in the U.S., and 2AP content is 0.152ppm.
The 2AP content of 3 10 parts of foreign materials of table
Application of the 8.2AP in soybean heredity group
Middle yellow 35 (2AP content is 0) Ji Shisheng is identified simultaneously using this method to come into leaves the something lost of (2AP content is 0.0814)
328 parts of materials of strain of group are passed, discovery different strains in progeny population have differences, and 2AP content distribution range is 0-
0.09ppm.These results of study are that theoretical basis has been established in the positioning of scent gene.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. the method for quickly analyzing -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group using GC-MS method, including with
Lower step:
(1) ultrasonic extraction is carried out after mixing soybean bean powder or florescence blade, ethyl alcohol and anhydrous sodium sulfate, by gained extract liquor
It is centrifuged after being mixed with NaCl, by 0.22 μm of membrane filtration of gained supernatant, solution to be measured is obtained, to described to be measured
Solution carries out GC-MS detection, obtains GC-MS testing result, the GC-MS testing result includes 2- acetyl group -1- pyrrolin
Appearance time and peak area；
(2) peak area and scheduled standard curve obtained according to the step (1), obtains 2- acetyl group-in solution to be measured
The concentration of 1- pyrrolin；The standard curve using the concentration of 2- acetyl group -1- pyrrolin standard solution as abscissa, with 2- acetyl
The peak area that base -1- pyrrolin standard solution carries out GC-MS detection is ordinate；
GC-MS testing conditions independently include: in the step (1) and (2)
Chromatographic condition: chromatographic column is An Jielong DB-WAX capillary column: 30m × 0.25mm × 0.25 μm, column temperature temperature program are
60 DEG C of holdings 2min, 10 DEG C/min rise to 100 DEG C, then rise to 230 DEG C with 30 DEG C/min, keep 5min；Constant linear velocity
41.2cm/s, injector temperature are 250 DEG C；Carrier gas is high-purity helium；Splitless injecting samples, sample volume are 1 μ L；
Mass Spectrometry Conditions: electron impact ion source, ion source temperature are 200 DEG C；Ionization energy is 70eV；Interface temperature is 220
℃；Full scan mode, scanning range are m/z 35~500.
2. detection method according to claim 1, which is characterized in that soybean bean powder or florescence leaf in the step (1)
The amount ratio of piece, ethyl alcohol and anhydrous sodium sulfate is (0.4~0.8g): (1.5~3mL): (0.1~0.2g).
3. detection method according to claim 1 or 2, which is characterized in that the time of ultrasonic extraction is in the step (1)
20~30min.
4. detection method according to claim 1, which is characterized in that soybean bean powder or florescence leaf in the step (1)
The mass ratio of piece and NaCl are (0.4~0.8): (0.1~0.2).
5. detection method according to claim 1, which is characterized in that 2- acetyl group -1- pyrrolin mark in the step (2)
The concentration of quasi- solution is 4~0.25ppm.
6. detection method according to claim 1, which is characterized in that soybean is through Tube Mill at beans in the step (1)
Powder or soybean bloom blade are by shredding processing.
7. detection method according to claim 1, which is characterized in that the 2- that GC-MS is detected in the step (1) and (2)
The appearance time of acetyl group -1- pyrrolin is 7.016min.
8. detection method according to claim 1 or claim 7, which is characterized in that when GC-MS is detected in the step (1) and (2)
With fragments characteristic ion m/z 83 be object ion, m/z 69 and m/z111 are with reference to ion.
9. a kind of quickly analyze the method for -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group big using GC-MS method
Application in beans breeding, which is characterized in that soybean is examined using detection method described in claim 1~8 any one
It surveys, screening obtains the high soybean of -1 pyrrolin content of 2- acetyl group and carries out breeding.
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