CN106442788B - The method of discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on pigment - Google Patents
The method of discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on pigment Download PDFInfo
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- CN106442788B CN106442788B CN201610868794.2A CN201610868794A CN106442788B CN 106442788 B CN106442788 B CN 106442788B CN 201610868794 A CN201610868794 A CN 201610868794A CN 106442788 B CN106442788 B CN 106442788B
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Abstract
The method of discrimination of fresh tobacco leaves sample quality in the tobacco metabolism group based on pigment that the present invention provides a kind of, this method is using the ratio of carrotene and content of chlorophyll b in fresh tobacco leaves sample as index, selection for metabolism group qualified samples, which provides foundation, to be differentiated to metabolism group fresh tobacco leaves sample quality.Compared with prior art, the present invention have the advantages that it is easy to operate quickly, result is accurate and reliable, amount of samples is few, can be that the analysis of tobacco metabolic characteristics and related tobacco metabolism group research be offered reference.
Description
Technical field
The present invention relates to analytical chemistry field, fresh tobacco leaves sample in a kind of differentiation tobacco metabolism group is particularly related to
Whether quality meets the method for discrimination of metabolism group research needs.
Background technology
Plant Metabolome be by investigate plant it is stimulated or disturbance later stage metabolite variation or its at any time
Variation, to study a kind of technology of plant.In the flow of metabolism group edge analysis, the acquisition of typical sample be it is primary simultaneously
And step of crucial importance, obtain qualified samples, with the metabolism state of the reflection sample of objective, be metabonomic analysis into
Capable basis and premise.The acquisition of plant fresh leaf sample needs to be quenched at low temperature to stop its physiological activity to ensure that it stops
The only metabolism state in sampling, standing procedure are after plucking a plant sample, and tinfoil package is placed in -196 DEG C of liquid nitrogen frozens, uses
It is analyzed with dry powder sample after fresh tobacco leaves are analyzed or are lyophilized.It is picked to this section of process before analysis in sample, needs to ensure
Fresh tobacco leaves are preserved in liquid nitrogen and are ground under the conditions of liquid nitrogen, and no it will cause the enzymatic brownings of tobacco leaf.
Pigment content and quality of tobacco are closely related in tobacco leaf.One side tobacco leaf exterior quality（Such as color）With tobacco leaf pigment
Content is directly related；Another aspect pigment, especially the carrotene precursor substance important as aroma components, to tobacco
Aroma quality and perfume quantity have direct effect.Some researches show that carotenoid is the important as precursors of flue-cured tobacco aroma component, in cigarette
In leaf neutral flavor constituents, very big a collection of compound is the catabolite of carotenoid, such as Megastigmatrienone, the different Buddhist of oxidation
Your ketone, dorinone, alpha, beta-lonone, dihydroactinidiolide etc. are all especially important fragrance matters in tobacco.However it is brown
The tobacco sample of change, color can become brown from bud green, but enzymatic browning react slight sample by merely look at sample outside
Shade is seen to be difficult to accurately differentiate whether sample is normal.
Invention content
The present invention carry exactly for the above-mentioned prior art in the presence of shortcoming and for a kind of to tobacco metabolism group
Middle fresh tobacco leaves sample quality carries out sentencing method for distinguishing.The present invention utilizes the ratio of carrotene and content of chlorophyll b in fresh tobacco leaves
Value differentiates the fresh tobacco leaves quality in metabolism group research, for metabolism group research sample quality differentiation provide according to
According to the method is easy to operate quickly, result is accurate and reliable.
The purpose of the present invention can be realized by following technique measures：
The method of discrimination of fresh tobacco leaves sample quality is new by detecting in the tobacco metabolism group based on pigment of the present invention
Carrotene and content of chlorophyll b in fresh tobacco leaf, calculate its ratio, are carried out to fresh tobacco leaves sample quality in tobacco metabolism group
Differentiate；It is as follows：
A, fresh tobacco leaves freeze-drying 0.2 ~ 0.4g of sample is weighed, 25 ~ 50mL extractants are added, ultrasonic extraction 20 under ice bath ~
40 min take extract liquor to be filtered through 0.45 μm of mocromembrane filter, and filtrate is packed into brown chromatogram bottle, wait for that HPLC is analyzed；
B, the condition of liquid chromatogram is：Chromatographic column Waters Nova-Pak-C18（3.9 × 150mm, 4 μm）；Column temperature 30
℃；0.5 mL/min of column flow rate；10 μ L of sample size；Mobile phase A：Isopropanol；Mobile phase B：The aqueous solution of 80% acetonitrile（V/
V）；Equilibration time 6min；Gradient elution：0 min, 100%B；40 min, 0%B；Chlorophyll b Detection wavelength 428nm, other plastids
Pigment Detection wavelength is 448nm；Absorption spectrum using retention time combination standard specimen is qualitative, quantified by external standard method；
C, the content of carrotene and chlorophyll b is calculated by standard curve.
Heretofore described fresh tobacco leaves freeze-drying sample is the fresh tobacco leaves of crop field picking, and fortune is preserved through -196 DEG C of liquid nitrogen containers
It is defeated, after liquid nitrogen frozen grinds and is lyophilized, for use tobacco slag is preserved in -80 DEG C of refrigerators；The extractant is the third of a concentration of 90%
Ketone solution.
The present invention establishes using the ratio of carrotene and content of chlorophyll b as index and distinguishes metabolism group normal specimens and brown
Change the standard of sample, i.e., the ratio of carrotene and content of chlorophyll b is qualified samples 0.19 or more in sample to be tested, is used
In tobacco metabonomic analysis, the ratio of carrotene and content of chlorophyll b is below for rotten failed test sample 0.19,
It is not useable for tobacco metabonomic analysis.
Beneficial effects of the present invention are as follows：
By the analysis and research of a large amount of fresh tobacco leaves samples, fresh tobacco leaves are found in case of reactions such as enzymatic brownings,
The content of pigment can vary widely, and the ratio of carrotene and content of chlorophyll b is substantially reduced, this sample would be unavailable for
Metabonomic analysis, therefore using the ratio of carrotene and content of chlorophyll b as a standard to fresh in tobacco metabolism group
Tobacco sample quality is differentiated.Compared with prior art, the present invention have it is easy to operate quickly, result is accurate and reliable, sample is used
Few advantage is measured, can be that the analysis of tobacco metabolic characteristics and related tobacco metabolism group research are offered reference.
Specific implementation mode
The present invention is described further below with reference to embodiment：
Embodiment 1
Pigment content in fresh tobacco leaves sample is measured using HPLC methods, it is found that the carrotene of browning sample and leaf are green
Plain b content ratios are substantially reduced.
Sample to be tested include 7 kinds in the provinces such as Fujian, Guizhou, Hunan, Henan, Yunnan, 20 places of production fresh cigarette
127 parts of leaf sample（Each point has 5-7 biology to repeat）.Including 92 parts of qualified samples（Sample in acquisition, transport and pre-
- 80 DEG C are strict controlled in processing procedure）, 35 parts of brown stain samples（It is complete due to liquid nitrogen deficiency etc. in storage and transport process
Brown stain）.
Sample analysis step is as described below：
1）The fresh tobacco leaves picked by crop field preserve transport, liquid nitrogen frozen grinding in -196 DEG C of liquid nitrogen containers, and low temperature is lyophilized, -
80 DEG C of refrigerator preservations wait for subsequently carrying out metabonomic analysis.It is accurate to weigh fresh tobacco leaves freeze-drying sample 0.2g or so（It is accurate to 0.
1mg）It is accurate that 25mL extractants are added in 50mL triangular flasks（A concentration of 90% acetone soln）, ultrasonic extraction 20 under ice bath
Min takes appropriate extract liquor to be filtered through 0.45 μm of mocromembrane filter.Filtrate is packed into 2mL brown chromatogram bottles, waits for that HPLC is analyzed.
2）The condition of liquid chromatogram is：Chromatographic column Waters Nova-Pak-C18（3.9 × 150mm, 4 μm）；Column temperature 30
℃；0.5 mL/min of column flow rate；10 μ L of sample size；Mobile phase A：Isopropanol；Mobile phase B：The aqueous solution of 80% acetonitrile（V/
V）；Equilibration time 6min；Gradient elution：0 min, 100%B；40 min, 0%B.Chlorophyll b Detection wavelength 428nm, other plastids
Pigment Detection wavelength is 448nm.Absorption spectrum using retention time combination standard specimen is qualitative, quantified by external standard method.
Depict carrotene（36.42min, 0.10-5.0 μ g/mL）With chlorophyll b（27.24min, 0.5-10.0 μ g/
mL）Standard curve.Regression analysis is carried out with the peak area response under various concentration, obtains standard curve regression equation Y（Hu Luo
Bu Su）=375.53X-1.31, Y（Chlorophyll b）=80.62X+3.07, related coefficient are respectively 0.9997 and 0.9991.
The ratio of tobacco sample information and carrotene and content of chlorophyll b in 1 embodiment 1 of table
| Sample number into spectrum | The place of production | Kind | Repeat number | Carrotene/chlorophyll b | It is whether qualified |
| HZ10 | Henan Xuchang | Zhongyan-100 | 5 | 0.04-0.06 | It is unqualified |
| C23Y | Guizhou Bijie | Cloud 97 | 6 | 0 | It is unqualified |
| HK10 | Henan Xuchang | K326 | 6 | 0.13-0.16 | It is unqualified |
| C07C | Fujian Longyan | Cloud 87 | 6 | 0 | It is unqualified |
| C24B | Guizhou the south of Guizhou Province | Your cigarette 2 | 6 | 0 | It is unqualified |
| C29K | Hunan Zhangjiajie | K326 | 6 | 0 | It is unqualified |
| C01K | Kunming, Yunnan | K326 | 5 | 0.22-0.25 | It is qualified |
| C02K | Yunnan Yuxi | K326 | 6 | 0.32-0.34 | It is qualified |
| C03C | Qujing of Yunnan | Cloud 87 | 6 | 0.26-0.29 | It is qualified |
| C04Y | Honghe, Yunnan | Cloud 97 | 6 | 0.29-0.32 | It is qualified |
| C05Y | Yunnan Pu'er | Cloud 87 | 6 | 0.25-0.33 | It is qualified |
| C06Y | Dali | Cloud 87 | 5 | 0.23-0.25 | It is qualified |
| C09Y | Southwest Guizhou | Cloud 97 | 6 | 0.25-0.28 | It is qualified |
| C12C | Henan table mountain | Zhongyan-100 | 6 | 0.32-0.35 | It is qualified |
| C13C | Nanyang, henan | Zhongyan-100 | 5 | 0.24-0.27 | It is qualified |
| C14C | Henan Zhumadian | Zhongyan-100 | 6 | 0.23-0.26 | It is qualified |
| C15K | Guangdong Shaoguan | K326 | 6 | 0.25-0.29 | It is qualified |
| C17K | Chenzhou, Hunan Province | K326 | 6 | 0.25-0.27 | It is qualified |
| C25N | Tongren district Guizhou Province | Southern river 3 | 6 | 0.26-0.30 | It is qualified |
| C26Y | Enshi | Cloud 87 | 6 | 0.31-0.40 | It is qualified |
| YH10 | Dali | The big gold dollar of safflower | 5 | 0.22-0.24 | It is qualified |
| GK10 | Zunyi, guizhou | K326 | 6 | 0.24-0.29 | It is qualified |
By testing above, serious qualitative change is found（Browning）The ratio of carrotene and content of chlorophyll b in sample is notable
It reduces.The present invention as differentiation metabolism group normal specimens and goes bad the ratio of carrotene and content of chlorophyll b unqualified
The standard of sample.The ratio of carrotene and content of chlorophyll b is qualified samples 0.19 or more i.e. in sample to be tested, can be used
In tobacco metabonomic analysis, the ratio of carrotene and content of chlorophyll b is below for rotten failed test sample 0.19,
It is not useable for metabonomic analysis.
Claims (3)
1. the method for discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on pigment, it is characterised in that：Pass through
Carrotene and content of chlorophyll b in fresh tobacco leaves are detected, its ratio is calculated, to fresh tobacco leaves sample matter in tobacco metabolism group
Amount is differentiated；Criterion：Using the ratio of carrotene and content of chlorophyll b as index, establishes and distinguish metabolism group normal sample
The standard of product and browning sample, i.e., the ratio of carrotene and content of chlorophyll b is qualified sample 0.19 or more in sample to be tested
Product, for tobacco metabonomic analysis, the ratio of carrotene and content of chlorophyll b is below unqualified for what is gone bad 0.19
Sample is not useable for tobacco metabonomic analysis；It is as follows：
A, fresh tobacco leaves freeze-drying 0.2 ~ 0.4g of sample is weighed, 25 ~ 50mL extractants are added, ultrasonic extraction 20 ~ 40 under ice bath
Min takes extract liquor to be filtered through 0.45 μm of mocromembrane filter, and filtrate is packed into brown chromatogram bottle, waits for that HPLC is analyzed；
B, the condition of liquid chromatogram is：Chromatographic column Waters Nova-Pak-C18, specification are：3.9 × 150mm, 4 μm；Column temperature 30
℃；0.5 mL/min of column flow rate；10 μ L of sample size；Mobile phase A：Isopropanol；Mobile phase B：The aqueous solution of 80% acetonitrile（V/
V）；Equilibration time 6min；Gradient elution：0 min, 100%B；40 min, 0%B；Chlorophyll b Detection wavelength 428nm, other plastids
Pigment Detection wavelength is 448nm；Absorption spectrum using retention time combination standard specimen is qualitative, quantified by external standard method；
C, the content of carrotene and chlorophyll b is calculated by standard curve.
2. sentencing method for distinguishing according to claim 1, it is characterised in that：The fresh tobacco leaves freeze-drying sample is crop field picking
Fresh tobacco leaves preserve transport through -196 DEG C of liquid nitrogen containers, after liquid nitrogen frozen grinds and is lyophilized, for use cigarette are preserved in -80 DEG C of refrigerators
Ye Mo.
3. sentencing method for distinguishing according to claim 1, it is characterised in that：The extractant is a concentration of 90% acetone soln.
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| CN106932512B (en) * | 2017-03-08 | 2019-03-29 | 云南中烟工业有限责任公司 | A kind of cigarette composition quality trends analysis method based on characteristic component non-volatile in pipe tobacco |
| CN106932515B (en) * | 2017-04-24 | 2019-11-01 | 山东省食品药品检验研究院 | The analysis method of true and false Rizhao Green Tea is distinguished based on UHPLC series connection high resolution mass spectrum application metabonomic technology |
| CN108872108A (en) * | 2018-07-02 | 2018-11-23 | 湖北省农业科学院中药材研究所 | Plant young leaflet tablet pigment detection method |
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