CN111220738A - Method for evaluating storage stability of water-removed substances of Anxi Tieguanyin spring tea - Google Patents
Method for evaluating storage stability of water-removed substances of Anxi Tieguanyin spring tea Download PDFInfo
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Abstract
The invention relates to a method for evaluating the storage stability of a water-removing substance of Anxi Tieguanyin spring tea, which extracts tea hydrolat by a steam distillation method, detects the content of 10 characteristic aroma components in the hydrolat by a gas chromatography-mass spectrometry (GC-MS) method and judges the stability of the water-removing substance of the Anxi Tieguanyin spring tea by the tolerance range of the content of the characteristic aroma components. The method has the advantages of high reliability, strong representativeness and good operability.
Description
Technical Field
The invention relates to the field of flavors and fragrances, in particular to a method for evaluating the storage stability of a water-removing substance of Anxi Tieguanyin spring tea.
Background
In recent years, with the consumption trend of individuation, diversification and continuous upgrading, a plurality of innovative cigarette products, such as cigarette products with different fragrances, such as various fruity fragrances, wine fragrances, tea fragrances and the like, are introduced in the tobacco industry. The cigarette products characterized by tea aroma include "september (golden brick)" series, "Taishan" full series and "Jiaozi (hard dragon and phoenix foretell good luck)" and so on. The tea essences used by different brands of cigarettes are different in source, for example, the essences used by the 'seven wolfs (gold brick)' series are extracts from Tieguanyin tea raw materials. Fujian tea has excellent quality and various varieties, tea leaves and tea flowers have unique aroma style and aroma, and cigarette developers screen characteristic tea leaves and tea flowers and extract aroma substances in the tea leaves and tea flowers by modern extraction and separation technology to obtain tea essences in various forms, such as hydrolat, essential oil and the like. The use of the tea essence integrates the aroma style and characteristic aroma of tea leaves and tea tree flowers into the seven wolf cigarette product, not only strengthens the style characteristic of the cigarette product, but also endows the cigarette product with deep cultural background.
In order to obtain high quality tea materials, picking and preliminary processing of tea materials such as tea leaves and tea flowers must be completed in a short time (generally, a week), and daily production capacity of a tea aroma extraction line is limited, and it is difficult to complete production processes such as extraction, concentration and the like of all the materials in such a short time. Therefore, the tea material after primary processing must be frozen in a freezer. Similar to agricultural products such as tobacco leaves and the like, the frozen storage can only greatly slow down the alcoholization rate of the tea raw materials, can not completely stop the alcoholization process of the raw materials, and the prolonging of the alcoholization time causes the change of the internal quality of the tea raw materials and possibly changes the quality of the tea essence. Therefore, it is necessary to monitor the storage quality stability of the tea raw materials to ensure the quality of the raw materials and further ensure the quality stability of the tea essence.
Disclosure of Invention
The invention aims to provide a method for evaluating the storage stability of a water-removed substance of Anxi Tieguanyin spring tea, which comprises the steps of extracting tea hydrolat, freeze-drying, redissolving, separating by chromatographic column chromatography, grouping according to the aroma characteristics of each fraction, qualitatively analyzing groups representing the main aroma characteristics of the tea by a solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS), screening 41 main aroma components with characteristic aroma and carrying out quantitative analysis, and measuring the olfactory threshold of each component in an ethanol medium to obtain a corresponding aroma vitality value (OAV). On this basis, 10 characteristic flavor components in which the OAV was high and the content reached the detection requirement were selected as the target compounds. Extracting tea hydrolat of different storage time, firstly carrying out sensory evaluation to obtain the tea hydrolat with basically consistent sensory evaluation, then extracting characteristic aroma components in the tea hydrolat by ethyl acetate vortex oscillation, analyzing by a gas chromatography-mass spectrometry (GC-MS) method, and establishing a tea raw material storage quality stability evaluation method by taking the content tolerance range of the characteristic aroma components as an evaluation index.
Therefore, the invention provides a method for evaluating the storage stability of the water-removed substances of the Anxi Tieguanyin spring tea, which comprises the following steps:
(1) adding 3-5 times (such as 3, 4 or 5 times) of water into the water-removed extract of Anxi Tieguanyin spring tea, performing steam distillation, collecting hydrolat, and stopping collecting when the hydrolat weight is 30-40% of water weight;
(2) detecting the hydrolat obtained in the step (1) by adopting GC-MS to obtain the following 10 components in the hydrolat: geraniol, benzaldehyde, linalool oxide (furan type), linalool, phenethyl alcohol, geraniol, indole, jasmone, dihydroactinidiolide, and methyl jasmonate;
(3) comparing the contents of the 10 components measured in the step (2) with the corresponding contents:
0.300-0.500 mu g/g of leaf alcohol; 0.190-0.266 mug/g of benzaldehyde; linalool oxide (furan type) 1.500-1.850 μ g/g; linalool 3.000-3.900 μ g/g; 1.400-1.900 mu g/g of phenethyl alcohol; geraniol 1.200-1.800 μ g/g; 2.400-4.400 mu g/g of indole; jasmone 0.570-0.750 microgram/g; dihydroactinidiolide 0.022-0.027 μ g/g; 0.060-0.300 mu g/g of methyl jasmonate;
(4) and (3) judging that the water-removed substances of the Anxi Tieguanyin spring tea are deteriorated when at least 4 of the 10 ingredient contents measured in the step (2) are not in the corresponding ingredient content range in the step (4).
In some embodiments, the contents of the ingredients in step (3) are: 0.390-0.495 mu g/g of leaf alcohol; 0.190-0.266 mug/g of benzaldehyde; linalool oxide (furan type) 1.500-1.850 μ g/g; linalool 3.000-3.900 μ g/g; 1.400-1.900 mu g/g of phenethyl alcohol; geraniol 1.222-1.800 μ g/g; indole 2.430-4.400 μ g/g; jasmone 0.570-0.710 μ g/g; dihydroactinidiolide 0.022-0.027 μ g/g; 0.083-0.101 mu g/g methyl jasmonate.
In some embodiments, the GC-MS gas chromatography conditions in step (2) are selected from one or more of the following:
i) a chromatographic column: agilent DB-5MS (60 m.times.0.25 mm.times.0.25 μm);
ii) a carrier gas: he;
iii) column flow rate: 1 mL/min;
iv) injection port temperature: 280 ℃;
v) sample size: 1 mu L of the solution;
vi) sample injection mode: split-flow sample injection with a split-flow ratio of 5: 1;
v) temperature rising mode: temperature programming, keeping at 50 ℃ for 1min, heating to 300 ℃ at 3 ℃/min, and keeping for 6 min.
In some embodiments, the mass spectrometric conditions in GC-MS in step (2) are selected from one or more of the following:
i) MS transmission line temperature: 280 ℃;
ii) EI ion source temperature: 280 ℃;
iii) quadrupole temperature: 150 ℃;
iv) EI ionization energy: 70 eV;
v) scan mode: full scan + selective ion scan.
In some embodiments, the anxi tieguanyin is red anxi tieguanyin.
In some embodiments, the source of the Anxi Tieguanyin tea is Anxi Xiang Huacun.
In some embodiments, the deterioration of the blanched material of the Anxi Tieguanyin spring tea is determined when 4, 5, 6, 7, 8, 9, or 10 of the 10 ingredient contents determined in step (2) are not within the corresponding ingredient content range determined in step (3).
Interpretation of terms:
in the invention, the 'Xianghua' is located in the west of Anxi, is a Tieguanyin producing area and mainly comprises Xianghuacun, Dongkun village, Xiangdi village, Dunqinghua and Baibancun. In some embodiments, Xianghuacun is specifically identified.
In the present invention, unless otherwise specified, "enzyme deactivation" refers to a process of deactivating or inactivating enzymes in fresh leaves at a certain temperature. The water-removing method comprises the following steps: roller fixation, pan frying fixation, hot air fixation, steam fixation, microwave fixation or steam-hot air mixed fixation, etc. The term "enzyme-deactivating substance" refers to a substance which has undergone enzyme deactivation treatment. The tea leaves obtained by the above treatment are the "enzyme-deactivating substances" described herein.
In the present invention, the term "gas chromatography" or "GC" refers to a chromatographic separation method in which a sample mixture is evaporated and injected into a stream of carrier gas (e.g., nitrogen or helium) moving through a column containing a stationary phase (composed of a liquid or a particulate solid) and separated into its constituent compounds according to the affinity of the compounds for the stationary phase.
In the present invention, the term "mass spectrometry" or "MS" refers to an analytical technique for identifying a compound by its mass. The basic principle of mass spectrometry is to ionize each component in a sample in an ion source to generate charged ions with different charge-mass ratios, and the charged ions form an ion beam under the action of an accelerating electric field and enter a mass analyzer. In the mass analyzer, the mass is determined by dispersing the generated opposite velocities by an electric field and a magnetic field, and focusing them to obtain mass spectra. MS techniques typically include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. The compound may be ionized and detected by any suitable method. A "mass spectrometer" typically includes an ionization source and an ion detector.
Advantageous effects of the invention
The invention provides a method for evaluating the storage stability of a water-removing substance of Anxi Tieguanyin spring tea, which extracts tea hydrolat by a steam distillation method, detects the content of 10 characteristic aroma components in the hydrolat by a gas chromatography-mass spectrometry (GC-MS) method and judges the stability of the water-removing substance of the Anxi Tieguanyin spring tea by the tolerance range of the content of the characteristic aroma components. The method has the advantages of high reliability, strong representativeness and good operability.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Taking the enzyme-deactivated substances of the Anxi Tieguanyin spring tea which is freshly put in a warehouse as a reference, taking the enzyme-deactivated substances of the tea which are stored for 6, 13, 24, 31, 39, 70 and 82 days respectively (respectively corresponding to the first batch to the eighth batch), and extracting tea hydrolat to obtain 9 hydrolat samples. First, the olfactory discrimination evaluation was carried out, and there was no significant difference in sensory quality among 9 batches of the hydrolat samples. And then extracting and GC-MS detecting to obtain the content of the characteristic aroma components, counting the standard deviation of the content of the characteristic aroma components under different storage times, and taking the tolerance range as an index for evaluating the storage stability so as to evaluate the storage stability of the water-removed substances of the Anxi Tieguanyin spring tea.
(1) Extraction of hydrolat (laboratory)
Weighing 1.5kg of tea fixation substance into a stainless steel steaming layer, and placing in an evaporation kettle. Weighing 6.0kg of boiled water, and uniformly spraying the boiled water on the tea raw material twice without soaking the raw material in water. Setting the heating temperature of the evaporation kettle to be 125-150 ℃, the temperature of the raw materials to be 100-110 ℃, and keeping the condensed water at room temperature. The collection was started from the first drop of distillate condensate and stopped when the weight was about 2.0 kg.
(2) Selection of characteristic aroma components
The tea hydrolat is lyophilized and concentrated, and is redissolved with methanol, filtered, the clear liquid is separated by gel chromatography (GPC), and 117 fractions are collected in total. The olfactory characteristics of the fractions are evaluated, the fractions with similar aroma characteristics are combined, the fractions without obvious aroma characteristics are removed, and the fractions can be divided into 11 components, and the grouping and combining conditions and the aroma characteristics of the components are described in table 1.
TABLE 1 evaluation of aroma characteristics of the fractions after gel chromatography
2) Qualitative analysis of flavor component
The preliminary analysis was performed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). The components 5, 6 and 7 have fresh, natural and rich tea aroma characteristics, can completely represent the benign characteristics of the tea hydrolat, and simultaneously remove some bad smell generated in the storage and treatment processes of the hydrolat; the analysis results show that the aroma components of the components 5, 6 and 7 are relatively similar, so that the components 5, 6 and 7 are combined, and the combined components are subjected to deep analysis by adopting various types of SPME fiber heads. And summarizing analysis results to obtain 100 volatile components in the combined distillate of the tea hydrolat.
3) Quantitative analysis of main flavor component
And further screening the aroma characteristics, commercialization, safety and the like, purchasing standard samples, and performing retention time comparison and aroma evaluation by GC-MS (gas chromatography-mass spectrometry), and finally determining 41 aroma components in the tea hydrolat (Table 2), wherein the aroma components can be divided into eight types of aroma notes of flower aroma, fruit aroma, milk aroma, green aroma, bean aroma, sweet aroma, fat wax aroma and cocoa aroma according to the aroma characteristics of the standard samples, wherein the aroma components comprise 16 types of flower aroma compounds, 13 types of fruit aroma compounds, 3 types of milk aroma compounds, 5 types of green aroma compounds and 1 type of bean aroma, sweet aroma, fat wax aroma and cocoa aroma compounds.
TABLE 2 tea hydrolat aroma composition and aroma description
Wherein the GC-MS detection method comprises the following specific steps:
preparation of internal standard solution
Weighing about 0.25g of styrofoam propionate into a 25mL brown volumetric flask, and using absolute ethyl alcohol to fix the volume to obtain an internal standard stock solution with the concentration of about 10 mg/mL. Accurately transferring 0.25mL of internal standard stock solution into a 25mL brown volumetric flask, and using absolute ethyl alcohol for constant volume to serve as an internal standard working solution with the concentration of about 100 mu g/mL.
Preparation of sample standard solution
0.25g of the solid standard was weighed into a beaker, dissolved completely by adding a small amount of ethyl acetate and transferred into a brown flask. Then 0.25g of liquid standard substance is respectively transferred into the brown volumetric flask, and ethyl acetate is used for constant volume to obtain a standard stock solution with the concentration of about 10 mg/mL. Then accurately transferring 0.25mL of the standard stock solution into a 25mL brown volumetric flask, and fixing the volume by ethyl acetate to obtain a first-class standard solution with the concentration of about 100 mu g/mL. Accurately transferring 0.25mL of the primary standard solution into a 25mL brown volumetric flask, and fixing the volume by ethyl acetate to obtain a secondary standard solution with the concentration of about 1 mu g/mL.
Preparation of sample standard working solution
1 and 5mL of the secondary standard solution were transferred to a 10mL brown volumetric flask, and 0.3mL of the internal standard working solution was added to obtain standard working solutions having concentrations of about 0.1, 0.5. mu.g/mL. Then 0.1, 0.2, 0.5, 1.0 and 2.0mL of the primary standard solution were transferred to a 10mL brown volumetric flask, and 0.3mL of the internal standard working solution was added to obtain standard working solutions with concentrations of about 1, 2, 5, 10 and 20. mu.g/mL.
Detection method
And respectively injecting the standard working solution and the pure dew extract liquid sample into a gas chromatography-mass spectrometer for detection. The gas chromatography conditions used were as follows: a chromatographic column: agilent DB-5MS (60m x 0.25.25 mm x 0.25 μm); carrier gas: he; sample introduction amount: 1 mu L of the solution; sample inlet temperature: 280 ℃; sample introduction mode: split-flow sample injection, split-flow ratio 5: 1; column flow rate: 1 mL/min; column oven temperature program: keeping the temperature at 50 ℃ for 1min, heating to 300 ℃ at 3 ℃/min, and keeping the temperature for 6 min. The mass spectrometry conditions used were as follows: MS transmission line temperature: 280 ℃, EI ion source temperature: 280 ℃, quadrupole temperature: 150 ℃; EI ionization energy: 70 eV; scanning mode: full scan + selective ion scan.
And carrying out regression analysis on the corresponding concentrations of the aroma components and the internal standard according to the quantitative ion peak areas to obtain a standard curve, wherein the correlation coefficient is more than or equal to 0.994. And substituting the quantitative ion peak area ratio of the aroma component and the internal standard into the standard curve to obtain the content of the aroma component in the tea leaf hydrolat sample. The retention time of each fragrance component, the parameters of the qualitative and quantitative ions and the contents are shown in Table 3.
TABLE 3 retention time and selection ions of fragrance ingredients and internal standard compounds
4) Determination of olfactory threshold and OAV value of main aroma component
Olfactory thresholds and corresponding aroma activity values (OAV) of the corresponding components in ethanol media were determined. Olfaction threshold and aroma activity value (OAV value) measurement method: respectively preparing the standard substance of each characteristic aroma component into 1.0mg/mL ethanol solution, and then respectively diluting for nine times in an equal time step by step to obtain the series concentration standard solution of each component. For the series of concentration standard solutions of each component, according to the method of GB/T22366-2008 "estimation of optimal estimation threshold", an aroma evaluator identified the standard solution from the standard solution and 2 blank liquids (ethanol and water) by aroma evaluation, and the geometric mean of the highest concentration of each evaluator identification error and the adjacent higher first-order concentration thereof was taken as the BET value of the corresponding component evaluated by the evaluator. For each ingredient, the geometric mean of BET values of 10 evaluators was calculated as the aroma threshold (T) of the ingredient, and then the aroma activity value (OAV) of each ingredient was calculated by the following formula, and the results are shown in table 4.
OAV＝C/T
Wherein C is the content of the flavor component (. mu.g/mL) in the sample.
Table 4 characterization results of main aroma components
Selecting the following 10 components from the detection results as key indexes of subsequent quality evaluation: geraniol, jasmone, dihydroactinidiolide, and methyl jasmonate.
(3) Tolerance range of characteristic fragrance component content
Taking tea leaves de-enzyming substances which are put in a fresh warehouse as a reference, taking the tea leaves de-enzyming substances (corresponding to the first batch to the eighth batch respectively) with the storage time of 6 days, 13 days, 24 days, 31 days, 39 days, 70 days and 82 days, and extracting tea leaf hydrolat according to the method in the step (1) to obtain hydrolat samples of 9 batches. The olfactory discrimination evaluation is carried out firstly, and the sensory quality of 9 batches of the hydrolat samples has no significant difference. Then, the hydrolat samples were extracted and analyzed by GC-MS for the content of each characteristic flavor component, and the standard deviation was calculated (Table 5).
The extraction method comprises the following steps:
15.0g of tea leaf was weighed into a plastic centrifuge tube, 3mL of ethyl acetate was added, along with 90 μ L of internal standard working solution. Vortexing at 2000rpm for 5min, shaking for 2 times, centrifuging at 8000rpm for 5min to promote layering, and collecting supernatant to chromatographic bottle for detection.
TABLE 59 content of characteristic flavor components of the blanched tea leaves of batch
The tolerance ranges of the 10 characteristic aroma components are calculated as follows:
0.300-0.500 mu g/g of leaf alcohol;
0.190-0.266 mug/g of benzaldehyde;
linalool oxide (furan type) 1.500-1.850 μ g/g;
linalool 3.000-3.900 μ g/g;
1.400-1.900 mu g/g of phenethyl alcohol;
geraniol 1.200-1.800 μ g/g;
2.400-4.400 mu g/g of indole;
jasmone 0.570-0.750 microgram/g;
dihydroactinidiolide 0.022-0.027 μ g/g;
0.060-0.130 mu g/g of methyl jasmonate.
Example 2
The hydrolat of the tea leaves which were stored for 94, 109 and 147 days was prepared under the same extraction, extraction and detection conditions as in example 1, and the contents of characteristic aroma components obtained by GC-MS detection were shown in Table 6, where significant differences were found by olfactory discrimination. As can be seen from the table, the content of only a part of compounds is within the content tolerance range, such as phenethyl alcohol, methyl jasmonate and the like, and the content of other compounds is greatly changed and exceeds the tolerance range.
TABLE 6 contents of characteristic flavor components of hydrolat of tea fixation product at different storage times
Conclusion
The stability evaluation method comprises the steps of screening 10 characteristic aroma components from the extracted tea water-removing substance hydrolat, establishing a content tolerance range of the characteristic aroma components of the water-removing substance hydrolat by extracting the water-removing substance hydrolat different storage times and detecting the content of the characteristic aroma components, and evaluating the hydrolat of the water-removing substance hydrolat at different storage times by combining olfactory discrimination evaluation so as to evaluate the storage stability of the water-removing substance. Similarly, the method for establishing the tolerance range of the content of the characteristic aroma components and combining the olfactory discrimination evaluation can also be used as a storage stability evaluation method of fresh tea and finished tea.
Although specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that, based upon the overall teachings of the disclosure, various modifications and alternatives to those details could be developed and still be encompassed by the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (7)
1. A method for evaluating the storage stability of a water-removing substance of Anxi Tieguanyin spring tea comprises the following steps:
(1) adding 3-5 times (such as 3, 4 or 5 times) of water into the water-removed extract of Anxi Tieguanyin spring tea, performing steam distillation, collecting hydrolat, and stopping collecting when the hydrolat weight is 30-40% of water weight;
(2) detecting the hydrolat obtained in the step (1) by adopting GC-MS to obtain the following 10 components in the hydrolat: geraniol, benzaldehyde, linalool oxide (furan type), linalool, phenethyl alcohol, geraniol, indole, jasmone, dihydroactinidiolide, and methyl jasmonate;
(3) comparing the contents of the 10 components measured in the step (2) with the corresponding contents:
0.300-0.500 mu g/g of leaf alcohol; 0.190-0.266 mug/g of benzaldehyde; linalool oxide (furan type) 1.500-1.850 μ g/g; linalool 3.000-3.900 μ g/g; 1.400-1.900 mu g/g of phenethyl alcohol; geraniol 1.200-1.800 μ g/g; 2.400-4.400 mu g/g of indole; jasmone 0.570-0.750 microgram/g; dihydroactinidiolide 0.022-0.027 μ g/g; 0.060-0.130 mu g/g of methyl jasmonate;
(4) and (3) judging that the water-removed substances of the Anxi Tieguanyin spring tea are deteriorated when at least 4 of the 10 ingredient contents measured in the step (2) are not in the corresponding ingredient content range in the step (4).
2. The method of claim 1, wherein the contents of the components in step (3) are as follows: 0.390-0.495 mu g/g of leaf alcohol; 0.190-0.266 mug/g of benzaldehyde; linalool oxide (furan type) 1.500-1.850 μ g/g; linalool 3.000-3.900 μ g/g; 1.400-1.900 mu g/g of phenethyl alcohol; geraniol 1.222-1.800 μ g/g; indole 2.430-4.400 μ g/g; jasmone 0.570-0.710 μ g/g; dihydroactinidiolide 0.022-0.027 μ g/g; 0.083-0.101 mu g/g methyl jasmonate.
3. The method of claim 1 or 2, wherein the GC-MS gas chromatography conditions in step (2) are selected from one or more of the following:
i) a chromatographic column: agilent DB-5MS (60 m.times.0.25 mm.times.0.25 μm);
ii) a carrier gas: he;
iii) column flow rate: 1 mL/min;
iv) injection port temperature: 280 ℃;
v) sample size: 1 mu L of the solution;
vi) sample injection mode: split-flow sample injection with a split-flow ratio of 5: 1;
v) temperature rising mode: temperature programming, keeping at 50 ℃ for 1min, heating to 300 ℃ at 3 ℃/min, and keeping for 6 min.
4. The method of any one of claims 1-3, wherein the mass spectrometric conditions in step (2) are selected from one or more of the following:
i) MS transmission line temperature: 280 ℃;
ii) EI ion source temperature: 280 ℃;
iii) quadrupole temperature: 150 ℃;
iv) EI ionization energy: 70 eV;
v) scan mode: full scan + selective ion scan.
5. The method of any one of claims 1-4, wherein the Anxi Tieguanyin is Red Anxi Tieguanyin.
6. The method of any one of claims 1-5, wherein said Anxi Tieguanyin habitat is Anxi Huacun.
7. The method according to any one of claims 1 to 6, wherein the deterioration of the enzyme-deactivated substance of Tie-Guanyin spring tea is judged when 4, 5, 6, 7, 8, 9 or 10 of the contents of 10 ingredients measured in step (2) are out of the contents of the corresponding ingredients in step (3).
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