CN108051522B - Method for quickly identifying tea plant germplasm suitable for making flower and fruit flavor type tea - Google Patents
Method for quickly identifying tea plant germplasm suitable for making flower and fruit flavor type tea Download PDFInfo
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Abstract
The invention belongs to the field of tea plant germplasm resource identification and utilization and new variety breeding, and particularly relates to a rapid identification method of tea plant germplasm suitable for making flower and fruit flavor type tea. The method comprises the following steps: 1) picking fresh leaves in the current year of tea tree planting, and extracting glycoside compounds in the fresh leaves to obtain a fresh leaf crude glycoside extracting solution; 2) measuring the content of geranyl primeveroside, benzyl alcohol primeveroside and benzyl alcohol primeveroside in the fresh-leaf crude glucoside extracting solution; 3) when the content of benzyl alcohol primeveroside and the content of phenethyl alcohol primeveroside in the fresh-leaf crude glycoside extracting solution are more than or equal to 50 mu M/100g dry weight, and the content of geranyl primeveroside is more than or equal to 100 mu M/100g dry weight, the tea tree germplasm is judged to be flower and fruit aroma type tea suitable for tea tree germplasm. The whole identification and analysis process only needs 1 working day, and compared with a direct identification method (6 years), the identification period is greatly shortened.
Description
Technical Field
The invention belongs to the field of tea plant germplasm resource identification and utilization and new variety breeding, and particularly relates to a rapid identification method of tea plant germplasm suitable for making flower and fruit flavor type tea.
Background
The aroma is one of important factors for evaluating the quality of the tea, the aroma of the tea is particularly optimal in flower and fruit aroma, and the flower and fruit aroma type tea is always favored by the tea consumption market. The formation of the flower and fruit fragrance characteristics of tea leaves is mainly influenced by tea plant germplasm, cultivation and processing technology, wherein the tea plant germplasm is a foundation. At present, the tea plant germplasm of tea with the flower and fruit flavor is mainly identified by a direct identification method, wherein a tea sample is prepared by picking fresh tea leaves and then the tea sample is subjected to sensory evaluation so as to determine the suitability of the germplasm. The direct identification method requires field planting of tea plant germplasm, and the quality identification result usually needs more than 6 years (6 years) from planting to repeated identification (3 years) in the period from normal growth to mineable period (3 years) of tea plants and years. The direct identification method has the defects of long identification period, complex identification procedure and large consumption of manpower, material resources and financial resources.
Terpene alcohols and aromatic alcohols in tea have flower and fruit fragrance and are important components of tea fragrance. For example, linalool has the floral notes lilac, lily of the valley and rose, as well as the scent notes of costustoot and fruit; nerolidol has a sweet, fresh and delicate neroli smell and also has a fragrance like rose, lily of the valley and apple flowers; phenethyl alcohol has a fresh and sweet rose scent, and the like. After the terpene alcohol and aromatic alcohol fragrant substances are synthesized in tea trees, a small part of the terpene alcohol and aromatic alcohol fragrant substances exist in a free state, most of the terpene alcohol and aromatic alcohol are combined with monosaccharide, disaccharide and the like to form glycoside compounds which are stored in cells, and after the volatile terpene alcohol and aromatic alcohol are combined with sugar, the water solubility is increased, the stability is enhanced, the biological activity is reduced or disappeared, so that the fresh tea trees can not smell flowers and fruits. The presence of the glycoside may be advantageous, on the one hand, for its transport in vivo and, on the other hand, may be in a less toxic or detoxified form to avoid its toxicity to the plant itself. During the tea processing process, glucoside accumulated in the body is hydrolyzed under the action of endogenous glycosidase to release volatile terpene alcohols and aromatic alcohols, so that the flower and fruit fragrance is shown. Terpene alcohols and aromatic alcohols in tea leaves mainly exist in fresh tea leaves in the form of primeveroside, and in the aspect of aroma formation, primeveroside in fresh tea leaves plays a more important role than glucoside and is a main aroma precursor substance in tea leaves. In the earlier stage, solid-phase microextraction and high-efficiency liquid chromatography-mass spectrometry are adopted to determine linalyl sakura glycoside, geranyl sakura glycoside, benzyl alcohol sakura glycoside and phenethyl alcohol sakura glycoside in fresh leaves of 120 Fujian tea germplasm, and the results show that the contents of geranyl sakura glycoside, benzyl alcohol sakura glycoside and phenethyl alcohol sakura glycoside in fresh leaves of tea germplasm suitable for preparing flower and fruit flavor type tea are obviously higher than those of common tea germplasm, and the contents of 3 kinds of glycosides in fresh leaves of tea germplasm suitable for preparing flower and fruit flavor type tea meet the following rules: the total content of the 3 glycosides is more than or equal to 150 mu M/100g dry weight (wherein the benzyl alcohol primeveroside content and the phenethyl alcohol primeveroside content are more than or equal to 50 mu M/100g dry weight, and the geranyl primeveroside content is more than or equal to 100 mu M/100g dry weight). Therefore, whether the tea plant germplasm is suitable for preparing the tea with high flower and fruit fragrance can be directly judged by measuring the content of the 3 kinds of primeveroside in the fresh tea plant germplasm leaves.
Disclosure of Invention
The invention aims to provide a method for quickly identifying tea plant germplasm suitable for making flower and fruit fragrance type tea aiming at the defects of the prior art. The tea plant germplasm suitable for preparing the flower and fruit fragrance type tea can be selected through simple experimental operation and analysis, the whole identification and analysis process only needs 1 working day, and the identification period is greatly shortened compared with a direct identification method (6 years).
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for quickly identifying tea plant germplasm suitable for making flower and fruit flavor type tea comprises the following specific steps:
1) extraction of crude glycoside compounds from fresh tea plant leaves
Picking 2g of first-round, one-bud and two-leaf fresh leaves of tea plant germplasm in spring, placing the fresh leaves in a freezing storage tube, storing the fresh leaves with dry ice, conveying the fresh leaves to a laboratory, and freezing and drying the fresh leaves;
grinding the freeze-dried tea leaves in a mortar, weighing 50.0mg of the tea leaves, performing ultrasonic extraction for 10min at normal temperature by using 4 mL of methanol for 3 times, centrifuging for 10min at the rotating speed of 3000r/min after each extraction, and combining the supernatants for 3 times;
③ adding 5nmol of 4-isopropyl benzyl alcohol-beta-D-glucoside into the combined supernatant, and then concentrating under reduced pressure at 30 ℃ until the mixture is dried;
fourthly, dissolving the extract into 20mL of purified water, and passing through an Agilent AccuBOND ODS-C18 solid phase extraction small column under a negative pressure state;
leaching the small column with 15mL of water, and eluting with 60mL of 70% (v/v) methanol aqueous solution;
sixthly, collecting 70% methanol eluent, carrying out negative pressure rotary evaporation to remove the methanol, and then, freezing and drying part of the aqueous solution and dissolving the part of the aqueous solution in 500 mu L of pure water;
seventhly, repeating the steps from the previous step to the sixth step for 3 times.
Liquid chromatography-mass spectrometry analysis and quantification of glycoside standard substance
Preparing geranyl primeveroside, benzyl alcohol primeveroside and phenethyl alcohol primeveroside standard substance solutions with the concentrations of 5 muM, 25M, 50M and 100 muM respectively, wherein each standard substance solution contains 5 muM of 4-isopropylbenzyl alcohol-beta-D-glucoside as an internal standard, the sampling amount is 10 muL each time, and the step is repeated for 3 times;
the analyzer is: LC/MS 2010A LC MS (Shimadzu, Japan) used CAPCELLPAK C18 as the column. The liquid phase conditions were: the column temperature is 40 ℃, the flow rate is 0.2mL/min, the mobile phase A is 5% (v/v) formic acid water solution, and the B is acetonitrile. The elution gradient was: keeping the volume fraction of the mobile phase B at 9%, maintaining for 30min, then rising to 24% within 5min, keeping for 30min, then rising to 90% within 0.1min, returning to 9% within 0.1min after maintaining for 5min, balancing for 15min, and injecting;
③ the mass spectrum adopts electrospray ionization (ESI) ionization mode, detects ions in anion mode, and adopts [ M + COO ] in single ion detection scan mode (SIM mode)]-As an observation ion for analysis;
and fourthly, obtaining a total ion map (shown in figure 1), mass spectrum information and a regression equation (shown in table 1) of each glucoside standard substance by LC-MS analysis.
TABLE 1 Retention time, Mass Spectrometry information and regression equation for glycoside standards
In the regression equation, Y represents the ratio of the mass spectrum intensity of the standard substance to the intensity of the internal standard, and X represents the concentration (μ M) of the corresponding standard substance.
Liquid chromatography-mass spectrometry analysis and quantification of glycoside substances in fresh tea leaves
The crude glycoside of tea tree germplasm is 10 microliter each time, and the step is repeated for 3 times.
The analyzer is: LC/MS 2010A LC MS (Shimadzu, Japan) used CAPCELLPAK C18 as the column. The liquid phase conditions were: the column temperature is 40 ℃, the flow rate is 0.2mL/min, the mobile phase A is 5% (v/v) formic acid water solution, the mobile phase B is acetonitrile, and the elution gradient is as follows: keeping the volume fraction of the mobile phase B at 9%, maintaining for 30min, then rising to 24% within 5min, maintaining for 30min, then rising to 90% within 0.1min, returning to 9% within 0.1min after maintaining for 5min, balancing for 15min, and injecting.
③ the mass spectrum adopts electrospray ionization (ESI) ionization mode, detects ions in anion mode, and adopts [ M + COO ] in single ion detection scan mode (SIM mode)]-The analysis was performed as an observation ion.
And fourthly, referring to the data in the table 1, determining the concentrations (x values) of the corresponding geranyl primeveroside, benzyl alcohol primeveroside and phenethyl alcohol primeveroside according to the retention time of the geranyl primeveroside, benzyl alcohol primeveroside and the intensity ratio (Y value) of the mass spectrum intensity of the corresponding glycoside in the germplasm to be detected to the internal standard, thereby calculating the relative contents of the geranyl primeveroside, the benzyl alcohol primeveroside and the phenethyl alcohol primeveroside in the fresh leaves of the germplasm to be detected.
Determination of suitability of flower and fruit fragrant tea for tea tree seed quality
Judging a standard: the total content of the 3 glycosides is more than or equal to 150 mu M/100g dry weight (wherein the content of benzyl alcohol primeveroside plus phenethyl alcohol primeveroside is more than or equal to 50 mu M/100g dry weight, and the content of geranyl primeveroside is more than or equal to 100 mu M/100g dry weight).
Secondly, if the content of 3 glucosides in the tea plant germplasm meets the standard, the tea plant germplasm is judged to be suitable for preparing the tea plant germplasm by the flower and fruit fragrant tea.
Compared with the prior art, the invention has the following advantages:
compared with a direct identification method, the method can obviously shorten the identification period (only 1 working day is needed, and the direct identification method needs 6 years), and the accuracy rate can reach more than 90%; the waste of manpower, material resources and financial resources in the tea tree germplasm adaptability identification process can be reduced; secondly, because the sampling of the fresh leaves is trace (1-2 g), the damage to the tree body of the tea tree can be ignored, and the influence on the subsequent cuttage propagation can not be caused; finally, the technology is additionally applied in the new variety breeding process of the tea trees, so that the directional breeding of the flower-fruit flavor type tea adaptive variety can be realized to a certain extent, and the blindness of the new variety breeding process is reduced. The technology can be used as a novel method for identifying the germplasm of the flower and fruit fragrance type tea adaptive tea tree.
Drawings
FIG. 1 Total ion plot of glycoside standards (Y-axis represents peak intensity, X-axis represents retention time).
Detailed Description
For further disclosure, but not limitation, the present invention is described in further detail below with reference to examples.
A method for quickly identifying tea plant germplasm suitable for making flower and fruit flavor type tea comprises the following specific steps:
1. extraction of tea tree germplasm fresh leaf glycoside compound
Firstly, picking 2g of first-round, one-bud and two-leaf fresh leaves of tea plant germplasm in spring, placing the fresh leaves in a freezing storage tube, storing the fresh leaves with dry ice, conveying the fresh leaves to a laboratory, and freezing and drying the fresh leaves.
② grinding the freeze-dried tea in a mortar, weighing 50.0mg, extracting with 4 mL methanol under ultrasonic at normal temperature for 10min for 3 times, centrifuging at 3000r/min for 10min after each extraction, and mixing the supernatant for 3 times.
③ adding 5nmol of 4-isopropylbenzyl alcohol-beta-D-glucoside into the combined supernatant, and then concentrating under reduced pressure at 30 ℃ until the mixture is dried.
Fourthly, the extract is dissolved in 20mL of purified water and passes through an Agilent AccuBOND ODS-C18 solid phase extraction column under the negative pressure state.
Fifthly, the small column is firstly leached by 15mL of water and then eluted by 60mL of 70% (v/v) methanol aqueous solution.
Sixthly, collecting 70% methanol eluent, and after the methanol is evaporated by negative pressure rotation, partially freezing and drying the aqueous solution and dissolving the aqueous solution in 500 mu L of pure water.
Seventhly, repeating the steps from the previous step to the sixth step for 3 times.
Liquid chromatography-mass spectrometry analysis and quantification of glycoside substances in fresh tea leaves
The crude glycoside of tea tree germplasm is 10 microliter each time, and the step is repeated for 3 times.
The analyzer is: LC/MS 2010A LC MS (Shimadzu, Japan) used CAPCELLPAK C18 as the column. The liquid phase conditions were: the column temperature is 40 ℃, the flow rate is 0.2mL/min, the mobile phase A is 5% (v/v) formic acid water solution, the mobile phase B is acetonitrile, and the elution gradient is as follows: keeping the volume fraction of the mobile phase B at 9%, maintaining for 30min, then rising to 24% within 5min, maintaining for 30min, then rising to 90% within 0.1min, returning to 9% within 0.1min after maintaining for 5min, balancing for 15min, and injecting.
③ the mass spectrum adopts electrospray ionization (ESI) ionization mode, detects ions in anion mode, and adopts [ M + COO ] in single ion detection scan mode (SIM mode)]-The analysis was performed as an observation ion.
And fourthly, referring to the data in the table 1, determining the concentrations (x values) of the corresponding geranyl primeveroside, benzyl alcohol primeveroside and phenethyl alcohol primeveroside according to the retention time of the geranyl primeveroside, benzyl alcohol primeveroside and the intensity ratio (Y value) of the mass spectrum intensity of the corresponding glycoside in the germplasm to be detected to the internal standard, thereby calculating the relative contents of the geranyl primeveroside, the benzyl alcohol primeveroside and the phenethyl alcohol primeveroside in the fresh leaves of the germplasm to be detected.
Determination of suitability of flower and fruit fragrant tea for tea tree seed quality
Judging a standard: the total content of the 3 glycosides is more than or equal to 150 mu M/100g dry weight (wherein the content of benzyl alcohol primeveroside plus phenethyl alcohol primeveroside is more than or equal to 50 mu M/100g dry weight, and the content of geranyl primeveroside is more than or equal to 100 mu M/100g dry weight).
Secondly, if the content of 3 glucosides in the tea plant germplasm meets the standard, the tea plant germplasm is judged to be suitable for preparing the tea plant germplasm by the flower and fruit fragrant tea.
Example 1 determination of Chuntao flavor of New tea plant
The quality of oolong tea with the new variety of tea trees, namely the spring peach aroma, is continuously identified for 3 years by using a direct identification method (see table 2-4), the aroma and taste scores of 3 years are higher than those of a control variety yellow , and the direct identification result shows that the spring peach aroma of the new variety of tea trees is tea tree germplasm suitable for preparing flower and fruit aroma type tea.
Table 2.2012 sensory evaluation table for tea sample
Sensory code evaluation of Fujian province tea quality supervision and inspection station.
Table 3.2013 sensory evaluation table for tea sample
Sensory code evaluation of Fujian province tea quality supervision and inspection station.
Table 4.2014 tea sense examination and appraisal table
Sensory code evaluation of Fujian province tea quality supervision and inspection station.
The content of 3 glycosides of the spring peach aroma of the new tea plant variety is obtained according to the steps 1 to 2 (shown in table 5), and then compared with a judgment standard, the variety completely meets the judgment standard that the total content of the 3 glycosides is more than or equal to 150 muM/100 g dry weight (wherein, the content of benzyl alcohol primrose glycoside and the content of phenethyl alcohol primrose glycoside are more than or equal to 50 muM/100 g dry weight, and the content of geranyl primrose glycoside is more than or equal to 100 muM/100 g dry weight), and the spring peach aroma of the new tea plant variety is judged to be a flower and fruit type tea suitable variety, which shows that the identification result of the method is consistent with the result of the direct identification method.
Example 2 determination of variety of early spring Hao tea Tree
The early spring tea is a good tea tree variety which is examined and made by Fujian province and is suitable for preparing red tea and green tea, and is a tea tree variety which is not suitable for preparing flower and fruit fragrance type tea.
The content of 3 glycosides of the variety is obtained according to the steps 1 to 2 (shown in table 5), and then the variety is compared with a judgment standard, the variety does not meet the judgment standard of that the total content of the 3 glycosides is more than or equal to 150 mu M/100g dry weight (wherein, the content of benzyl alcohol primrose glycoside + the content of phenethyl alcohol primrose glycoside is more than or equal to 50 mu M/100g dry weight, and the content of geranyl primrose glycoside is more than or equal to 100 mu M/100g dry weight), and the early spring is judged to be not a flower and fruit flavor type tea leaf suitable variety, which indicates that the identification result applying the method is consistent with the result of a direct identification method.
Example 3 determination of tea plant variety of Fuding white tea
The Fuding white tea is a national approved tea tree fine variety suitable for preparing red tea and green tea, and is a tea tree variety unsuitable for preparing flower and fruit flavor type tea.
The content of 3 glycosides of the variety is obtained according to the steps 1 to 2 (shown in table 5), and then the content is compared with a judgment standard, the variety does not meet the judgment standard of that the total content of the 3 glycosides is more than or equal to 150 mu M/100g dry weight (wherein the content of benzyl alcohol primeveroside + the content of phenethyl alcohol primeveroside is more than or equal to 50 mu M/100g dry weight, and the content of geranyl primevergreen tea is more than or equal to 100 mu M/100g dry weight), and the fuding white tea is judged to be not a flower and fruit flavor type tea suitable variety, which shows that the identification result applying the method is consistent with the result of a direct identification method.
TABLE 5 glycoside content of tea tree variety to be tested (unit: μ M/100g dry weight)
Firstly: benzyl alcohol, primrose glycoside; secondly, the step of: phenethyl alcohol primrose glycoside; ③: geranyl primrose glycoside.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (3)
1. A method for quickly identifying tea plant germplasm suitable for making flower and fruit flavor type tea is characterized by comprising the following steps: the method comprises the following steps:
1) picking fresh leaves in the current year of tea tree planting, and extracting glycoside compounds in the fresh leaves to obtain a fresh leaf crude glycoside extracting solution;
2) measuring the content of geranyl primeveroside, benzyl alcohol primeveroside and benzyl alcohol primeveroside in the fresh-leaf crude glucoside extracting solution;
3) when the content of benzyl alcohol primeveroside and the content of phenethyl alcohol primeveroside in the fresh-leaf crude glycoside extracting solution are more than or equal to 50 mu M/100g dry weight, and the content of geranyl primeveroside is more than or equal to 100 mu M/100g dry weight, judging that the tea plant germplasm is flower and fruit aroma type tea suitable for preparing the tea plant germplasm;
the step 2) is specifically as follows:
③ the mass spectrum adopts electrospray ionization mode, detects ions in anion mode, and adopts [ M + COO ] in single ion detection scanning mode]-As an observation ion for analysis;
establishing regression equations of the intensity ratios of the three glycoside mass spectrum intensities and the internal standard and the concentration of the glycoside respectively; substituting the mass spectrum intensity of the three glycosides obtained in the step (c) and the intensity ratio of the internal standard into a regression equation, thereby calculating the relative contents of geranyl primeveroside, benzyl alcohol primeveroside and phenethyl alcohol primeveroside in the fresh leaves of the germplasm to be detected.
2. The method for rapidly identifying tea plant germplasm suitable for flower and fruit flavored tea leaves according to claim 1, which is characterized by comprising the following steps of: the step 1) is specifically as follows:
⑦ or more
～
Repeating for 3 times to obtain crude glycoside extract of fresh leaves.
3. The method for rapidly identifying tea plant germplasm suitable for flower and fruit flavored tea leaves according to claim 1, which is characterized by comprising the following steps of: the method for establishing the regression equation of the three glycosides comprises the following steps:
③ the mass spectrum adopts electrospray ionization mode, detects ions in anion mode, and adopts [ M + COO ] in single ion detection scanning mode]-As an observation ion for analysis;
fourthly, obtaining a total ion map, mass spectrum information and a regression equation of each glucoside standard substance through LC-MS analysis, wherein the regression equation of the geranyl primrose glucoside is as follows: y =0.6041x + 1.1490; the regression equation for benzyl alcohol primeveroside is: y =0.2574x + 1.1043; the regression equation for the phenyl ethyl alcohol primrose glycoside is: y =0.4765x + 2.7871.
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