Determination of theabrownin content in Liubao tea

Abstract

The invention discloses a method for determining theabrownin content in Liubao tea, and belongs to the technical field of tea component determination. The method can accurately determine theabrownin content in Liubao tea, and the method has good repeatability, recovery rate, linear relationship and sensitivity, and has high specificity for determining theabrownin in Liubao tea. The method comprises the following preparation steps: (1) taking a plurality of first-grade Liubao teas and mixing them to prepare a theabrownin reference substance, and calibrating the purity of theabrownin, and using the calibrated theabrownin standard substance to prepare a theabrownin concentration-absorbance curve of a standard Liubao tea sample; (2) taking a Liubao tea sample to be determined, soaking and extracting it, adding ethanol, mixing and standing it, and obtaining a precipitate of the sample to be determined after centrifugation; (3) dissolving and diluting the precipitate of the sample to be determined, and putting it into an ultraviolet spectrophotometer to determine the absorbance of the sample to be determined; (4) using the theabrownin concentration-absorbance curve of the standard Liubao tea sample to calculate the theabrownin concentration corresponding to the absorbance of the sample to be determined, and then calculating the theabrownin content.

Description

Technical Field

[0001] The invention relates to the technical field of tea component determination, and more specifically, to a method for determining the content of theabrownin in Liubao tea. Background Art

[0002] Liubao tea refers to a dark tea variety with a unique betel nut aroma, made from fresh leaves of large-leaf tea trees in Liubao Town, Wuzhou, Guangxi, China, through five processes of withering, rolling, piling, re-rolling, and drying. It is a post-fermented tea. Liubao tea is dark brown in color, with a bright red soup, a mellow and smooth taste, and a pure aroma. The older it is, the more fragrant it is. The storage years have a great influence on the aroma, flavor, and taste of Liubao tea, and the storage years have become an important factor affecting the price of Liubao tea in the market.

[0003] Different storage years will affect the quality of Liubao tea, as well as tea polyphenols, amino acids, theaflavins, thearubigins, theabrownins and other ingredients in the tea. The theabrownin content in Liubao tea with different aging times ranges from 9.49% to 16.29%. With the increase of aging time, the theabrownin content in Liubao gradually increases, and the increase is obvious.

[0004] It can be seen that theabrownin content is one of the important criteria for evaluating the quality of Liubao tea. In the national standard, only the spectrophotometric method for the determination of theabrownin content in black tea in NY / T3675-2020 is used to determine theabrownin in black tea. However, the matrix of black tea is quite different from that of Liubao tea. If the determination method of the national standard NY / T 3675-2020 is used to determine theabrownin content in Liubao tea, the Roberts empirical coefficient in the standard is not applicable to the determination of theabrownin in Liubao tea. The result obtained is too different from the actual situation, and the theabrownin content in Liubao tea cannot be accurately known, which in turn affects the evaluation of tea quality. Therefore, it is urgent to invent a method that can effectively determine theabrownin content in Liubao tea so as to more accurately evaluate the quality of Liubao tea. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the theabrownin content in Liubao tea. The method can accurately determine the theabrownin content in Liubao tea. The method has good repeatability, recovery rate, linear relationship and sensitivity, and has high specificity for determining the theabrownin in Liubao tea.

[0006] The technical solution of the present invention is as follows:

[0007] The method for determining theabrownin content in Liubao tea comprises the following preparation steps:

[0008] (1) Mixing a plurality of different batches of Grade 1 Liubao tea to prepare a theabrownin reference substance, calibrating the purity of theabrownin, and using the calibrated theabrownin standard substance to prepare a theabrownin concentration-absorbance curve of a standard Liubao tea sample;

[0009] (2) soaking, extracting, filtering, adding anhydrous ethanol, mixing and standing, and centrifuging to obtain a precipitate of the test product;

[0010] (3) dissolving and diluting the precipitate of the test product obtained in step (2) with water, and placing it in an ultraviolet spectrophotometer to measure the absorbance of the test product;

[0011] (4) Using the theabrownin concentration-absorbance curve of the standard Liubao tea sample, the theabrownin concentration corresponding to the absorbance of the test sample obtained in step (3) is calculated, and then the theabrownin content is calculated based on the volume of the diluted solution.

[0012] Furthermore, in the step (2), the Liubao tea sample to be tested is soaked in boiling water, with 2 g of the Liubao tea sample to be tested added to every 100 mL of boiling water. After soaking, the sample is sealed and extracted in a boiling water bath for 40 minutes.

[0013] Furthermore, in the step (2), the added anhydrous ethanol reagent accounts for 90% of the total amount, and after addition, the mixture is placed in a refrigerator at 4°C and allowed to stand for 3 hours.

[0014] Furthermore, in the step (2), the static solution is centrifuged at 4°C for 15 minutes at a centrifugal speed of 9000 r / min.

[0015] Furthermore, in the step (3), when the dissolved and diluted solution is placed in an ultraviolet spectrophotometer for measurement, the measurement wavelength is 280±2nm and the colorimetric thickness is 1cm.

[0016] Furthermore, in the step (3), if the absorbance of the sample to be tested is not in the range of 0.1 to 0.7, the dissolved and diluted solution is further diluted and then placed in an ultraviolet spectrophotometer to measure the absorbance of the sample to be tested until the absorbance of the sample to be tested is in the range of 0.1 to 0.7.

[0017] Furthermore, the step (1) comprises the following steps:

[0018] (1.1) taking equal portions of first-grade Liubao tea from multiple different batches and mixing them to obtain a standard Liubao tea sample;

[0019] (1.2) Soaking the standard Liubao tea sample obtained in step (1.1) in boiling water, extracting it in a water bath, filtering and concentrating it to obtain a standard concentrate;

[0020] (1.3) adding anhydrous ethanol to the standard concentrate obtained in step (1.2), mixing and allowing to stand, and centrifuging to obtain a standard precipitate;

[0021] (1.4) adding ethyl acetate reagent to the standard precipitate obtained in step (1.3), mixing and standing, centrifuging and drying to obtain crude theabrownin;

[0022] (1.5) dissolving the crude theabrownin obtained in step (1.4) filtering and freeze-drying to obtain the refined theabrownin;

[0023] (1.6) The theabrownin concentration-absorbance curve method is used to calculate the theabrownin content in the Liubao tea sample using the theabrownin concentrate obtained in step (1.5).

[0024] Furthermore, in the step (1.6), before determining the absorbance of the theabrownin concentrate at different concentrations, it is also necessary to determine the residual amounts of ethanol and ethyl acetate therein using headspace-gas chromatography-mass spectrometry to determine the actual content of theabrownin in the theabrownin concentrate.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The method for determining the content of theabrownin in Liubao tea of ​​the present invention comprises the following steps: taking a plurality of different batches of grade 1 Liubao tea and mixing them to prepare a theabrownin reference substance, and calibrating the purity of theabrownin; using the calibrated theabrownin standard substance to prepare a theabrownin concentration-absorbance curve of a standard Liubao tea sample; taking a Liubao tea sample to be determined, soaking, extracting and filtering, adding anhydrous ethanol, mixing and standing, and obtaining a precipitate of the product to be determined after centrifugation; dissolving and diluting the precipitate of the product to be determined with water, and placing it in an ultraviolet spectrophotometer to determine the absorbance of the product to be determined; using the theabrownin concentration-absorbance curve of the standard Liubao tea sample to obtain the theabrownin concentration corresponding to the absorbance of the product to be determined, and then obtaining the theabrownin content according to the volume of the diluted solution. The determination method of the present invention can accurately determine the content of theabrownin in Liubao tea, and the repeatability, recovery rate, linear relationship and sensitivity of the method are all good, and the method has high specificity for determining theabrownin in Liubao tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 This is the theabrownin concentration-absorbance curve of the standard Liubao tea sample. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but does not constitute any limitation to the present invention.

[0030] The method for determining theabrownin content in Liubao tea of ​​the present invention comprises the following preparation steps:

[0031] (1) A plurality of different batches of Grade 1 Liubao tea were mixed to prepare a theabrownin reference substance, and the purity of theabrownin was calibrated. The theabrownin concentration-absorbance curve of the standard Liubao tea sample was prepared using the calibrated theabrownin standard substance.

[0032] The following steps are involved:

[0033] (1.1) Take 1 equal portion of each of multiple batches of Grade 1 Liubao tea and mix them to obtain standard Liubao tea samples. The selected Liubao tea is multiple batches of Grade 1 Liubao tea that have been evaluated by tea tasters. 150 g of each batch of sample Liubao tea is weighed on an electronic analytical balance and mixed evenly.

[0034] (1.2) Soak the standard Liubao tea sample obtained in step (1.1) in boiling water and extract it in a water bath, filter and concentrate it to obtain a standard concentrate.

[0035] The ratio of the added standard Liubao tea sample to boiling water was 1:10, and the extraction was sealed in a boiling water bath for 40 minutes, and the extraction times were 2 times. After the extraction was completed, the tea soup was filtered with gauze while hot (the filter receiving container was placed in an ice bath), and the collected tea soup was concentrated using a balanced concentrate.

[0036] (1.3) Add anhydrous ethanol to the standard concentrate obtained in step (1.2), mix and stand, and centrifuge to obtain a standard precipitate. The amount of anhydrous ethanol added accounts for 90% of the total solution. After mixing evenly, place in a 4°C refrigerator and stand for 3 hours. Centrifuge the solution at 4°C for 15 minutes at a centrifugal speed of 9000 r / min.

[0037] (1.4) Add ethyl acetate reagent to the standard precipitate obtained in step (1.3), mix and stand, centrifuge and dry to obtain the crude theabrownin. The amount of ethyl acetate added accounts for 90% of the total solution. After mixing evenly, place in a 4°C refrigerator and stand for 3 hours. Centrifuge the standing solution at 4°C for 15 minutes at a centrifugal speed of 9000r / min. After the centrifugation, the precipitate obtained is dried in an oven at 65°C±2°C for 1.5 hours to obtain the crude theabrownin.

[0038] (1.5) The crude theabrownin obtained in step (1.4) is dissolved, filtered, and freeze-dried to obtain the refined theabrownin.

[0039] Among them, 5 g of the crude theabrownin is added to every 100 mL of ultrapure water, and after dissolution, it is filtered through a filter membrane (>50000Da filter membrane), and then freeze-dried to obtain the fine theabrownin.

[0040] (1.6) The theabrownin concentration-absorbance curve method is used to calculate the theabrownin content in the Liubao tea sample using the theabrownin concentrate obtained in step (1.5).

[0041] Before measuring the absorbance of theabrownin products at different concentrations, it is also necessary to use headspace-gas chromatography-mass spectrometry to measure the residual amounts of ethanol and ethyl acetate to find out the actual content of theabrownin in theabrownin products. The actual content of theabrownin in theabrownin products is: 1-(ethanol%+ethyl acetate%). The obtained theabrownin products need to be sealed and stored in a refrigerator at 2-8°C away from light.

[0042] The detection method of ethanol and ethyl acetate residual solvents in theabrownin products is:

[0043] Preparation of standard theabrownin assay solution: Accurately weigh 0.50g of theabrownin concentrate into a 5mL volumetric flask, dissolve it in water and dilute to the mark, and pipette 3.00mL into a 20mL headspace bottle for assay.

[0044] Chromatographic conditions: Column: DB-WAX capillary column (30m×0.25mm×0.25μm)

[0045] Inlet temperature: 250 °C, split ratio: 20:1;

[0046] Column temperature: initial temperature 40°C, hold time 5 min, increase to 200°C at 20°C / min, hold time 3 min, increase to 240°C at 60°C / min, hold time 1 min; column flow rate (He): 1.2 mL / min;

[0047] Ion source temperature: 230°C, electron energy: 70eV

[0048] Monitoring mode: selected ion monitoring (SIM), ethanol monitoring ions: 30, 31, 45, 46, ethyl acetate monitoring ions: 43, 45, 61, 70, 88.

[0049] Headspace sampler: heating box temperature 90 °C, quantitative loop temperature 100 °C, transfer line temperature 120 °C, sample bottle equilibration 15 min, injection duration 0.5 min.

[0050] (2) Take the Liubao tea sample to be tested, soak, extract and filter, add anhydrous ethanol, mix and let stand, and obtain the precipitate of the test product after centrifugation.

[0051] The Liubao tea sample to be tested was immersed in boiling water, and the weight of the Liubao tea sample to be tested was 2 g per 100 mL of boiling water. After immersion, the sample was sealed and extracted in a boiling water bath for 40 min. The added anhydrous ethanol reagent accounted for 90% of the total amount, and was placed in a 4°C refrigerator for 3 hours after addition. The solution that had been allowed to stand was centrifuged at 4°C for 15 min at a centrifugal speed of 9000 r / min.

[0052] (3) The precipitate of the test product obtained in step (2) is dissolved and diluted with water, and placed in an ultraviolet spectrophotometer to measure the absorbance of the test product.

[0053] Among them, the precipitate of the test product is dissolved with water and transferred to a 100mL volumetric flask, and the volume is adjusted to the scale. Accurately pipette 0.50mL of the above dilution into a 50mL volumetric flask, and the volume is adjusted to the scale with water. Shake well, and the diluted test solution is placed in an ultraviolet spectrophotometer for measurement. The measured wavelength is 280±2nm, the colorimetric thickness is 1cm, and ultrapure water is used as the blank for zero adjustment.

[0054] If the measured absorbance of the sample is not within the range of 0.1 to 0.7, the diluted solution is further diluted and then placed in an ultraviolet spectrophotometer to measure the absorbance of the sample until the measured absorbance of the sample is within the range of 0.1 to 0.7.

[0055] (4) Using the theabrownin concentration-absorbance curve of the standard Liubao tea sample, the theabrownin concentration corresponding to the absorbance of the test sample obtained in step (3) is calculated, and the theabrownin content is calculated by substituting the volume of the diluted solution into the theabrownin concentration.

[0056] The determination method of the present invention uses rated Liubao tea to prepare the reference substance of theabrownin, uses ethanol and ethyl acetate to further purify theabrownin, and uses headspace-gas chromatography-mass spectrometry to determine the residual solvent in the purification process. The headspace-gas chromatography-mass spectrometry can achieve high sensitivity for the detection of residual solvents than gas chromatography, and the detection ability for trace residues is higher than that of gas phase, and the mass spectrum anti-interference ability is stronger than that of gas phase. The qualitative analysis using mass-to-charge ratio can exclude the factors that the detection target cannot be detected due to the interference of chromatographic peaks in gas phase method. The headspace-gas chromatography-mass spectrometry uses a headspace sampler for sample injection. When the temperature of the heating box is set to be only above the boiling point of the residue, it can ensure that the remaining substances above the boiling point are not volatilized and do not enter the detection system, thereby reducing pollution. The gas chromatography determination method will also vaporize the remaining substances except the target at the same time, enter the system, cause system pollution, such as the chromatographic peaks cannot be further separated, thereby causing the measurement result to be wrong.

[0057] The purity of theabrownin is calibrated by detecting the amount of residual solvent, and the theabrownin after the calibration purity is used as a reference substance for determining theabrownin in Liubao tea to obtain the theabrownin concentration-absorbance curve of the standard Liubao tea sample. After obtaining the standard Liubao tea sample absorbance-theabrownin content curve table, the curve table is used to detect theabrownin in Liubao tea. The source of the reference substance on the matrix is ​​Liubao tea. Using the reference substance with the same matrix source for detection can further eliminate the factors that affect the measurement results due to different matrices. Therefore, the determination method of the present invention has good repeatability, recovery rate, linear relationship, and sensitivity, and has high specificity for determining theabrownin in Liubao tea.

[0058] Experimental example

[0059] 1. Obtain the theabrownin concentration-absorbance curve of the standard Liubao tea sample

[0060] (1) Take 30 different batches of Grade 1 Liubao tea that have been evaluated by tea tasters, weigh 150 g of each batch of Liubao tea on an electronic analytical balance, and mix these 30 batches of Liubao tea to obtain a standard Liubao tea sample.

[0061] (2) Soak the standard Liubao tea sample in boiling water at a ratio of 1:10, and extract in a sealed boiling water bath for 40 minutes, twice. After the extraction is completed, filter the tea soup with gauze while it is hot (the filter receiving container is placed in an ice bath), and concentrate the collected tea soup with a balanced concentrate to obtain a standard concentrate.

[0062] (3) Add anhydrous ethanol to the standard concentrate and let the mixture stand. The amount of anhydrous ethanol added accounts for 90% of the total solution. After mixing evenly, place it in a 4°C refrigerator and let it stand for 3 hours. Centrifuge the solution at 4°C for 15 minutes at a centrifugal speed of 9000 r / min to obtain a standard precipitate.

[0063] (4) Add ethyl acetate reagent to the standard precipitate and let the mixture stand, with the amount of ethyl acetate added accounting for 90% of the total solution. After mixing evenly, place the mixture in a 4°C refrigerator and let it stand for 3 hours. Centrifuge the solution at 4°C for 15 minutes at a centrifugal speed of 9000 r / min. After the centrifugation, bake the precipitate at 65°C±2°C in an oven for 1.5 hours to obtain the crude theabrownin.

[0064] (5) Dissolve and filter the crude theabrownin. Add 5 g of the crude theabrownin to every 100 mL of ultrapure water. After dissolution, filter through a filter membrane (>50,000 Da filter membrane), and then freeze-dry to obtain the refined theabrownin.

[0065] (6) The residual amounts of ethanol and ethyl acetate in the theabrownin concentrate were determined by headspace-gas chromatography-mass spectrometry to determine the actual content of theabrownin in the theabrownin concentrate. The actual content of theabrownin in the theabrownin concentrate was: 1-(ethanol%+ethyl acetate%).

[0066] The detection method of ethanol and ethyl acetate residual solvents in theabrownin products is:

[0067] Preparation of standard theabrownin assay solution: Accurately weigh 0.50g of theabrownin concentrate into a 5mL volumetric flask, dissolve it in water and dilute to the mark, and pipette 3.00mL into a 20mL headspace bottle for assay.

[0068] Chromatographic conditions: Column: DB-WAX capillary column (30m×0.25mm×0.25μm)

[0069] Inlet temperature: 250 °C, split ratio: 20:1;

[0070] Column temperature: initial temperature 40°C, hold time 5 min, increase to 200°C at 20°C / min, hold time 3 min, increase to 240°C at 60°C / min, hold time 1 min; column flow rate (He): 1.2 mL / min;

[0071] Ion source temperature: 230°C, electron energy: 70eV

[0072] Monitoring mode: selected ion monitoring (SIM), ethanol monitoring ions: 30, 31, 45, 46, ethyl acetate monitoring ions: 43, 45, 61, 70, 88.

[0073] Headspace sampler: heating box temperature 90 °C, quantitative loop temperature 100 °C, transfer line temperature 120 °C, sample bottle equilibration 15 min, injection duration 0.5 min.

[0074] The absorbance of theabrownin of different concentrations was measured to obtain theabrownin concentration-absorbance curve of standard Liubao tea sample, such as Figure 1 shown.

[0075] 2. Determination of theabrownin content in Liubao tea

[0076] Ten groups of Liubao tea leaves from different batches were taken and designated as Liubao tea 1-10 respectively. The theabrownin content in each group of Liubao tea was determined by the determination method of the present invention, the weighing method, the ultraviolet method in NY / T 3675-2020, and the indirect ultraviolet method.

[0077] The assay method of the present invention:

[0078] (1) Liubao tea 1 to 10 are respectively used as Liubao tea samples 1 to 10 to be tested, and each group of Liubao tea samples to be tested is subjected to the following operations: each group of Liubao tea samples to be tested is immersed in boiling water, and 2 g of the Liubao tea sample to be tested is added to each 100 mL of boiling water. After immersion, the samples are sealed and extracted in a boiling water bath for 40 min. The added anhydrous ethanol reagent accounts for 90% of the total amount, and after addition, the samples are placed in a 4°C refrigerator and allowed to stand for 3 hours. The solution that has been allowed to stand is centrifuged at 4°C for 15 min, and the centrifugal speed is 9000 r / min. After centrifugation, 10 groups of sediments of the tested products are obtained.

[0079] (2) The 10 groups of test sample precipitates obtained were subjected to the following operations: the test sample precipitates were dissolved with water and transferred to a 100 mL volumetric flask, and the volume was adjusted to the mark. 0.50 mL of the above dilution was accurately pipetted into a 50 mL volumetric flask, and the volume was adjusted to the mark with water. The diluted test solution was placed in an ultraviolet spectrophotometer for measurement at a wavelength of 280 ± 2 nm and a colorimetric thickness of 1 cm. Ultrapure water was used as a blank for zero adjustment, and the absorbance of the 10 groups of test samples was measured.

[0080] When measuring the absorbance of the sample to be tested, if the absorbance is not in the range of 0.1 to 0.7, the dissolved and diluted solution is further diluted and then placed in an ultraviolet spectrophotometer to measure the absorbance of the sample to be tested until the measured absorbance of the sample to be tested is in the range of 0.1 to 0.7.

[0081] (3) The theabrownin concentration-absorbance curve of the standard Liubao tea sample was used to determine the theabrownin concentrations corresponding to the absorbances of the 10 groups of test samples, and the theabrownin contents of the 10 groups of test samples were calculated, as shown in Table 1.

[0082] Table 110 Absorbance, theabrownin concentration and theabrownin content of Liubao tea

[0083] name Measured absorbance Concentration / (ug / mL) Sampling volume / g Theabrownin percentage Liubao Tea1 0.2698 48.7626 5.0121 9.7％ Liubao Tea 2 0.2787 50.5533 5.0211 10.1％ Liubao Tea 3 0.2946 53.7525 5.0312 10.7％ Liubao Tea 4 0.3014 55.1207 5.0144 11.0％ Liubao Tea 5 0.2945 53.7324 5.0333 10.7％ Liubao Tea 6 0.2844 51.7002 5.0345 10.3％ Liubao Tea 7 0.3544 65.7847 5.0122 13.1％ Liubao Tea 8 0.3824 71.4185 5.0344 14.2％ Liubao Tea 9 0.3788 70.6942 5.0612 14.0％ Liubao Tea 10 0.3648 67.8773 5.0944 13.3％

[0084] The theabrownin content in each group of Liubao tea was determined by weighing method, UV method in NY / T 3675-2020, and indirect UV method, as shown in Table 2.

[0085] Table 2 The content of theabrownin in 10 groups of Liubao tea determined by different methods

[0086]

[0087] According to Table 1 and Table 2, the theabrownin content of 10 groups of Liubao tea samples to be measured by weighing method is 9.7% to 13.9%, the theabrownin content of 10 groups of Liubao tea samples to be measured by ultraviolet method in NY / T 3675-2020 is 20.1% to 25.5%, the theabrownin content of 10 groups of Liubao tea samples to be measured by indirect ultraviolet method is 29.2% to 40.1%, and the theabrownin content of 10 groups of Liubao tea samples to be measured by the determination method of the present invention is 9.7% to 14.2%. The theabrownin content measured by the weighing method and the determination method of the present invention is close, the indirect ultraviolet method determination result is quite different from the weighing method determination result, and the NY / T 3675-2020 and the weighing method determination result are quite different. The reason is that the Roberts empirical coefficient in the NY / T 3675-2020 standard is not applicable to the determination of theabrownin in Liubao tea, and the matrix of Liubao tea and black tea is very different. In the indirect UV method, there is no theabrownin reference substance as a benchmark, so the error is large.

[0088] The determination method of the present invention determines theabrownin in Liubao tea samples by using the prepared theabrownin reference substance. Six samples are taken from each of the 10 groups of Liubao tea samples to be determined. The theabrownin content of each sample is determined using the determination method of the present invention, and the repeatability of the determination of these samples is calculated. Then, the spiked test and the quantitative limit test are performed on the Liubao tea samples to be determined. The determination results are shown in Tables 3 to 5, respectively.

[0089] Table 3 Theabrownin content of 10 groups of Liubao tea samples to be determined

[0090]

[0091] Table 4 The theabrownin spiked test results of Liubao tea samples to be determined

[0092]

[0093] Table 5 The quantitative limit test results of theabrownin in Liubao tea samples to be determined

[0094]

[0095] According to Tables 3 to 5, the theabrownin content of 6 samples taken out of 10 groups of Liubao tea samples to be determined is 9.7% to 14.2%, and the repeatability RSD of the determination of 6 samples in each group of Liubao tea samples to be determined is 2.3% to 4.2%. The recovery rate of the spiked test of the Liubao tea samples to be determined using theabrownin reference substance is 92.6% to 106.2% when the spiked amount ranges from 0.5% to 15%. The quantitative limit of theabrownin is 0.5% when the blank solution is added with theabrownin reference substance, and the linear correlation coefficient is 0.997. It can be seen that the determination method of the present invention has good repeatability, recovery rate, linear relationship and sensitivity, and has high specificity for determining theabrownin in Liubao tea.

[0096] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining theabrownin content in Liubao tea, characterized in that: The method comprises the following preparation steps: (1) Mixing a plurality of different batches of Grade 1 Liubao tea to prepare a theabrownin reference substance, calibrating the purity of theabrownin, and using the calibrated theabrownin standard substance to prepare a theabrownin concentration-absorbance curve of a standard Liubao tea sample; (2) soaking, extracting, filtering, adding anhydrous ethanol, mixing and standing, and centrifuging to obtain a precipitate of the test product; (3) dissolving and diluting the precipitate of the test product obtained in step (2) with water, and placing it in an ultraviolet spectrophotometer to measure the absorbance of the test product; (4) using the theabrownin concentration-absorbance curve of the standard Liubao tea sample to determine the theabrownin concentration corresponding to the absorbance of the test sample obtained in step (3), and then determining the theabrownin content based on the volume of the diluted solution; The step (1) comprises the following steps: (1.1) taking equal portions of first-grade Liubao tea from multiple different batches and mixing them to obtain a standard Liubao tea sample; (1.2) Soaking the standard Liubao tea sample obtained in step (1.1) in boiling water, extracting it in a water bath, filtering and concentrating it to obtain a standard concentrate; (1.3) adding anhydrous ethanol to the standard concentrate obtained in step (1.2), mixing and allowing to stand, and centrifuging to obtain a standard precipitate; (1.4) adding ethyl acetate reagent to the standard precipitate obtained in step (1.3), mixing and standing, centrifuging and drying to obtain crude theabrownin; (1.5) dissolving the crude theabrownin obtained in step (1.4) filtering and freeze-drying to obtain the refined theabrownin; (1.6) Using headspace-gas chromatography-mass spectrometry to measure the residual amounts of ethanol and ethyl acetate, the actual content of theabrownin in the theabrownin premium product was obtained, and the actual content of theabrownin in the theabrownin premium product was calculated. The theabrownin concentration-absorbance curve of the standard Liubao tea sample was prepared using the actual content of theabrownin in the theabrownin premium product; In the step (2), the added anhydrous ethanol reagent accounts for 90% of the total amount, and after addition, it is placed in a 4°C refrigerator and left to stand for 3 hours.

2. The method for determining theabrownin content in Liubao tea according to claim 1, characterized in that: In the step (2), the Liubao tea sample to be tested is soaked in boiling water, with 2 g of the Liubao tea sample to be tested added to every 100 mL of boiling water. After soaking, the sample is sealed and extracted in a boiling water bath for 40 minutes.

3. The method for determining theabrownin content in Liubao tea according to claim 1, characterized in that: In the step (2), the static solution is centrifuged at 4°C for 15 minutes at a centrifugal speed of 9000 r / min.

4. The method for determining theabrownin content in Liubao tea according to claim 1, characterized in that: In the step (3), when the dissolved and diluted solution is placed in an ultraviolet spectrophotometer for measurement, the measurement wavelength is 280±2nm and the thickness of the cuvette is 1cm.

5. The method for determining theabrownin content in Liubao tea according to claim 4, characterized in that: In the step (3), if the absorbance of the sample to be tested is not in the range of 0.1 to 0.7, the diluted solution is further diluted and then placed in an ultraviolet spectrophotometer to measure the absorbance of the sample to be tested until the absorbance of the sample to be tested is in the range of 0.1 to 0.7.

Images