CN106404959A - Screening method of Xiangyu pumpkin - Google Patents
Screening method of Xiangyu pumpkin Download PDFInfo
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- CN106404959A CN106404959A CN201610967084.5A CN201610967084A CN106404959A CN 106404959 A CN106404959 A CN 106404959A CN 201610967084 A CN201610967084 A CN 201610967084A CN 106404959 A CN106404959 A CN 106404959A
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Abstract
The invention relates to a screening method of Xiangyu pumpkin, comprising: detecting whether pumpkin pump contains the characteristic compound 2-acetyl-1-pyrroline by gas chromatography and mass spectrometry, and if yes, determining pumpkin containing the characteristic compound is Xiangyu pumpkin. Through mass researches and comparison with other pumpkin materials, it is found by the inventor that the characteristic substance of Xiangyu pumpkin is obtained 2-acetyl-1-pyrroline; by determining the characteristic substance, quantifiable substances can be provided for germplasm identification and screening of Xiangyu pumpkin; in addition, detection conditions optimized by the inventor can provide quick and accurate identification on the existence and content of the above characteristic compound in Xiangyu pumpkin.
Description
Technical field
The present invention relates to vegetable breeding field, more particularly to a kind of screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.
Background technology
Fructus Cucurbitae moschatae, as important garden crop a kind of in cucurbitaceous plant, has higher commercial value and economic worth,
It is one of important industrial crops of China.Wherein Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae possesses the features such as aromatic flavor, good quality because of it, is deep on market
One of small fruit type pumpkin variety popular with consumers, selling price is higher, has preferable economic benefit.
The fragrance of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae is the important indicator that its quality is constituted, and in Rhizoma Steudnerae Henryanae pumpkin variety Breeding Process, extremely closes
The problem of key is the precise Identification to aroma characteristics, and at present, the breeding daily life of a family, using sensory test method, relies primarily on subjectivity and smells
Feel and to carry out the judgement of aroma characteristics, but the method has certain limitation, lack the standard of objective judgement, detection people's
Odor threshold difference is different, and sensorial differences are larger, and qualification result is accurately fixed and reliability is difficult to ensure that, can be to the mirror of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae
Fixed, screening produces subjective impact, and can not make a kind of digitization, objective appraisal to the screening of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.Due to perfume (or spice)
Taste composition is complicated, and various flavor component interacts so that accurate, quick, the simple fragrance to Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae reflects
Surely become one of difficult problem of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae breeding.
And, yet there are no so far with regard to Fructus Cucurbitae moschatae fragrance matter detection technique and Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae characteristic chicken flavor compound
Report.
Content of the invention
Based on this it is necessary to be directed to the problems referred to above, provide a kind of screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.
Concrete technical scheme is as follows：
Whether a kind of screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae, first contain feature using gas chromatography-mass spectrography detection pumpkin fruit
Compound 2- acetyl group -1- pyrrolin, the Fructus Cucurbitae moschatae containing this feature compound is Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.
Wherein in some embodiments, the detecting step of described gas chromatography-mass spectrography is as follows：
Sample pretreatment：Take pumpkin fruit shave, liquid nitrogen freezing, preserve at -70～-80 DEG C, obtain sample, will before detection
Sample lyophilization, dehydration, add in liquid nitrogen and pulverize, after liquid nitrogen volatilization, obtain pulverized specimen；
Detection：Accurately weigh pulverized specimen described in 1～3g, put in 20ml ml headspace bottle, add 1 μ L concentration to be 4 μ g/ μ L
3- nonanone is ethanol as internal standard substance, solvent, tightens rapidly head space bottle cap, puts in 40～70 DEG C of water-baths, then insert rapidly
Enter solid-phase micro-extracting device, balance 2～30min, 40～70 DEG C of absorption 35～50min；After absorption, extracting head is inserted gas phase color
Spectrum-mass spectrometric injection port carries out desorption, and injector temperature is 270 DEG C, and the desorption time is 4.5min；Adopt after desorption
Described gas chromatograph-mass spectrometer carries out Data acquisition and issuance to the volatile ingredient of sample.
Wherein in some embodiments, the detecting step of described gas chromatography-mass spectrography is as follows：
Sample pretreatment：Take pumpkin fruit shave, liquid nitrogen freezing, preserve at -80 DEG C, obtain sample, by sample before detection
Lyophilization, is subsequently adding in liquid nitrogen and grinds to form fine powder, obtains pretreatment dry powdery sample after liquid nitrogen volatilization；
Detection：Accurately weigh pulverized specimen described in 1g, put in 20ml ml headspace bottle, add the 3- that 1 μ L concentration is 4 μ g/ μ L
Nonanone is ethanol as internal standard substance, solvent, tightens rapidly head space bottle cap, puts in 70 DEG C of water-baths, be then rapidly inserted into solid phase
Micro-extraction device, balances 2min, 70 DEG C of absorption 35min；The injection port after absorption, extracting head being inserted gas chromatograph-mass spectrometer enters
Row desorption, injector temperature is 270 DEG C, and the desorption time is 4.5min；Described gas chromatograph-mass spectrometer is adopted after desorption
Data acquisition and issuance is carried out to the volatile ingredient of sample.
Wherein in some embodiments, the GC conditions in described gas chromatography-mass spectrography detection are as follows：Gas phase color
Spectrum post DB-5MS；Carrier gas is helium, and flow velocity is 1.0ml/min；Sample introduction pattern is SPME hand sampling；Injector temperature is 270
℃；Sequencing heats up：50 DEG C of holding 3min, subsequently rise to 250 DEG C with the speed of 5 DEG C/min, keep 5min；
Mass Spectrometry Conditions are as follows：EI ion source；Ionization voltage 70eV；230 DEG C of ion source temperature；150 DEG C of level Four bar temperature；Connect
280 DEG C of temperature of mouth；Scan pattern is entirely to sweep, m/z 35-450.
Wherein in some embodiments, the specifications parameter of described chromatographic column is 60m × 0.32mm × 0.25 μm.
Wherein in some embodiments, the lyophilization in described sample pretreatment step is the temperature at -45～-55 DEG C
And lyophilizing 46～50 hours under conditions of pressure≤100pa.
Wherein in some embodiments, the particle diameter of described dry powdery sample is ＜ 0.5mm.
The present invention compares the advantage of prior art and has the beneficial effect that：
Inventor passes through numerous studies, using Gas chromatographyMass spectrometry, commonly southern by Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae and other
Melon compares, and obtains the property material composition of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae：2- acetyl group -1- pyrrolin, chemical formula is C6H9NO, is otherwise known as
Ancient morpholine, is weak yellow liquid, is a kind of natural Studies of The Aromatic Substances, is soluble in hot water, ethanol and ether, features described above material composition
It is defined as the material that the evaluation and screening offer of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae can quantify.Further, inventor institute preferably testing conditions, can
Fast and accurately to identify the presence or absence of features described above compound in Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.
Brief description
Fig. 1 numbers the total ion current figure of the sample being YJ-1 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 2 numbers the total ion current figure of the sample being YJ-2 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 3 numbers the total ion current figure of the sample being YJ-3 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 4 numbers the total ion current figure of the sample being YJ-4 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 5 numbers the total ion current figure of the sample being YJ-5 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 6 numbers the total ion current figure of the sample being YJ-6 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 7 is the total ion current figure that the sample being EY-1 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 8 is the total ion current figure that the sample being EY-2 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Fig. 9 is the total ion current figure that the sample being EY-3 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 10 is the total ion current figure that the sample being EY-4 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 11 is the total ion current figure that the sample being EY-5 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 12 is the total ion current figure that the sample being EY-6 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 13 numbers the total ion current figure of the sample being YJ-7 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 14 is the total ion current figure that the sample being BX-1 numbered by non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
The total ion current figure of the sample for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae numbering YJ-8 for the Figure 15；
Figure 16 numbers the total ion current figure of the sample being HS-1 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 17 numbers the total ion current figure of the sample being HS-2 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 18 numbers the total ion current figure of the sample being HS-3 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 19 numbers the total ion current figure of the sample being HS-4 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 20 numbers the total ion current figure of the sample being HS-5 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae；
Figure 21 numbers the total ion current figure of the sample being HS-6 for Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.
Specific embodiment
Further illustrate the present invention by the following examples.
In the embodiment of the present invention specimen in use be the Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae of Vegetables Inst., Guangdong Academy of Agricultural Sciences's selection-breeding with
And non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae, device therefor, reagent are commercially available mill run, wherein：
Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae sample is the Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material of Liang Lei Vegetables Inst., Guangdong Academy of Agricultural Sciences selection-breeding, is many
For selfing homozygous inbred lines material, character is stable, and sample number into spectrum is YJ-1～YJ-8 and HS-1～HS-6.
Non- Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae sample be the selection-breeding of Liang Lei Vegetables Inst., Guangdong Academy of Agricultural Sciences not have Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae fragrant
The Fructus Cucurbitae moschatae material of taste character, is inbreeding of more generation homozygous inbred lines material, and character is stable, number respectively be EY-1～EY-6 and
BX-1.
Gas chromatograph-mass spectrometer is 7890A-7000C gas chromatograph-mass spectrometer, public purchased from U.S. Agilent
Department.
50/30 μm of DVB/CAR/PDMS extracting head of solid-phase micro-extracting device, purchased from Supleco company of the U.S..
Alkane standard substance (C7-C40) it is purchased from sigma.
Experimental example 1 Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae characteristic compounds determine
Choose the class Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material that numbering is YJ-1～YJ-6, sample from 6 ripe melons altogether, in addition choose
One class number be EY-1～EY-6 non-6 melons of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material, follow the steps below detection：
Above-mentioned pumpkin fruit is sampled, shave, then rapid liquid nitrogen freezing, places -80 DEG C of ultra cold storage freezers pending.
The Fructus Cucurbitae moschatae sample of preservation is carried out lyophilization (- 50 DEG C, pressure≤100pa, lyophilizing 48 hours), lyophilization
Afterwards, Fructus Cucurbitae moschatae sample is added in liquid nitrogen and pulverize, obtain the pulverized specimen (particle diameter ＜ 0.5mm) of drying after liquid nitrogen volatilization, use
In late detection.
Accurately weigh 1g pulverized specimen, put in 20ml ml headspace bottle, add 1 μ L 3- nonanone (4 μ g/ μ L) as internal standard substance,
Solvent is ethanol, tightens rapidly head space bottle cap, puts in 70 DEG C of water-baths.It is rapidly inserted into (50/30 μm of solid-phase micro-extracting device
DVB/CAR/PDMS extracting head, Supleco company of the U.S.), balance 2min, 70 DEG C of absorption 35min.After absorption, extracting head is inserted
The chromatographic injection port entering in Agilent 7890A gas chromatogram series connection 7000C mass spectrograph carries out desorption, and injector temperature is
270 DEG C, the desorption time is 4.5min.Using Agilent 7890A gas chromatogram series connection 7000C mass spectrograph, sample volatility is become
Divide and carry out Data acquisition and issuance.
Gas phase condition：Gas chromatographic column DB-5MS (60m × 0.32mm × 0.25 μm)；Carrier gas is helium, and flow velocity is
1.0ml/min；Sample introduction pattern is SPME hand sampling；Injector temperature is 270 DEG C；Sequencing heats up：50 DEG C of holding 3min, with
Rise to 250 DEG C with the speed of 5 DEG C/min afterwards, keep 5min.Mass Spectrometry Conditions：EI ion source；Ionization voltage 70eV；Ion source temperature
230 DEG C of degree；150 DEG C of level Four bar temperature；280 DEG C of interface temperature；Scan pattern is entirely to sweep, m/z 35-450.
The data collecting is analyzed.Total ion current figure according to each sample can tentatively judge the difference between YJ and EY
Iso compounds (Fig. 1～12).Using Masshunter qualitative analyses software (Agilent Masshunter Qualitative
Analysis is B.07.00) deconvolution, RT window proportionality coefficient 100.00 are carried out to the full total number evidence collecting；Exclusion m/z
28；Snr threshold 2.0；Extract window：Left side m/z variable quantity 0.3amu, right side m/z variable quantity 0.7amu；Absolute peak height >=
300；Definitely peak area >=1000.
Data file after deconvolution is introduced directly into MPP analysis software (Mass Profile after being converted into .cef file
Professional Software Package 13.1) it is analyzed.
Compound tentatively filters：Minimum absolute abundance 2000counts；Retention time scope 4.5-46min；Mass number model
Enclose 35.0-450.0；Fragment ion number >=5.
Compound alignment parameters：Retention time deviation range 0.05min；Matching attribute 0.3；Lowest resolution 0.2；Baseline
It is set as the half of all samples.
Compound screening assays parameter：Occur 60% at least in a treatment group；Variation at least in one group of sample
Coefficient<30%；Significance test (ANOVA) p<0.05.
Using Masshunter qualitative analyses software, difference compound is examined, according to alkane standard substance (C7-C40)
The authentic communication providing calculates retention index, is carried out according to compound retention index and NIST MS analysis result qualitative, is used
Data base be NIST 14, the qualitative compound of reservation average >=80 point at least in a group.
Qualitative results according to difference compound are thus obtain the characteristic compounds 2- acetyl group -1- pyrroles of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae
Quinoline, this kind of compound is all not detected by the sample of non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material, and in Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material totally 6 samples
In can detect, and in all kinds of volatile compounds detecting, content is higher (asking for an interview table 1 and Fig. 1～12).Follow-up
Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae Breeding Process in, by detecting the presence or absence of this characteristic compounds and height identifying, to screen Rhizoma Steudnerae Henryanae Semen Cucurbitae
Matter.
Table 1：Peak in Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae YJ-1～YJ-6 and non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae EY-1～EY-6 for the 2- acetyl group -1- pyrrolin
Area comparison
Can be seen that YJ-1～YJ-6 sample from table 1 and Fig. 1～6 sample total ion current figure in 11.75 points of retention time
The unimodal of larger peak area can be detected near clock, be compound 2- acetyl group -1- pyrrolin through qualitative analyses, rather than Rhizoma Steudnerae Henryanae
Fructus Cucurbitae moschatae EY1～EY6 germplasm can not detect this compound (asking for an interview Fig. 7～12).Tentatively conclude that this compound is Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae
Odor characteristic compound.
Experimental example 2
For the characteristic compounds 2- acetyl group -1- pyrrolin of confirmatory experiment example 1 gained Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae, choosing numbering is YJ-7
A class Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material and an other class number be BX-1 non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material being tested：
Experimental procedure is as follows：
Respectively with YJ-7 and BX-1 material as object of study, above-mentioned pumpkin fruit is sampled, shave, then rapid liquid nitrogen
Freezing, places -80 DEG C of ultra cold storage freezers pending.
The Fructus Cucurbitae moschatae sample of preservation is carried out lyophilization, dehydration.After to be dried, Fructus Cucurbitae moschatae sample added in liquid nitrogen and grinds to form
Powder, obtains the powder sample that Fructus Cucurbitae moschatae is dried, for late detection after liquid nitrogen volatilization.
Accurately weigh 1g pulverized specimen, put in 20ml ml headspace bottle, add 1 μ L 3- nonanone (4 μ g/ μ L) as internal standard substance,
Tighten rapidly head space bottle cap, put in 70 DEG C of water-baths.It is rapidly inserted into solid-phase micro-extracting device, balance 2min, 70 DEG C of absorption
35min.Subsequently, the extracting head insertion Agilent 7890A gas chromatogram series connection mass spectrometric chromatographic injection port of 7000C is carried out
Desorption, injector temperature is 270 DEG C, and the desorption time is 4.5min.Using Agilent 7890A gas chromatogram series connection 7000C
Mass spectrograph carries out Data acquisition and issuance to sample volatile ingredient.
Gas phase condition：Gas chromatographic column DB-5MS (60m × 0.32mm × 0.25 μm)；Carrier gas is helium, and flow velocity is
1.0ml/min；Sample introduction pattern is SPME hand sampling；Injector temperature is 270 DEG C；Sequencing heats up：50 DEG C of holding 3min, with
Rise to 250 DEG C with the speed of 5 DEG C/min afterwards, keep 5min.Mass Spectrometry Conditions：EI ion source；Ionization voltage 70eV；Ion source temperature
230 DEG C of degree；150 DEG C of level Four bar temperature；280 DEG C of interface temperature；Scan pattern is entirely to sweep, m/z 35-450.
The data collecting is analyzed.Using Masshunter qualitative analyses software (Agilent Masshunter
Qualitative Analysis is B.07.00) deconvolution, RT window proportionality coefficient are carried out to the full total number evidence collecting
100.00；Exclusion m/z 28；Snr threshold 2.0；Extract window：Left side m/z variable quantity 0.3amu, right side m/z variable quantity
0.7amu；Definitely peak height >=300；Definitely peak area >=1000.
First according to appearance difference, determine in two class materials have discrepant compound, be primarily upon the presence or absence of peak (figure
13, Figure 14).Using Masshunter qualitative analyses software, difference compound is identified, according to alkane standard substance (C7-C40)
The authentic communication providing calculates retention index, is carried out according to compound retention index and NIST MS analysis result qualitative, is used
Data base be NIST 14.
(retention time is about to be equally not detected by compound 2- acetyl group -1- pyrrolin in non-Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae sample BX-1
For 11.75 minutes), the characteristic compounds of checking Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae fragrance are 2- acetyl group -1- pyrrolin further.
Experimental example 3
For the characteristic compounds 2- acetyl group -1- pyrrolin of confirmatory experiment example 1 gained Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae, to number as YJ-8
Fructus Cucurbitae moschatae material is sample, in different extraction temperature, is tested again under the conditions of equilibration time and adsorption time：
Above-mentioned pumpkin fruit is sampled, shave, then rapid liquid nitrogen freezing, places -80 DEG C of ultra cold storage freezers pending.
The Fructus Cucurbitae moschatae sample of preservation is carried out lyophilization, dehydration.After to be dried, Fructus Cucurbitae moschatae sample added in liquid nitrogen and grinds to form
Powder, obtains the powder sample that Fructus Cucurbitae moschatae is dried, for late detection after liquid nitrogen volatilization.
Accurately weigh 1g pulverized specimen, put in 20ml ml headspace bottle, add 1 μ L 3- nonanone (4 μ g/ μ L) as internal standard substance,
Tighten rapidly head space bottle cap, put in 50 DEG C of water-baths.It is rapidly inserted into solid-phase micro-extracting device, balance 5min, 50 DEG C of absorption
40min.Subsequently, the extracting head insertion Agilent 7890A gas chromatogram series connection mass spectrometric chromatographic injection port of 7000C is carried out
Desorption, injector temperature is 270 DEG C, and the desorption time is 4.5min.Using Agilent 7890A gas chromatogram series connection 7000C
Mass spectrograph carries out Data acquisition and issuance to sample volatile ingredient.
Gas phase condition：Gas chromatographic column DB-5MS (60m × 0.32mm × 0.25 μm)；Carrier gas is helium, and flow velocity is
1.0ml/min；Sample introduction pattern is SPME hand sampling；Injector temperature is 270 DEG C；Sequencing heats up：50 DEG C of holding 3min, with
Rise to 250 DEG C with the speed of 5 DEG C/min afterwards, keep 5min.Mass Spectrometry Conditions：EI ion source；Ionization voltage 70eV；Ion source temperature
230 DEG C of degree；150 DEG C of level Four bar temperature；280 DEG C of interface temperature；Scan pattern is entirely to sweep, m/z 35-450.
There is simple spike (asking for an interview Figure 15) in total ion current graph discovery in retention time at 11.75 minutes first.Then to adopting
The data collecting is analyzed.Using Masshunter qualitative analyses software (Agilent Masshunter Qualitative
Analysis is B.07.00) deconvolution, RT window proportionality coefficient 100.00 are carried out to the full total number evidence collecting；Exclusion m/z
28；Snr threshold 2.0；Extract window：Left side m/z variable quantity 0.3amu, right side m/z variable quantity 0.7amu；Absolute peak height >=
300；Definitely peak area >=1000.
Using Masshunter qualitative analyses software, compound is identified, according to alkane standard substance (C7-C40) provide
Authentic communication calculate retention index, qualitative, to be used number is carried out according to compound retention index and NIST MS analysis result
It is NIST 14 according to storehouse.
Shown according to qualitative results, the compound measuring for 11.75 minutes in retention time is 2- acetyl group -1- pyrrolin,
Illustrate that this compound, under different extraction temperature, equilibration time and adsorption time, can be arrived by stable detection, further
The characteristic compounds of checking Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae fragrance are 2- acetyl group -1- pyrrolin.
Embodiment 1
A kind of screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae, comprises the following steps：
Sample pretreatment：Using a class Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae material as HS-1-HS-6 for the numbering as test sample, totally 6 supply examination
Mature fruit, Proper Sampling Period is in June, 2016.Take pumpkin fruit shave, liquid nitrogen freezing, preserve at -80 DEG C；The south that will preserve
Melon sample carries out lyophilization (- 50 DEG C, pressure≤100pa, lyophilizing 48 hours), after being dried, Fructus Cucurbitae moschatae sample is added in liquid nitrogen
Pulverize, after liquid nitrogen volatilization, obtain the pulverized specimen (＜ 0.5mm) that Fructus Cucurbitae moschatae is dried, for late detection.
Detection：Accurately weigh 1g pulverized specimen, put in 20ml ml headspace bottle, add 1 μ L 3- nonanone (4 μ g/ μ L) as interior
Mark thing, solvent is ethanol, tightens rapidly head space bottle cap, puts in 70 DEG C of water-baths.It is rapidly inserted into solid-phase micro-extracting device, balance
2min, 70 DEG C of absorption 35min.Subsequently, chromatograph in extracting head insertion Agilent 7890A gas chromatogram series connection 7000C mass spectrograph
The injection port of instrument carries out desorption, and injector temperature is 270 DEG C, and the desorption time is 4.5min.Using Agilent 7890A gas phase
Chromatographic tandem 7000C mass spectrograph carries out Data acquisition and issuance to sample volatile ingredient.
Gas phase condition：Gas chromatographic column DB-5MS (60m × 0.32mm × 0.25 μm)；Carrier gas is helium, and flow velocity is
1.0ml/min；Sample introduction pattern is SPME hand sampling；Injector temperature is 270 DEG C；Sequencing heats up：50 DEG C of holding 3min, with
Rise to 250 DEG C with the speed of 5 DEG C/min afterwards, keep 5min.Mass Spectrometry Conditions：EI ion source；Ionization voltage 70eV；Ion source temperature
230 DEG C of degree；150 DEG C of level Four bar temperature；280 DEG C of interface temperature；Scan pattern is entirely to sweep, m/z35-450.
The data collecting is analyzed.Total ion current figure (asking for an interview Figure 16～21) first according to each repeated sample,
It can be found that about there was simple spike to occur at 11.75 minutes, and this compound separates preferably.Subsequently, fixed using Masshunter
Property analysis software (Agilent Masshunter Qualitative Analysis is B.07.00) to 6 for examination sample collections
To the full total number according to carrying out deconvolution, RT window proportionality coefficient 100.00；Exclusion m/z 28；Snr threshold 2.0；Extract window
Mouthful：Left side m/z variable quantity 0.3amu, right side m/z variable quantity 0.7amu；Definitely peak height >=300；Definitely peak area >=1000.
Using Masshunter qualitative analyses software, 6 are examined for the compound in sample basis, and according to alkane
Standard substance (C7-C40) authentic communication that provides calculates retention index, entered according to compound retention index and NIST MS analysis result
Row is qualitative, and the data base being used is NIST 14, qualitative compound average >=80 point of reservation.
According to qualitative results thus further determining that the spy that the compound occurring at 11.75 minutes is Rhizoma Steudnerae Henryanae Pumpkin Germplasm HS
Levy compound 2- acetyl group -1- pyrrolin.And this compound appearance retention time is stable, can be good with other compounds
Separate (asking for an interview Figure 16-21).
Each technical characteristic of embodiment described above can arbitrarily be combined, for making description succinct, not to above-mentioned reality
The all possible combination of each technical characteristic applied in example is all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all it is considered to be the scope of this specification record.
Embodiment described above only have expressed the several embodiments of the present invention, and its description is more concrete and detailed, but simultaneously
Can not therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
Say, without departing from the inventive concept of the premise, some deformation can also be made and improve, these broadly fall into the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (7)
1. whether a kind of screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae is it is characterised in that first adopt gas chromatography-mass spectrography detection pumpkin fruit
Containing characteristic compounds 2- acetyl group -1- pyrrolin, the Fructus Cucurbitae moschatae containing this feature compound is Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae.
2. the screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae according to claim 1 is it is characterised in that described gas chromatography-mass spectrography
Detecting step is as follows：
Sample pretreatment：Take pumpkin fruit shave, liquid nitrogen freezing, preserve at -70～-80 DEG C, obtain sample, by sample before detection
Lyophilization, dehydration, add in liquid nitrogen and pulverize, after liquid nitrogen volatilization, obtain pulverized specimen；
Detection：Accurately weigh pulverized specimen described in 1～3g, put in 20ml ml headspace bottle, add the 3- nonyl that 1 μ L concentration is 4 μ g/ μ L
Ketone is ethanol as internal standard substance, solvent, tightens rapidly head space bottle cap, puts in 40～70 DEG C of water-baths, is then rapidly inserted into solid
Phase extraction apparatus, balance 2～30min, 40～70 DEG C of absorption 35～50min；After absorption, extracting head is inserted gas chromatogram-matter
The injection port of spectrometer carries out desorption, and injector temperature is 270 DEG C, and the desorption time is 4.5min；Adopt described after desorption
Gas chromatograph-mass spectrometer carries out Data acquisition and issuance to the volatile ingredient of sample.
3. the screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae according to claim 2 is it is characterised in that described gas chromatography-mass spectrography
Detecting step is as follows：
Sample pretreatment：Take pumpkin fruit shave, liquid nitrogen freezing, preserve at -80 DEG C, obtain sample, before detection, sample is freezed
It is dried, is subsequently adding in liquid nitrogen and grinds to form fine powder, after liquid nitrogen volatilization, obtain pretreatment dry powdery sample；
Detection：Accurately weigh pulverized specimen described in 1g, put in 20ml ml headspace bottle, add the 3- nonanone that 1 μ L concentration is 4 μ g/ μ L
As internal standard substance, solvent is ethanol, tightens rapidly head space bottle cap, puts in 70 DEG C of water-baths, is then rapidly inserted into the micro- extraction of solid phase
Take device, balance 2min, 70 DEG C of absorption 35min；The injection port after absorption, extracting head being inserted gas chromatograph-mass spectrometer is solved
Absorption, injector temperature is 270 DEG C, and the desorption time is 4.5min；Described gas chromatograph-mass spectrometer is adopted to sample after desorption
The volatile ingredient of product carries out Data acquisition and issuance.
4. the screening technique of the Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae according to any one of claims 1 to 3 it is characterised in that described gas chromatogram-
GC conditions in mass spectrography detection are as follows：Gas chromatographic column DB-5MS；Carrier gas is helium, and flow velocity is 1.0ml/min；Enter
Original mold formula is SPME hand sampling；Injector temperature is 270 DEG C；Sequencing heats up：50 DEG C of holding 3min, subsequently with 5 DEG C/min
Speed rise to 250 DEG C, keep 5min；
Mass Spectrometry Conditions are as follows：EI ion source；Ionization voltage 70eV；230 DEG C of ion source temperature；150 DEG C of level Four bar temperature；Interface temperature
280 DEG C of degree；Scan pattern is entirely to sweep, m/z 35-450.
5. the screening technique of Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae according to claim 4 is it is characterised in that the specifications parameter of described chromatographic column is
60m×0.32mm×0.25μm.
6. the screening technique of the Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae according to any one of claims 1 to 3 is it is characterised in that described specimen preprocessing
Lyophilization in reason step is lyophilizing 46～50 hours under conditions of -45～-55 DEG C of temperature and pressure≤100pa.
7. the screening technique of the Rhizoma Steudnerae Henryanae Fructus Cucurbitae moschatae according to any one of claims 1 to 3 is it is characterised in that described dry powdery
The particle diameter of sample is ＜ 0.5mm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109444314A (en) * | 2018-11-28 | 2019-03-08 | 中国农业科学院作物科学研究所 | The method and application of -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group are quickly analyzed using GC-MS method |
| CN110836942A (en) * | 2019-11-29 | 2020-02-25 | 浙江省农业科学院 | Method for analyzing smelly components in fruit and vegetable products |
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| CN109444314A (en) * | 2018-11-28 | 2019-03-08 | 中国农业科学院作物科学研究所 | The method and application of -1 pyrrolin content of soybean odor characteristic compound 2- acetyl group are quickly analyzed using GC-MS method |
| CN110836942A (en) * | 2019-11-29 | 2020-02-25 | 浙江省农业科学院 | Method for analyzing smelly components in fruit and vegetable products |
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