A marker compound for identifying the origin of black tea, a screening method and a method for identifying the origin of black tea

Abstract

The application discloses a kind of marker compounds and screening method for distinguishing black tea origin and a kind of black tea origin identification method, belong to tea distinguishing technical field, the application first filters out kaempferol 3-O-(galloyl)-glucoside as the marker for distinguishing black tea origin as marker.It is stable to exist and higher in content in Sri Lanka black tea, while in the representative black tea in China, the content is significantly lower or difficult to detect, and the difference has stability and significance.The identification method of the application comprises: preparing standard extract of the black tea sample to be tested, and using high performance liquid chromatography-mass spectrometry to qualitatively and quantitatively analyze the extract.The scientific and accurate discrimination of the geographical origin of black tea can be realized by detecting the presence or absence of the marker compound or the amount of the marker compound.

Description

Technical Field

[0001] This invention belongs to the field of tea identification technology, specifically relating to a marker compound and identification method for identifying the origin of black tea. Background Technology

[0002] Black tea, as one of the most consumed tea categories globally, is beloved by consumers for its unique flavor and rich nutritional benefits. Both Chinese and foreign (such as Indian, Sri Lankan, and Kenyan) black teas hold significant positions in the market, but significant differences exist in their quality characteristics and market value—differences stemming from a combination of factors including tea variety, cultivation environment, and processing techniques. In terms of variety, Chinese black tea primarily uses small-leaf varieties with relatively small leaves, while foreign black teas often use large-leaf varieties, resulting in even larger leaves. Regarding cultivation environment, China's tea-producing regions are vast with significant altitude variations, greatly influencing the quality of black tea. Foreign tea-growing regions have significantly different cultivation environments and harvesting methods, heavily influenced by local tea culture, leading to differences in tea quality. In terms of processing techniques, Chinese black tea inherits traditional techniques, combining large-scale production with handcrafted refinement, primarily using the Gongfu black tea processing method. Foreign black tea, influenced by the plantation system of the colonial era, emphasizes large-scale, standardized production to meet mass market demands, primarily using the CTC (Crush, Tear, Burn) processing method.

[0003] This difference in quality and value makes the accurate identification of Chinese and foreign black teas of significant practical importance in tea trade, quality control, and consumer rights protection. In actual market circulation, unscrupulous merchants sometimes pass off low-value foreign black teas as high-value premium Chinese black teas (such as Qimen black tea and Dianhong black tea), seriously disrupting market order and harming the legitimate rights and interests of consumers and legitimate businesses. Therefore, establishing scientific, accurate, and efficient methods for identifying Chinese and foreign black teas has become a pressing technical issue that the tea industry needs to address.

[0004] Currently, domestic and international identification techniques for black tea are mainly divided into two categories: traditional sensory evaluation and modern instrumental analysis. Traditional sensory evaluation relies on professional tea tasters to evaluate the appearance, liquor color, aroma, taste, and infused leaves of tea using sight, smell, taste, and touch. This method is simple to operate and low in cost, but it is highly subjective—the identification results are greatly affected by the tea taster's experience, physiological state, and environmental factors. Repeatability and accuracy are difficult to guarantee, and it cannot meet the needs of rapid screening of large batches of samples. Furthermore, it is difficult to accurately distinguish subtle differences between Chinese and foreign black teas.

[0005] To overcome the shortcomings of traditional methods, modern instrumental analysis techniques have been gradually applied to the identification of black tea, mainly including gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), hyperspectral imaging, and electronic noses. Each of these techniques has its limitations. The core issue is that current technologies have not yet clearly identified specific markers for the identification of Chinese and foreign black teas, nor have they established a systematic identification scheme based on these markers. Studies have shown that Chinese and foreign black teas differ significantly in key quality components. These distinguishing components contain specific markers that can be used for precise identification, but current technologies have failed to systematically screen, verify, and confirm these potential markers, making it impossible to achieve efficient differentiation between Chinese and foreign black teas through these markers.

[0006] Therefore, screening and identifying specific markers that can distinguish between Chinese and foreign black tea, and developing a marker-based method for identifying Chinese and foreign black tea that is easy to operate, cost-effective, and has a high accuracy rate, is of great significance for standardizing the tea market and ensuring the healthy development of the industry. It has also become a research hotspot and core need for those skilled in the art. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a marker compound for identifying the origin of black tea, as well as an objective, quantifiable, and convenient method for doing so. This application, for the first time, uses the content of kaempferol 3-O-(galloyl)-glucoside as a core indicator. This compound is stably present and present in high amounts in major foreign black teas such as those from Sri Lanka, while its content is significantly low or undetectable in representative Chinese black teas (such as Qimen black tea and Zhengshan Xiaozhong). This difference is stable and significant, making it an ideal marker for identifying the origin of black tea.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A marker compound for identifying the origin of black tea, wherein the marker is kaempferol-3-O-(galloyl)-glucoside, with the following structural formula: .

[0009] As mentioned above, if the content of kaempferol-3-O-(galloyl)-glucoside in tea is <12 mg / g, then the tea is determined to be produced in China; if the content of kaempferol-3-O-(galloyl)-glucoside is >27 mg / g, then the tea is determined to be produced in Sri Lanka.

[0010] This invention provides a method for screening marker compounds to determine the origin of black tea, wherein the marker is kaempferol-3-O-(galloyl)-glucoside, and specifically includes the following steps: Step A: Multi-stage extraction of black tea samples: First, the black tea is subjected to ultrasonic extraction and drying. The dried matter is then reconstituted with water. Then, multi-stage extraction is performed with dichloromethane and ethyl acetate. The extract is dried and redissolved in methanol aqueous solution to obtain the aqueous layer and ethyl acetate layer extracts, respectively. Step B: Perform UHPLC-DAD-MS analysis on the aqueous and ethyl acetate extracts, respectively; Step C: Use Abf Converter software to convert the mass spectrometry data, and use MS-DIAL software to deconvolve, denoise, and align the mass spectrometry data, outputting a DAD spectral dataset. Use Agilent Mass Hunter Workstation software to extract the full-scan spectra of the compounds in the 190-640 nm range. Correct the retention time in the DAD spectral dataset according to ∆T, and delete absorbance values ​​below 100 mAU in the dataset, so that the DAD absorption spectrum is divided into colorless and colored time periods. Use the readxl package in R studio to compare the variables in the aligned LC-MS dataset and the DAD dataset one by one, and filter and delete the LC-MS variables in the colorless time period. OPLS regression analysis was performed using SIMCA-P 18.0 software. In the model, filtered LC-MS data and DAD spectral data were used as X and Y variables, respectively, and substances with VIP values ​​greater than 2 were identified as key coloring substances in black tea. Step D: Identify the VIP substances based on retention time, molecular weight, and secondary mass spectrometry fragments. Among them, Kaempferol 3-O-(galloyl)-glucoside (m / z 599) was identified as the key chromogenic substance with a VIP value greater than 2.

[0011] As described above, in step A, a black tea sample is weighed and extracted with 70% methanol-water using ultrasonic extraction. After centrifugation, the supernatant is collected. The extraction step is repeated twice. The supernatant is collected and diluted to a final volume. The extract is then processed using a nitrogen blower and a freeze dryer to obtain dry matter. Subsequently, the extract is redissolved with pure water and then redissolved using a multi-stage extraction process with dichloromethane and ethyl acetate, with each extraction being repeated three times. The water, ethyl acetate, and dichloromethane layers are dried separately, and the precise weight of each component is recorded. Then, 70% methanol-water is added to redissolve the water and ethyl acetate layers, ensuring that the concentration of the redissolved extract is equivalent to 20 mg / mL of the original black tea.

[0012] As described above, in step B of the screening method, an Agilent 1290 UHPLC-Q-TOF-MS equipped with DAD was used for analysis, wherein the detection range of DAD is 190-640 nm; the chromatographic column is C20.18 The chromatographic column was 2.1 × 50 mm and 1.7 μm; the flow rate was 0.3 mL / min; the temperature was 40℃; the mobile phase A was 0.1% formic acid in water; and the mobile phase B was 0.1% formic acid in acetonitrile. The elution gradient for the ethyl acetate layer sample was as follows: 0 min, 2% mobile phase B; 0–5 min, 2%–15% mobile phase B; 5–20 min, 15%–16% mobile phase B; 20–22 min, 16%–18% mobile phase B; 22–27 min, 18%–20% mobile phase B; 27–29 min, 20%–28% mobile phase B; 29–33 min, 28% mobile phase B; 33–35 min, 28%–30% mobile phase B; 35–37 min, 30%–50% mobile phase B; 37–39 min, 50%–98% mobile phase B; 39–41 min, 98%–2% mobile phase B; 41–45 min, 2% mobile phase B. The injection volume was 2 μL. The elution gradient for the aqueous layer samples was as follows: 0-5 min, 2%-10% mobile phase B; 5-25 min, 10%-20% mobile phase B; 25-30 min, 20% mobile phase B; 30-35 min, 20%-50% mobile phase B; 35-37 min, 50%-98% mobile phase B; 37-39 min, 98% mobile phase B; 39-41 min, 98%-2% mobile phase B; 41-45 min, 2% mobile phase B. The injection volume was 5 μL, the acquisition range was m / z 100-1500, the scanning mode was negative ion, and the scanning rate was 2 spectra / sec. DAD conditions: peak width of 0.1 min (2.5 Hz), scan range of 190-640 nm, spectral bandwidth of 2 nm, and acquired signals at 278, 360, 380, 400, 420, 460, 520 and 620 nm.

[0013] This invention also provides a method for identifying the origin of black tea, using the aforementioned marker compound for identifying the origin of black tea, with the following specific steps: S1. Prepare the test solution and reference solution for the black tea sample to be tested; S2. The content of kaempferol-3-O-(galloyl)-glucoside in the test solution was determined by UHPLC-QQQ-MS using the external standard method; S3. Determine the type of black tea based on the content of kaempferol-3-O-(galloyl)-glucoside: If the content of kaempferol-3-O-(galloyl)-glucoside is ≤0.5mg / g, it is determined to be black tea produced in China; if the content of kaempferol-3-O-(galloyl)-glucoside is ≥5.0mg / g, it is determined to be black tea produced outside of China.

[0014] The method for determining the origin of black tea as described above, wherein the preparation method of the detection solution in step S1 is as follows: weigh a dry tea sample quantitatively, add 70% methanol-water (v / v) and vortex mix, extract ultrasonically at 40°C, centrifuge at room temperature and extract the supernatant, repeat the extraction step three times, combine the supernatants, and adjust the volume with pure water to make the sample concentration suitable.

[0015] As described above, in the method for determining the origin of black tea, the preparation method of the reference solution in step S1 is as follows: Kaempferol-3-O-(galloyl)-glucoside solution is prepared using 70% methanol-water solution at concentrations of 5, 10, 20, 50, 100, and 500 ng / mL, and a concentration-peak area standard curve is established.

[0016] The method for determining the origin of black tea as described above uses a sample concentration of 10 mg / mL.

[0017] As described above, the chromatographic conditions for high-performance liquid chromatography (HPLC) in step S2 are as follows: column temperature 40℃, flow rate 0.3 mL / min, injection volume 2 μL, gradient elution: mobile phase A is 0.1% formic acid water, mobile phase B is 0.1% formic acid acetonitrile, and the elution gradient is: 0 min, 2% mobile phase B; 0-5 min, 2%-15% mobile phase B; 5-15 min, 15%-21% mobile phase B; 15-17 min, 21%-50% mobile phase B; 17-19 min, 50%-98% mobile phase B; 19-21 min, 98% mobile phase B; 21-23 min, 98%-2% mobile phase B; 23-25 ​​min, 2% mobile phase B.

[0018] As described above, in the method for determining the origin of black tea, the mass spectrometry in step S2 uses the MRM mode, with the mother ion at m / z 599 and the daughter ion at m / z 447.

[0019] Compared to existing technologies, the identification technology for Chinese and foreign black tea based on specific biomarkers protected in this application has significant advantages. The identification method of this invention includes: preparing a standard extract of the black tea sample to be tested, and performing qualitative and quantitative analysis of the extract using high-performance liquid chromatography-mass spectrometry. By detecting the presence or absence of the biomarker compound, or comparing its concentration with a preset threshold, the geographical origin (China or foreign) of the black tea can be scientifically and accurately determined. This invention fills the gap in the existing technology for identifying Chinese and foreign black teas due to the lack of highly specific chemical biomarkers. This method has high sensitivity and good reproducibility, providing strong technical support for the origin certification, quality control, and market supervision of black tea products.

[0020] This application identifies a highly specific and reliable marker compound for determining the origin of black tea. Through systematic screening and verification, this application has identified a specific marker that can accurately distinguish between Chinese and foreign black teas. This marker originates from the essential differences in chemical composition caused by differences in varietal, terroir, and processing techniques between Chinese and foreign black teas, rather than non-specific differences caused by environmental interference or random errors. Using this as the basis for identification can fundamentally avoid the subjectivity of traditional sensory evaluation and the lack of specificity of existing instrumental analysis, significantly improving the accuracy and repeatability of the identification results, far superior to existing technologies.

[0021] The identification method described in this application is simple, efficient, and widely applicable. Based on identified specific biomarkers, this application eliminates the need for complex and expensive large-scale instruments, and simplifies the operational steps. Attached Figure Description

[0022] Figure 1 The colors of the aqueous and ethyl acetate solutions of different grades of Sri Lankan black tea (20 mg / mL) are shown. Figure 2 Scatter plots of PCA and OPLS models for extracts from the aqueous and ethyl acetate layers of different grades of Sri Lankan black tea; Figure 3 It is Kaempferol 3- O Baseline diagram of -(galloyl)-glucoside; Figure 4 It is Kaempferol 3- O Primary mass spectrum and molecular weight of (galloyl)-glucoside; Figure 5 It is Kaempferol 3- O Secondary mass spectrum of (galloyl)-glucoside; Figure 6 Kaempferol 3- is found in Sri Lankan and Chinese black tea. O Results of β-(galloyl)-glucoside content detection; Figure 7 Kaempferol 3- is a major black tea variety from tea-producing countries. O Results of galloyl-glucoside content detection. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Marker confirmation Sri Lankan black tea of ​​different grades (from different regions) was collected, and key coloring substances were analyzed using established chromomics methods. (1) Multi-stage extraction of black tea samples: Weigh 1 g of black tea sample and add 15 mL of 70% methanol-water (v / v). Extract by ultrasonication at 40℃ for 40 min, followed by centrifugation at 8000 rpm for 10 min. Collect the supernatant. Repeat the extraction process twice. Collect the supernatant and bring the volume to 25 mL, achieving a concentration of 40 mg / mL. The extract was dried using a nitrogen evaporator and a freeze dryer. The solution was then reconstituted with pure water and subjected to multi-stage extraction with dichloromethane and ethyl acetate, with each extraction repeated three times. The water, ethyl acetate, and dichloromethane extracts were then dried using a nitrogen evaporator and a freeze dryer, and the precise weight of each component was recorded. Different volumes of 70% methanol-water (v / v) were then added to redissolve the water and ethyl acetate extracts, ensuring the redissolved extract concentration was equivalent to 20 mg / mL of the original black tea.

[0025] (2) UHPLC-DAD-MS analysis: Analysis was performed using an Agilent 1290 UHPLC-Q-TOF-MS equipped with a DAD (Distillation Acid Detector), with a detection range of 190–640 nm. A C18 column (2.1 × 50 mm, 1.7 μm) was used. The flow rate was 0.3 mL / min, the temperature was 40 °C, and the mobile phase consisted of 0.1% formic acid in water (v / v) (phase A) and 0.1% formic acid in acetonitrile (v / v) (phase B).

[0026] The elution gradient for the ethyl acetate layer samples was as follows: 0 min, 2% B; 0–5 min, 2%–15% B; 5–20 min, 15%–16% B; 20–22 min, 16%–18% B; 22–27 min, 18%–20% B; 27–29 min, 20%–28% B; 29–33 min, 28% B; 33–35 min, 28%–30% B; 35–37 min, 30%–50% B; 37–39 min, 50%–98% B; 39–41 min, 98%–2% B; 41–45 min, 2% B. The injection volume was 2 μL.

[0027] The elution gradient for the aqueous samples was as follows: 0-5 min, 2%-10% B; 5-25 min, 10%-20% B; 25-30 min, 20% B; 30-35 min, 20%-50% B; 35-37 min, 50%-98% B; 37-39 min, 98% B; 39-41 min, 98%-2% B; 41-45 min, 2% B. The injection volume was 5 μL, the acquisition range was m / z 100-1500, and the scanning mode was negative ion. The scan rate was 2 spectra / sec.

[0028] DAD conditions: peak width of 0.1 min (2.5 Hz), scan range of 190-640 nm, spectral bandwidth of 2 nm, and acquired signals at 278, 360, 380, 400, 420, 460, 520 and 620 nm.

[0029] (3) Data analysis: After converting the mass spectrometry data using Abf Converter software, MS-DIAL (version 5.3) software was used to perform deconvolution, noise reduction, and alignment on the mass spectrometry data.

[0030] Full-scan spectra of compounds in the 190-640 nm range were extracted using Agilent Mass Hunter Workstation software (version 10.0). The DAD absorption spectra of all samples were segmented with a time step of approximately 0.007 min, aligned, and output as a DAD spectral dataset. Subsequently, retention times in the DAD spectral dataset were corrected according to ∆T, and absorbance values ​​below 100 mAU were removed to eliminate instrument noise and low-absorbance substances, thus dividing the DAD absorption spectra into colorless and colored time periods. Finally, the variables in the aligned LC-MS dataset and the DAD dataset were compared one by one using the readxl package (version 1.4.3) in R studio, and LC-MS variables in the colorless time periods were filtered out and removed.

[0031] OPLS regression analysis was performed using SIMCA-P software. In chromomics, filtered LC-MS and DAD spectral data were used as X and Y variables, respectively. Substances with VIP values ​​greater than 2 were identified as key chromogenic substances.

[0032] (4) Identification of key VIP substances According to the retention time ( Figure 3 ), molecular weight ( Figure 4 ) and secondary mass spectrometry fragments ( Figure 5 The VIP substances were identified. Kaempferol 3-O-(galloyl)-glucoside (m / z 599) was identified as the key chromogenic substance with a VIP value greater than 2.

[0033] Detection of Kaempferol-3-O-(Galloyl)-Glucoside Content in Black Tea from Different Countries and Origins Example 1

[0034] Fifteen different types of black tea from China and Sri Lanka (from different regions of China and Sri Lanka) were used for separate extraction: 100 mg of dried tea sample was weighed from each source, added to 4 mL of 70% methanol-water (v / v), and vortexed to mix. After ultrasonic extraction at 40°C, the sample was centrifuged at 8000 rpm for 10 min at room temperature, and the supernatant was extracted. This extraction step was repeated three times. The supernatants were then combined, and the volume was adjusted to 10 mL with pure water, resulting in a final sample concentration of 10 mg / mL.

[0035] Preparation of reference standards: Kaempferol-3-O-(galloyl)-glucoside solution was prepared using 70% methanol-water (v / v) at concentrations of 5, 10, 20, 50, 100, and 500 ng / mL, and a concentration-peak area standard curve was established.

[0036] Extracts from black tea samples from different sources were analyzed by UHPLC-QQQ-MS: The chromatographic column was a Waters UPLC C10. 18 The chromatographic column was 2.1 × 50 mm, 1.7 μm; the mobile phase was 0.1% formic acid water (v / v) (phase A) and 0.1% formic acid acetonitrile (phase B); the column temperature was 40 °C; the flow rate was 0.3 mL / min; and the injection volume was 2 μL.

[0037] The elution gradient is: 0 min, 98% mobile phase A, 2% mobile phase B; 0-5 min, 98%-85% mobile phase A, 2%-15% mobile phase B; 5-15 min, 85%-79% mobile phase A, 15%-21% mobile phase B; 15-17 min, 79%-50% mobile phase A, 21%-50% mobile phase B; 17-19 min, 50%-2% mobile phase A, 50%-98% mobile phase B; 19-21 min, 2% mobile phase A, 98% mobile phase B; 21-23 min, 2%-98% mobile phase A, 98%-2% mobile phase B; 23-25 ​​min, 98% mobile phase A, 2% mobile phase B.

[0038] The mass spectrometer used MRM mode, with the parent ion at m / z 599 and the daughter ion at m / z 447.

[0039] The content of Kaempferol 3-O-(galloyl)-glucoside was calculated based on the test results. The experimental results are as follows: Figure 6 As shown.

[0040] The average content of Kaempferol 3-O-(galloyl)-glucoside in Sri Lankan black tea was 44.15 mg / g, and the five lowest contents were 27.15, 27.46, 29.87, 35.66, and 39.41 mg / g. The average content of Kaempferol 3-O-(galloyl)-glucoside in black tea from China was 7.54 mg / g. The five highest values ​​were 11.22, 10.26, 8.70, 8.21, and 7.95 mg / g.

[0041] Based on the above results and Figure 6 As can be seen, the content of Kaempferol 3-O-(galloyl)-glucoside in Chinese black tea is significantly lower than that in Sri Lankan black tea. Therefore, if the content of Kaempferol 3-O-(galloyl)-glucoside in black tea is greater than 27 mg / g, it can be identified as Sri Lankan black tea; if the content of Kaempferol 3-O-(galloyl)-glucoside in black tea is less than 12 mg / g, it can be identified as Chinese black tea.

[0042] Example 2 Extracts were made from black teas from China, Sri Lanka, Kenya, India, and Türkiye, respectively. 100 mg of dried tea sample was weighed from each source, added to 4 mL of 70% methanol-water (v / v), and vortexed to mix. After ultrasonic extraction at 40°C, the sample was centrifuged at 8000 rpm for 10 min at room temperature, and the supernatant was extracted. This extraction step was repeated three times. The supernatants were then combined, and the volume was adjusted to 10 mL with pure water, resulting in a final sample concentration of 10 mg / mL.

[0043] Preparation of reference standards: Kaempferol-3-O-(galloyl)-glucoside solution was prepared using 70% methanol-water (v / v) at concentrations of 5, 10, 20, 50, 100, and 500 ng / mL, and a concentration-peak area standard curve was established.

[0044] Extracts from black tea samples from different sources were analyzed by UHPLC-QQQ-MS: The chromatographic column was a Waters UPLC C10. 18 The chromatographic column was 2.1 × 50 mm, 1.7 μm; the mobile phase was 0.1% formic acid water (v / v) (phase A) and 0.1% formic acid acetonitrile (phase B); the column temperature was 40 °C; the flow rate was 0.3 mL / min; and the injection volume was 2 μL.

[0045] The elution gradient is: 0 min, 98% mobile phase A, 2% mobile phase B; 0-5 min, 98%-85% mobile phase A, 2%-15% mobile phase B; 5-15 min, 85%-79% mobile phase A, 15%-21% mobile phase B; 15-17 min, 79%-50% mobile phase A, 21%-50% mobile phase B; 17-19 min, 50%-2% mobile phase A, 50%-98% mobile phase B; 19-21 min, 2% mobile phase A, 98% mobile phase B; 21-23 min, 2%-98% mobile phase A, 98%-2% mobile phase B; 23-25 ​​min, 98% mobile phase A, 2% mobile phase B.

[0046] The mass spectrometer used MRM mode, with the parent ion at m / z 599 and the daughter ion at m / z 447.

[0047] The content of Kaempferol 3-O-(galloyl)-glucoside was calculated based on the test results. The experimental results are as follows: Figure 7 As shown in the figure, the content of Kaempferol 3-O-(galloyl)-glucoside in Chinese black tea is significantly lower than that in Sri Lankan black tea, and the content in Sri Lankan black tea is greater than 40 mg / g. Furthermore, black teas from countries such as Kenya, India, and Turkey also contain a certain amount of Kaempferol 3-O-(galloyl)-glucoside, but their content is lower than that in Sri Lankan black tea and higher than that in Chinese black tea.

Claims

1. A marker compound for identifying the origin of black tea, characterized in that: The biomarker is kaempferol-3-O-(galloyl)-glucoside, with the following structural formula: 。 2. The marker compound for identifying the origin of black tea according to claim 1, characterized in that: If the content of kaempferol-3-O-(galloyl)-glucoside in tea is <12 mg / g, the tea is determined to be produced in China. If the content of kaempferol-3-O-(galloyl)-glucoside is >27 mg / g, the tea is determined to be produced in Sri Lanka.

3. A method for screening marker compounds to determine the origin of black tea, characterized in that: Includes the following steps: Step A: Multi-stage extraction of black tea samples: First, the black tea is subjected to ultrasonic extraction and drying. The dried matter is then reconstituted with water. Then, multi-stage extraction is performed with dichloromethane and ethyl acetate. The extract is dried and redissolved in methanol aqueous solution to obtain the aqueous layer and ethyl acetate layer extracts, respectively. Step B: Perform UHPLC-DAD-MS analysis on the aqueous and ethyl acetate extracts, respectively; Step C: Use Abf Converter software to convert the mass spectrometry data, and use MS-DIAL software to deconvolve, denoise, and align the mass spectrometry data, outputting a DAD spectral dataset. Use Agilent Mass Hunter Workstation software to extract the full-scan spectra of the compounds in the 190-640 nm range. Correct the retention time in the DAD spectral dataset according to ∆T, and delete absorbance values ​​below 100 mAU in the dataset, so that the DAD absorption spectrum is divided into colorless and colored time periods. Use the readxl package in R studio to compare the variables in the aligned LC-MS dataset and the DAD dataset one by one, and filter and delete the LC-MS variables in the colorless time period. OPLS regression analysis was performed using SIMCA-P software. In the model, filtered LC-MS data and DAD spectral data were used as X and Y variables, respectively, and substances with VIP values ​​greater than 2 were identified as key coloring substances in black tea. Step D: Identify the VIP substances based on retention time, molecular weight, and secondary mass spectrometry fragments. Among them, Kaempferol3-O-(galloyl)-glucoside (m / z 599) was identified as the key chromogenic substance with a VIP value greater than 2.

4. The method for screening marker compounds for identifying the origin of black tea according to claim 3, characterized in that: In step A, a black tea sample was weighed and extracted with 70% methanol-water using ultrasonic extraction. After centrifugation, the supernatant was collected. The extraction process was repeated twice. The supernatant was collected and diluted to a final volume. The extract was then processed using a nitrogen blower and a freeze dryer to obtain dry matter. The extract was then redissolved with pure water and subjected to multi-stage extraction with dichloromethane and ethyl acetate, with each extraction being repeated several times. The water, ethyl acetate, and dichloromethane extracts were dried separately, and the precise weight of each component was recorded. Then, 70% methanol-water was added to redissolve the water and ethyl acetate extracts, ensuring that the concentration of the redissolved extract was equivalent to 20 mg / mL of the original black tea.

5. The method for screening marker compounds for identifying the origin of black tea according to claim 3, characterized in that: In step B, analysis was performed using an Agilent 1290 UHPLC-Q-TOF-MS equipped with a DAD, where the detection range of the DAD was 190-640 nm; the chromatographic column was C10. 18 The chromatographic column was 2.1 × 50 mm and 1.7 μm; the flow rate was 0.3 mL / min; the temperature was 40℃; the mobile phase A was 0.1% formic acid in water; and the mobile phase B was 0.1% formic acid in acetonitrile. The elution gradient for the ethyl acetate layer sample was as follows: 0 min, 2% mobile phase B; 0-5 min, 2%-15% mobile phase B; 5-20 min, 15%-16% mobile phase B; 20-22 min, 16%-18% mobile phase B; 22-27 min, 18%-20% mobile phase B; 27-29 min, 20%-28% mobile phase B; 29-33 min, 28% mobile phase B; 33-35 min, 28%-30% mobile phase B; 35-37 min, 30%-50% mobile phase B; 37-39 min, 50%-98% mobile phase B; 39-41 min, 98%-2% mobile phase B; 41-45 min, 2% mobile phase B. The injection volume was 2 μL. The elution gradient for the aqueous layer samples was as follows: 0-5 min, 2%-10% mobile phase B; 5-25 min, 10%-20% mobile phase B; 25-30 min, 20% mobile phase B; 30-35 min, 20%-50% mobile phase B; 35-37 min, 50%-98% mobile phase B; 37-39 min, 98% mobile phase B; 39-41 min, 98%-2% mobile phase B; 41-45 min, 2% mobile phase B. The injection volume was 5 μL, the acquisition range was m / z 100-1500, the scanning mode was negative ion, and the scanning rate was 2 spectra / sec. DAD conditions: peak width of 0.1 min (2.5 Hz), scan range of 190-640 nm, spectral bandwidth of 2 nm, and acquired signals at 278, 360, 380, 400, 420, 460, 520 and 620 nm.

6. A method for determining the origin of black tea, characterized in that, The specific steps for detecting the marker compound for determining the origin of black tea as described in any one of claims 1-2 in a black tea sample are as follows: S1. Prepare the test solution and reference solution for the black tea sample to be tested; S2. The content of kaempferol-3-O-(galloyl)-glucoside in the test solution was determined by UHPLC-QQQ-MS using the external standard method; S3. Determine the type of black tea based on the content of kaempferol-3-O-(galloyl)-glucoside: If the content of kaempferol-3-O-(galloyl)-glucoside is <12 mg / g, it is determined to be black tea produced in China; if the content of kaempferol-3-O-(galloyl)-glucoside is >27 mg / g, it is determined to be black tea produced in Sri Lanka.

7. The method for determining the origin of black tea according to claim 6, characterized in that: The preparation method of the detection solution in step S1 is as follows: Weigh the dry tea sample quantitatively, add 70% methanol-water (v / v) and vortex to mix, extract by ultrasonication at 40℃, centrifuge at room temperature and extract the supernatant, repeat the extraction step three times, combine the supernatants, and adjust the volume with pure water to make the sample concentration suitable; The preparation method of the reference solution is as follows: Prepare kaempferol-3-O-(galloyl)-glucoside solution with 70% methanol-water (v / v) at concentrations of 5, 10, 20, 50, 100 and 500 ng / mL, and establish a concentration-peak area standard curve.

8. The method for determining the origin of black tea according to claim 7, characterized in that, The sample concentration was 10 mg / mL.

9. The method for determining the origin of black tea according to claim 6, characterized in that, The chromatographic conditions for high performance liquid chromatography in step S2 are as follows: column temperature 40℃, flow rate 0.3 mL / min, injection volume 2 μL, gradient elution: mobile phase A is 0.1% formic acid water, mobile phase B is 0.1% formic acid acetonitrile, and the elution gradient is: 0 min, 2% mobile phase B; 0-5 min, 2%-15% mobile phase B; 5-15 min, 15%-21% mobile phase B; 15-17 min, 21%-50% mobile phase B; 17-19 min, 50%-98% mobile phase B; 19-21 min, 98% mobile phase B; 21-23 min, 98%-2% mobile phase B; 23-25 ​​min, 2% mobile phase B.

10. The method for determining the origin of black tea according to claim 6, characterized in that, In step S2, the mass spectrometry uses MRM mode, with the parent ion at m / z 599 and the daughter ion at m / z 447.

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