CN108051522A - A kind of tea germplasm rapid identification method for fitting flowers and fruits odor type tealeaves processed - Google Patents
A kind of tea germplasm rapid identification method for fitting flowers and fruits odor type tealeaves processed Download PDFInfo
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Abstract
The invention belongs to Tea Germplasm identification and utilizations and breeding of new variety field, and in particular to a kind of tea germplasm rapid identification method for fitting flowers and fruits odor type tealeaves processed.This method comprises the following steps：1）Fresh leaf is picked then in tea tree planting, first extracts the glucoside compound in fresh leaf, fresh leaf raw sugar glycosides extracting solution is made；2）Measure geranyl primrose glucosides, benzyl alcohol primrose glucosides, the content of benzyl carbinol primrose glucosides in fresh leaf raw sugar glycosides extracting solution；3）When the benzyl alcohol primrose Glycosides Contents in fresh leaf raw sugar glycosides extracting solution+benzyl carbinol primrose Glycosides Contents >=50 μM/100g dry weights, and during geranyl primrose glucosides >=100 μM/100g dry weights, then judge that the tea germplasm fits tea germplasm processed for flowers and fruits odor type tealeaves.Entire identification and analytic process only need 1 working day, and greatly qualification cycle is shortened compared to Direct Identification method (6 years).
Description
Technical field
The invention belongs to Tea Germplasm identification and utilizations and breeding of new variety field, and in particular to the suitable flowers and fruits processed of one kind are fragrant
The tea germplasm rapid identification method of type tealeaves.
Background technology
Fragrance is one of important factor for evaluating tea leaf quality, and tea aroma is especially optimal with flowers and fruits perfume (or spice), and flowers and fruits odor type tealeaves is always
Favored be subject to Tea Consumption market.The formation of bluish dogbane fruity flavor characteristic is mainly by tea germplasm, cultivation and processing technology
It influences, wherein tea germplasm is basis.Currently the identification of the tea germplasm to fitting flowers and fruits odor type tealeaves processed, which mainly uses, directly reflects
Determine method, this method goes out Tea Samples by picking fresh leaves of tea plant processing and fabricating, then to Tea Samples carry out sensory review so as to
Determine the suitable property processed of the germplasm.Direct Identification method needs field planting tea germplasm, extremely can the productive life by tea tree normal growth
(3 years) and time repetitive identified (3 years), quality identification result generally requires (6 years) for many years from planting to obtaining.Direct Identification
Method is long there are qualification cycle, and evaluation program complexity is, it is necessary to the shortcomings that expending substantial amounts of human and material resources and financial resources.
Terpene alcohols and aromatic alcohol in tealeaves have flowers and fruits fragrant, be tea aroma important composition into
Point.For example, linalool not only has lilac, the lily of the valley and the fragrance of a flower of rose, but also there are Radix Aucklandiae, fruity breath；Nerolidol carries sweet tea
Clear soft and graceful flores aurantii breath, and have as the fragrance of rose, the lily of the valley and apple flower；Benzyl carbinol has fresh and sweet Rose Essentielle etc..Terpene
The important component of enol and aromatic series alcohols aroma substance as tea aroma, after being synthesized in tea tree body, small part is to swim
Amorph exists, and most and monose, disaccharides etc. combine to form glycoside compounds and be stored in cell, volatile terpenol
And after aromatic alcohols is combined with sugar, water solubility increase, stability enhancing, bioactivity is reduced or disappeared, therefore fresh leaves of tea plant one
As can't smell flowers and fruits perfume.On the one hand the presence of glucosides may be beneficial to it and transport in vivo, on the other hand may be with one kind
Low toxicity or the form of detoxification avoid it from causing to poison to plant itself.During tea processing, the glucosides of cylinder accumulation can be
It is hydrolyzed under the action of endogenous glycosidase, releases volatile terpene alcohols and aromatic series alcohols, so as to show flowers and fruits fragrance
Feature.Terpene alcohols and aromatic alcohol in tealeaves are mainly present in the form of primrose glucosides in fresh tea leaves,
In terms of fragrance formation, the primrose glucosides in fresh tea leaves plays the role of prior than glucoside, is main fragrance in tealeaves
Precursor substance.We use solid phase microextraction with efficient LC-MS technology in 120 Fujian tea germplasm fresh leafs early period
Linalyl primrose glucosides, geranyl primrose glucosides, benzyl alcohol primrose glucosides, benzyl carbinol primrose glucosides are determined, and are as a result sent out
Now the geranyl primrose glucosides in the tea germplasm fresh leaf of suitable flowers and fruits odor type tealeaves processed, benzyl alcohol primrose glucosides, benzyl carbinol primrose
The content of glucosides is significantly higher than general tea germplasm, and fits 3 kinds of glucosides in the tea germplasm fresh leaf of flowers and fruits odor type tealeaves processed
Content meets following rule：3 kinds of glucosides total content >=150 μM/100g dry weights (wherein, benzyl alcohol primrose Glycosides Contents+benzyl carbinol
Primrose Glycosides Contents >=50 μM/100g dry weights, geranyl primrose Glycosides Contents >=100 μM/100g dry weights).Therefore, measure is passed through
The content of above-mentioned 3 kinds of primrose glucosides can directly judge the high flowers and fruits aroma type tea of the whether suitable system of tea germplasm in tea germplasm fresh leaf
Leaf.
The content of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of tea germplasm for fitting flowers and fruits odor type tealeaves processed
Rapid identification method.The present invention can be chosen the tea germplasm for fitting flowers and fruits odor type tealeaves processed by simple experimental implementation and analysis
It elects, entire identification and analytic process only need 1 working day, and greatly mirror is shortened compared to Direct Identification method (6 years)
Fixed cycle.
Purpose to realize the present invention, the present invention adopt the following technical scheme that：
A kind of tea germplasm rapid identification method for fitting flowers and fruits odor type tealeaves processed, includes step in detail below：
1）The extraction of the fresh blade raw sugar glycosides compound of tea germplasm
1. picking tea germplasm spring first run two leaves and a bud fresh leaf 2g is put in cryopreservation tube, sent with dry ice preservation to laboratory, freezing
It is dry；
2. the tealeaves of freeze-drying is taken to be ground in mortar, 50.0mg is weighed, it is common with ultrasonic extraction 10min under 4 mL methanol room temperature
Extraction 3 times centrifuges 10min after having extracted every time with the rotating speed of 3000r/min, merges 3 supernatants；
3. to the 4- isopropylbenzyl alcohols-β-D-Glucose glycosides for merging addition 5nmol in supernatant, then depressurized at 30 DEG C dense
It is reduced to drying；
4. extract is dissolved in 20mL pure water, extracted under negative pressure state by Agilent AccuBOND ODS-C18 solid phases
Take pillar；
5. it is first then eluted with 15mL water wash pillars with 60mL70% (v/v) methanol aqueous solution；
6. collecting 70% meoh eluate, after negative pressure rotary evaporation falls methanol, aqueous solution is partly lyophilized and is dissolved in 500 μ L
In pure water；
7. above 2.~be 6. repeated 3 times.
, the LC-MS analysis of glycoside reference substance and quantitative
1. compound concentration is 5,25,50,100 μM respectively geranyl primrose glucosides, benzyl alcohol primrose glucosides, benzyl carbinol primeverose
Glycosides standard solution contains 5 μM of 4- isopropylbenzyl alcohols-β-D-Glucose glycosides in each standard solution as internal standard,
Each sample size is 10 μ L, which is repeated 3 times；
2. analytical instrument is：LC/MS 2010A LC-MS instrument (Shimadzu, Japan), chromatographic column is using CAPCELL PAK
C18 columns.Liquid-phase condition is：40 DEG C, flow velocity 0.2mL/min of column temperature, mobile phase A are the aqueous formic acid of 5% (v/v), and B is second
Nitrile.Gradient is：The volume fraction of Mobile phase B is first maintained at 9%, maintains 30 min, 24% is then risen in 5min and is kept
Then 30min rises to 90% in 0.1min, 9% is back in 0.1min after maintaining 5min, balances sample introduction after 15min；
3. mass spectrum uses electron spray ionisation (ESI) Ionization mode, ion is detected with ion mode, is examined in single ion
It surveys under scan pattern (SIM patterns) and uses [M+COO]-It is analyzed as observation ion；
4. analyzed by LC-MS, obtain the total ion figure (see Fig. 1) of each glucosides reference substance, Information in Mass Spectra and regression equation (see
Table 1).
Retention time, Information in Mass Spectra and the regression equation of 1. glucosides reference substance of table
* Y represents the mass spectral intensities of standard substance and interior target intensity ratio in regression equation, and X represents the concentration of corresponding standard substance
(μM)。
, the LC-MS analysis of glycoside substance and quantitative in fresh tea leaves
1. each sample size of raw sugar glycosides of tea germplasm is 10 μ L, which is repeated 3 times.
2. analytical instrument is：LC/MS 2010A LC-MS instrument (Shimadzu, Japan), chromatographic column is using CAPCELL
PAK C18 columns.Liquid-phase condition is：40 DEG C, flow velocity 0.2mL/min of column temperature, mobile phase A are the aqueous formic acid of 5% (v/v), and B is
Acetonitrile, gradient are：The volume fraction of Mobile phase B is first maintained at 9%, maintains 30 min, 24% is then risen in 5min and is protected
30min is held, 90% is then risen in 0.1min, 9% is back in 0.1min after maintaining 5min, balances sample introduction after 15min.
3. mass spectrum uses electron spray ionisation (ESI) Ionization mode, ion is detected with ion mode, isolated
[M+COO] is used under son detection scan pattern (SIM patterns)-It is analyzed as observation ion.
4. 1 data of reference table, according to geranyl primrose glucosides, benzyl alcohol primrose glucosides, benzyl carbinol primrose glucosides reference substance
Corresponding glucosides mass spectral intensities determine corresponding geranyl primrose with interior target intensity ratio (Y value) in retention time and germplasm to be measured
Glucosides, benzyl alcohol primrose glucosides, the concentration (x values) of benzyl carbinol primrose glucosides, so as to calculate geranyl in germplasm fresh leaf to be measured
Primrose glucosides, benzyl alcohol primrose glucosides, the relative amount of benzyl carbinol primrose glucosides.
, flowers and fruits odor type tealeaves fit the judgement of tea germplasm processed
1. criterion：3 kinds of glucosides total content >=150 μM/100g dry weights (wherein, benzyl alcohol primrose Glycosides Contents+benzyl carbinol cherry
Careless glucosides >=50 μM/100g dry weights, geranyl primrose glucosides >=100 μM/100g dry weights).
2. if the content of 3 kinds of glucosides in tea germplasm meets above-mentioned standard, judge that the tea germplasm is fragrant for flowers and fruits
Type tealeaves fits tea germplasm processed.
The present invention has the following advantages compared with the prior art：
The present invention is compared with Direct Identification method, and can significantly shortening qualification cycle first, (present invention only needs 1 working day, directly
Identification method needs 6 years), accuracy rate is up to more than 90%；Tea germplasm can be reduced and fit human and material resources, financial resources in property qualification process processed
Waste；Secondly as fresh leaf sampling is micro (1~2g), destroy can be ignored caused by tea tree tree body, therefore not
Numerous impact can be expanded to follow-up cuttage；Finally, additional application this technology can certain journey in new tea cultivar Breeding Process
Realize that flowers and fruits odor type tealeaves fits the directive breeding of property kind processed, reduces the blindness of breeding of new variety process on degree.The technology can
The new Identification Method of property tea germplasm processed is fitted as flowers and fruits odor type tealeaves.
Description of the drawings
Total ion figure of Fig. 1 glucosides reference substances（Y-axis represents peak intensity, and X-axis represents retention time）.
Specific embodiment
Further to disclose rather than limiting the present invention, below in conjunction with example, the present invention is described in further detail.
A kind of tea germplasm rapid identification method for fitting flowers and fruits odor type tealeaves processed, includes step in detail below：
1st, the extraction of tea germplasm fresh leaf piece glucoside compound
1. picking tea germplasm spring first run two leaves and a bud fresh leaf 2g is put in cryopreservation tube, sent with dry ice preservation to laboratory, freezing
It is dry.
2. the tealeaves of freeze-drying is taken to be ground in mortar, 50.0mg is weighed, with ultrasonic extraction under 4 mL methanol room temperature
10min, coextraction 3 times centrifuge 10min after having extracted every time with the rotating speed of 3000r/min, merge 3 supernatants.
3. to the 4- isopropylbenzyl alcohols-β-D-Glucose glycosides for merging addition 5nmol in supernatant, then subtract at 30 DEG C
Pressure is concentrated to dryness.
4. extract is dissolved in 20mL pure water, consolidated under negative pressure state by Agilent AccuBOND ODS-C18
Mutually extraction pillar.
5. it is first then eluted with 15mL water wash pillars with 60mL70% (v/v) methanol aqueous solution.
6. collecting 70% meoh eluate, after negative pressure rotary evaporation falls methanol, aqueous solution is partly lyophilized and is dissolved in
In 500 μ L pure water.
7. above 2.~be 6. repeated 3 times.
, the LC-MS analysis of glycoside substance and quantitative in fresh tea leaves
1. each sample size of raw sugar glycosides of tea germplasm is 10 μ L, which is repeated 3 times.
2. analytical instrument is：LC/MS 2010A LC-MS instrument (Shimadzu, Japan), chromatographic column is using CAPCELL
PAK C18 columns.Liquid-phase condition is：40 DEG C, flow velocity 0.2mL/min of column temperature, mobile phase A are the aqueous formic acid of 5% (v/v), and B is
Acetonitrile, gradient are：The volume fraction of Mobile phase B is first maintained at 9%, maintains 30 min, 24% is then risen in 5min and is protected
30min is held, 90% is then risen in 0.1min, 9% is back in 0.1min after maintaining 5min, balances sample introduction after 15min.
3. mass spectrum uses electron spray ionisation (ESI) Ionization mode, ion is detected with ion mode, isolated
[M+COO] is used under son detection scan pattern (SIM patterns)-It is analyzed as observation ion.
4. 1 data of reference table, according to geranyl primrose glucosides, benzyl alcohol primrose glucosides, benzyl carbinol primrose glucosides reference substance
Corresponding glucosides mass spectral intensities determine corresponding geranyl primrose with interior target intensity ratio (Y value) in retention time and germplasm to be measured
Glucosides, benzyl alcohol primrose glucosides, the concentration (x values) of benzyl carbinol primrose glucosides, so as to calculate geranyl in germplasm fresh leaf to be measured
Primrose glucosides, benzyl alcohol primrose glucosides, the relative amount of benzyl carbinol primrose glucosides.
, flowers and fruits odor type tealeaves fit the judgement of tea germplasm processed
1. criterion：3 kinds of glucosides total content >=150 μM/100g dry weights (wherein, benzyl alcohol primrose Glycosides Contents+benzyl carbinol cherry
Careless glucosides >=50 μM/100g dry weights, geranyl primrose glucosides >=100 μM/100g dry weights).
2. if the content of 3 kinds of glucosides in tea germplasm meets above-mentioned standard, judge that the tea germplasm is fragrant for flowers and fruits
Type tealeaves fits tea germplasm processed.
The judgement of embodiment 1, new tea cultivar spring peach perfume
The quality of Oolong tea (being shown in Table 2~4) of new tea cultivar spring peach perfume, 3 years perfume (or spice) are identified within continuous 3 years using Direct Identification method
Gas, flavour score are above control kind Huang Im, and Direct Identification is the result shows that new tea cultivar spring peach is fragrant for suitable flowers and fruits aroma type tea processed
The tea germplasm of leaf.
2. 2012 years Tea Samples sensory review's tables of table
* Fujian Province's tea quality supervision and inspection station sense organ password is evaluated.
3. 2013 years Tea Samples sensory review's tables of table
* Fujian Province's tea quality supervision and inspection station sense organ password is evaluated.
4. 2014 years tealeaves sensory review's tables of table
* Fujian Province's tea quality supervision and inspection station sense organ password is evaluated.
The content (shown in table 5) of 3 kinds of glucosides of new tea cultivar spring peach perfume is obtained according to above-mentioned steps 1 to step 2, then
By it compared with criterion, which complies fully with " 3 kinds of glucosides total content >=150 μM/100g dry weight (wherein, benzyl alcohol cherries
Careless Glycosides Contents+benzyl carbinol primrose glucosides >=50 μM/100g dry weights, geranyl primrose glucosides >=100 μM/100g dry weights) " sentence
Calibration is accurate, judges that new tea cultivar spring peach perfume (or spice) fits property kind processed for flowers and fruits odor type tealeaves, this explanation applies the mirror of the method for the present invention
It is consistent with the result of Direct Identification method to determine result.
The judgement of embodiment 2, early spring milli tea tree breed
Early spring milli is the tea tree improved seeds of the suitable red and green tea processed of Fujian Province's authorization, for the tea tree of uncomfortable flowers and fruits odor type tealeaves processed
Kind.
The content (shown in table 5) of its 3 kinds of glucosides is obtained according to above-mentioned steps 1 to step 2, then by itself and criterion ratio
Compared with the discrepancy of assortment closes " 3 kinds of glucosides total content >=150 μM/100g dry weights (wherein, benzyl alcohol primrose Glycosides Contents+benzyl carbinol
Primrose glucosides >=50 μM/100g dry weights, geranyl primrose glucosides >=100 μM/100g dry weights) " criterion, judge early spring milli
It is not that flowers and fruits odor type tealeaves fits property kind processed, this explanation is using the qualification result of the method for the present invention and the result one of Direct Identification method
It causes.
The judgement of embodiment 3, Fuding white tea tea tree breed
Fuding white tea is the tea tree improved seeds of the suitable red and green tea processed of country's authorization, for the tea of uncomfortable flowers and fruits odor type tealeaves processed
Set kind.
According to above-mentioned steps 1 to step 2 obtain its 3 kinds of glucosides content (shown in table 5), then by its with criterion
Compare, which closes " 3 kinds of glucosides total content >=150 μM/100g dry weights (wherein, benzyl alcohol primrose Glycosides Contents+benzene second
Alcohol primrose glucosides >=50 μM/100g dry weights, geranyl primrose glucosides >=100 μM/100g dry weights) " criterion, judge Fuding
Big white tea is not that flowers and fruits odor type tealeaves fits property kind processed, this explanation is using the qualification result of the method for the present invention and Direct Identification method
As a result it is consistent.
Glycosides Contents (the unit of 5. tea tree breed to be measured of table：μM/100g dry weights)
*①：Benzyl alcohol primrose glucosides；②：Benzyl carbinol primrose glucosides；③：Geranyl primrose glucosides.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (4)
1. a kind of tea germplasm rapid identification method for fitting flowers and fruits odor type tealeaves processed, it is characterised in that：Comprise the following steps：
1）Fresh leaf is picked then in tea tree planting, first extracts the glucoside compound in fresh leaf, fresh leaf raw sugar glycosides extracting solution is made；
2）Geranyl primrose glucosides, benzyl alcohol primrose glucosides, benzyl carbinol primrose glucosides in fresh leaf raw sugar glycosides extracting solution is measured to contain
Amount；
3）As the benzyl alcohol primrose Glycosides Contents in fresh leaf raw sugar glycosides extracting solution+benzyl carbinol primrose Glycosides Contents >=50 μM/100g
Dry weight, and during geranyl primrose glucosides >=100 μM/100g dry weights, then judge the tea germplasm for the flowers and fruits odor type tealeaves system of fitting
Tea germplasm.
2. the tea germplasm rapid identification method according to claim 1 for fitting flowers and fruits odor type tealeaves processed, it is characterised in that：Step
Rapid 1）Specially：
1. picking tea germplasm spring first run two leaves and a bud fresh leaf 2g is put in cryopreservation tube, sent with dry ice preservation to laboratory, freezing
It is dry；
2. the tealeaves of freeze-drying is taken to be ground in mortar, 50.0mg is weighed, it is common with ultrasonic extraction 10min under 4 mL methanol room temperature
Extraction 3 times centrifuges 10min after having extracted every time with the rotating speed of 3000r/min, merges 3 supernatants；
3. to the 4- isopropylbenzyl alcohols-β-D-Glucose glycosides for merging addition 5nmol in supernatant, then depressurized at 30 DEG C dense
It is reduced to drying；
4. the dried object of step 3. is dissolved in 20mL pure water, pass through Agilent AccuBOND ODS- under negative pressure state
C18 solid phase extraction columns；
5. it is first then eluted with 15mL water wash pillars with the methanol aqueous solution that 60mL volume fractions are 70%；
6. collecting 70% meoh eluate, after negative pressure rotary evaporation falls methanol, aqueous solution is partly lyophilized and is dissolved in 500 μ L
In pure water；
7. above 2.~be 6. repeated 3 times, obtain fresh leaf raw sugar glycosides extracting solution.
3. the tea germplasm rapid identification method according to claim 1 for fitting flowers and fruits odor type tealeaves processed, it is characterised in that：Step
Rapid 2）Specially：
1. each sample size of fresh leaf raw sugar glycosides extracting solution is 10 μ L, which is repeated 3 times；
2. being measured using LC/MS 2010A LC-MS instrument, chromatographic column is using CAPCELL PAK C18 columns；Liquid phase
Condition is：40 DEG C, flow velocity 0.2mL/min of column temperature, mobile phase A is the aqueous formic acid that volume fraction is 5%, and B is acetonitrile, elution
Gradient is：The volume fraction of Mobile phase B is first maintained at 9%, maintains 30 min, 24% is then risen in 5min and keeps 30min, so
90% is risen in 0.1min afterwards, 9% is back in 0.1min after maintaining 5min, balances sample introduction after 15min；
3. mass spectrum uses electron spray ionisation Ionization mode, ion is detected with ion mode, is swept in single ion detection
It retouches and uses [M+COO] under pattern-It is analyzed as observation ion；
4. three kinds of glucosides mass spectral intensities and the regression equation of interior target intensity ratio and the concentration of glucosides are established respectively；3. step is obtained
The mass spectral intensities of the three kinds of glucosides obtained are substituted into interior target intensity ratio in regression equation, fragrant in germplasm fresh leaf to be measured so as to calculate
Phyllopodium primrose glucosides, benzyl alcohol primrose glucosides, the relative amount of benzyl carbinol primrose glucosides.
4. the tea germplasm rapid identification method according to claim 3 for fitting flowers and fruits odor type tealeaves processed, it is characterised in that：Step
Suddenly 4. the method for building up of the regression equation of three kinds of glucosides is：
1. compound concentration is 5,25,50,100 μM respectively geranyl primrose glucosides, benzyl alcohol primrose glucosides, benzyl carbinol primeverose
Glycosides standard solution contains 5 μM of 4- isopropylbenzyl alcohols-β-D-Glucose glycosides in each standard solution as internal standard,
Each sample size is 10 μ L, which is repeated 3 times；
2. analytical instrument is LC/MS 2010A LC-MS instrument, chromatographic column is using CAPCELL PAK C18 columns；Liquid phase item
Part is：40 DEG C, flow velocity 0.2mL/min of column temperature, mobile phase A are the aqueous formic acid of volume fraction 5%, and B is acetonitrile；Gradient
For：The volume fraction of Mobile phase B is first maintained at 9%, maintains 30 min, 24% is then risen in 5min and keeps 30min, Ran Hou
90% is risen in 0.1min, 9% is back in 0.1min after maintaining 5min, balances sample introduction after 15min；
3. mass spectrum uses electron spray ionisation Ionization mode, ion is detected with ion mode, is swept in single ion detection
It retouches and uses [M+COO] under pattern-It is analyzed as observation ion；
4. being analyzed by LC-MS, the total ion figure of each glucosides reference substance, Information in Mass Spectra and regression equation, geranyl primrose are obtained
The regression equation of glucosides is：Y=0.6041x+1.1490；The regression equation of benzyl alcohol primrose glucosides is：Y=0.2574x+
1.1043；The regression equation of benzyl carbinol primrose glucosides is：Y=0.4765x+2.7871.
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| CN110749668A (en) * | 2019-10-10 | 2020-02-04 | 中国科学院华南植物园 | Marker and method for screening high-aroma tea tree resource suitable for oolong tea |
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