Application of age characteristic compound of fuzhuan brick tea and method for determining age of fuzhuan brick tea by using the compound

Abstract

The application belongs to the technical field of tea identification, and particularly relates to application of a characteristic compound of Fuzhuan brick tea year and a method for identifying the Fuzhuan brick tea year by using the compound. A linear equation of the content of N-ethyl-2-pyrrolidone-substituted Fuzhuan brick tea F and the Fuzhuan brick tea year is established, the quantitative result of the content of N-ethyl-2-pyrrolidone-substituted Fuzhuan brick tea F of an unknown Fuzhuan brick tea sample is determined, and the result is substituted into the linear equation, so that the estimated value of the Fuzhuan brick tea sample year is obtained. The method excludes the human subjective factors involved in the previous Fuzhuan brick tea year identification mainly based on tea evaluation, provides unified quantitative data, the result is objective, and the experimental operation and data analysis are rapid, simple and convenient.

Description

Technical Field

[0001] This invention belongs to the field of tea identification technology, specifically relating to the application of a compound characteristic of the age of Fu brick tea and a method for identifying the age of Fu brick tea using this compound. Background Technology

[0002] Fu brick tea is a representative category of traditional Chinese dark tea. Its key difference from other post-fermented teas lies in a unique "fermentation" process. This process involves artificially controlling the fermentation of *Aspergillus cristatus*, which gives Fu brick tea a mellow taste, a unique aroma, and various health benefits such as anti-obesity, anti-hyperglycemia, antibacterial, and anti-inflammatory properties.

[0003] Storage time (years) is crucial to the quality formation of Fu brick tea. Traditionally, it is believed that Fu brick tea requires long-term storage to enhance its flavor, accompanied by a series of physicochemical changes: the dry tea color changes from yellowish-brown to dark brown, the tea soup color changes from orange-yellow to orange-red, the taste changes from bitter and astringent to mellow, the green and astringent aroma gradually dissipates, and the floral and aged aromas gradually become more prominent. With increasing storage years, the market value of Fu brick tea significantly increases. However, currently, the identification of Fu brick tea from different years mainly relies on sensory evaluation, which is highly subjective and lacks objective chemical composition indicators. This has led to the phenomenon of younger teas being passed off as older teas in the market, seriously disrupting industry order and significantly hindering the development of the Fu brick tea industry. Therefore, there is an urgent need to establish an objective and accurate method to determine the storage year of Fu brick tea to ensure product quality and market regulation.

[0004] In our previous research, we discovered a new component in Fu brick tea: N-ethyl-2-pyrrolidone-substituted Fu brick element F (chemical structural formula shown in [link to chemical formula]). Figure 1 The content of N-ethyl-2-pyrrolidone-substituted Fuzhuanxin F in Fuzhuan tea gradually increases during storage and shows a good linear relationship with the storage year. Therefore, it is proposed to use the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanxin F in Fuzhuan tea as a discriminant for the age of Fuzhuan tea, for the identification and determination of unknown Fuzhuan tea years. Summary of the Invention

[0005] To address the problems in existing technologies, the present invention aims to provide a technical solution for the application of a characteristic compound of Fu brick tea's age and a method for determining the age of Fu brick tea using this compound. This method can provide effective technical support for determining the age of Fu brick tea on the market. The method has the advantages of simple operation, quantifiable data, objective results, and high accuracy.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the application of N-ethyl-2-pyrrolidone-substituted Fuzhuanxin F as a characteristic compound for determining the age of Fuzhuan tea.

[0007] The second aspect of this invention provides a method for determining the age of Fu brick tea based on characteristic compounds of the tea. A linear equation is established between the content of N-ethyl-2-pyrrolidone-substituted Fu brick tea and the age of the Fu brick tea. The estimated value of the age of the Fu brick tea sample is obtained by substituting the content of N-ethyl-2-pyrrolidone-substituted Fu brick tea F in an unknown Fu brick tea sample into the above linear equation.

[0008] Furthermore, the linear equation relating the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F in the Fuzhuan tea to the age of the Fuzhuan tea is: y=0.57665x-1.63945.

[0009] Furthermore, the discrimination method specifically includes the following steps: 1) Extraction of components in Fu brick tea: Collect Fu brick tea samples, grind them into powder, accurately weigh the Fu brick tea powder, extract the chemical components in it with methanol aqueous solution, and take the supernatant after high-speed centrifugation. 2) Quantitative analysis method for N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F, a characteristic compound of aged Fuzhuanzi tea, based on liquid chromatography-mass spectrometry (LC-MS): The peak area of ​​N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F in the sample was determined and substituted into a pre-established "content-peak area" standard curve [Y (content, unit μg / g) = X (peak area) / 4.65937 / 100000000000*200]. Based on the standard curve, the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F corresponding to the peak area of ​​the compound was calculated. 3) Establish a linear equation for determining the age of Fu brick tea using a series of Fu brick tea samples with known years; 4) Substitute the quantitative results of N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F in the unknown Fuzhuan tea sample into the linear equation for Fuzhuan tea age determination to obtain the estimated value of the Fuzhuan tea sample age.

[0010] Furthermore, the small amount of Fu brick tea powder in step 1) is 0.1 grams.

[0011] Furthermore, in step 1), the proportion of methanol-water solution is 50-90%, the extraction temperature is 50-100℃, and the extraction time is 10-50 minutes.

[0012] Furthermore, in step 1), the high-speed centrifugation speed is 5000-10000 rpm, and the centrifugation time is 2-10 minutes.

[0013] Furthermore, the quantitative analysis in step 2) is an external standard quantitative method, specifically, the mass spectrometry response value of the target compound is calculated based on a standard working curve to obtain its content.

[0014] Furthermore, in step 3), the linear equation for determining the age of Fu brick tea is established with the content of N-ethyl-2-pyrrolidone-substituted Fu brick tea element F in the Fu brick tea sample as the x variable and the year of the Fu brick tea sample as the y variable.

[0015] This invention identifies the age of Fu brick tea by using characteristic compounds of different ages, and has the following advantages: 1) The compound used as an indicator of the age of Fu brick tea is Fu brick element F, which is N-ethyl-2-pyrrolidone substituted and discovered for the first time in tea, and is original; 2) It eliminates the subjective human factors involved in the previous tea-based age determination of Fu brick tea, provides unified quantitative data, and the results are objective. It can be tested without the need for high-end professional tea-evaluation personnel, which greatly reduces the requirements for personnel. 3) Experimental operations and data analysis are quick, simple, and convenient. Attached Figure Description

[0016] Figure 1 It is the chemical structural formula of N-ethyl-2-pyrrolidone-substituted Fuzhuanin F-cThea; Figure 2 (A) is the extract ion chromatogram of N-ethyl-2-pyrrolidone-substituted Fuzhuanin F-cThea analyzed by liquid chromatography-mass spectrometry. Figure 2 (B) is the extracted ion mass spectrum of N-ethyl-2-pyrrolidone-substituted Fuzhuanin F-cThea analyzed by liquid chromatography-mass spectrometry. Figure 3 It is a linear equation for determining the age of Fu brick tea. Detailed Implementation

[0017] The embodiments of the present invention are described in detail below with reference to the appendix: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the scope of protection of the present invention is not limited to the following embodiments.

[0018] Example 1

[0019] Identification of the age of Fuzhuan tea: The content of Fuzhuanin F-cThea in Fuzhuan tea samples from known years is measured, and then a linear discriminant model is established with the year to identify the age of Fuzhuan tea samples from unknown years.

[0020] Detailed operation steps: 1) Collect 12 samples of Anhua Fu brick tea from different years, grind them into powder and store them at -20℃.

[0021] 2) Determination of the content of Fuzhuanin F-cThea in Fuzhuan tea samples: Accurately weigh 0.1 g of Fu brick tea sample powder into a centrifuge tube, add 20 mL of 70% methanol aqueous solution, mix thoroughly, and extract in a 70℃ water bath for 30 minutes, shaking once every 5 minutes. Centrifuge at 8000 rpm for 10 minutes, collect the supernatant and pass it through a 0.22 µm nylon membrane, then transfer 1 mL to a liquid chromatography vial for subsequent LC-MS analysis.

[0022] The chromatographic conditions for liquid chromatography-mass spectrometry (LC-MS) analysis were as follows: T3 column (100 mm × 2.1 mm, 1.8 μm, Waters Corporation, USA); mobile phase A was 0.1% formic acid-water solution, and mobile phase B was acetonitrile solution containing 0.1% formic acid; injection volume was 3 μL; and flow rate was 0.4 mL / min. -1 The column temperature was 40℃; the mobile phase linear gradient elution was as follows: 0 min, 2% B phase; 0.5 min, 2% B phase; 10 min, 15% B phase; 18 min, 40% B phase; 20 min, 90% B phase; 20.9 min, 90% B phase; 21 min, 2% B phase; 25 min, 2% B phase.

[0023] The mass spectrometry conditions for LC-MS were as follows: an electrospray ionization (ESI) source was used, and data acquisition was performed in both positive and negative ion modes. In positive ion mode, the capillary voltage was 3.5 kV; in negative ion mode, the capillary voltage was -3.5 kV. Other parameters remained consistent in both modes: capillary temperature 300 °C, auxiliary gas temperature 350 °C, auxiliary gas flow rate 10 L / min, and mass spectrometry scan range of mass-to-charge ratio (m / z) 80–1200.

[0024] The elution characteristics of Fuzhuanin F-cThea under these chromatographic-mass spectrometric conditions are as follows: Figure 2 As shown in Table 1, the content of Fuzhuanin F-cThea was determined using the external standard quantification method: the peak area of ​​the mass spectrometry response of Fuzhuanin F-cThea was substituted into a pre-established compound standard curve to calculate its content. The Fuzhuanin F-cThea content in the 12 Fuzhuan tea samples is shown in Table 1. The Fuzhuanin F-cThea content increased with the age of the Fuzhuan tea.

[0025] Table 1. Relationship between the age of Fuzhuan tea and its F-cThea content Year relative peak area Content (μg / g) 0 1776073.341 7.62 0 1768235.890 7.59 0 1707490.227 7.33 1 1060729.146 4.55 1 1370170.991 5.88 1 1412408.867 6.06 2 1254495.005 5.38 2 1327023.206 5.70 2 1317312.969 5.65 3 2430364.325 10.43 3 2205238.766 9.47 3 2265054.662 9.72 5 2479362.878 10.64 5 2590790.081 11.12 5 2500762.321 10.73 6 2749183.993 11.80 6 2710883.819 11.64 6 2751984.839 11.81 7 2856924.599 12.26 7 3058455.990 13.13 7 3112385.776 13.36 11 4301236.800 18.46 11 4351391.003 18.68 11 4282862.174 18.38 12 4482691.412 19.24 12 4489554.451 19.27 12 5842533.392 25.08 13 5974121.599 25.64 13 5862892.539 25.17 13 5852534.926 25.12 14 6829452.314 29.31 14 6932693.540 29.76 14 6970029.601 29.92 16 7539753.655 32.36 16 7347513.226 31.54 16 7160750.216 30.74 .

[0026] 3) Establishment of a linear equation for determining the age of Fu brick tea Using the content of Fuzhuanin F-cThea (μg / g) in the Fuzhuan tea samples as the x variable and the year of the Fuzhuan tea samples as the y variable, a linear regression was performed. The resulting linear equation for determining the year of Fuzhuan tea was y = 0.57665x - 1.63945. R 2 =0.93289 ( Figure 3 ).

[0027] 4) Identification and determination of the age of Fu brick tea Twelve representative Fu brick tea samples were purchased, with their year information kept confidential beforehand. The tea was then processed according to steps 1-4 of the instruction manual to determine its age. The experimental results showed that the estimated year matched the actual year well, with a discrimination error ranging from -0.88 to 0.94 years, demonstrating high accuracy, as shown in Table 2.

[0028] Table 2. Fuzhuanin F-cTe content, actual year, and predicted year in 12 Fuzhuan tea samples .

Claims

1. Application of N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F as a characteristic compound for determining the age of Fuzhuan tea.

2. A method for determining the age of Fu brick tea based on characteristic compounds of aged Fu brick tea, characterized in that, A linear equation was established between the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanxin F in Fuzhuan tea and the age of Fuzhuan tea. By measuring the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanxin F in unknown Fuzhuan tea samples and substituting it into the above linear equation, the estimated age of Fuzhuan tea samples was obtained.

3. The method for determining the age of Fu brick tea based on characteristic compounds of aged Fu brick tea as described in claim 2, characterized in that, The linear equation relating the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanxin F in Fuzhuan tea to the age of the Fuzhuan tea is: y = 0.57665x - 1.63945.

4. The method for determining the age of Fu brick tea based on characteristic compounds of aged Fu brick tea as described in claim 2, characterized in that, Specifically, the following steps are included: 1) Extraction of components in Fu brick tea: Collect Fu brick tea samples, grind them into powder, accurately weigh the Fu brick tea powder, extract the chemical components in it with methanol aqueous solution, and take the supernatant after high-speed centrifugation. 2) Quantitative analysis method for N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F, a characteristic compound of aged Fuzhuanzi tea, based on liquid chromatography-mass spectrometry (LC-MS): The peak area of ​​N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F in the sample was determined and substituted into a pre-established "content-peak area" standard curve [Y (content, unit μg / g) = X (peak area) / 4.65937 / 10000000000*200]. Based on the standard curve, the content of N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F corresponding to the peak area of ​​the compound was calculated. 3) Establish a linear equation for determining the age of Fu brick tea using a series of Fu brick tea samples with known years; 4) Substitute the quantitative results of N-ethyl-2-pyrrolidone-substituted Fuzhuanzi F in the unknown Fuzhuan tea sample into the linear equation for Fuzhuan tea age determination to obtain the estimated value of the Fuzhuan tea sample age.

5. The discrimination method as described in claim 2, characterized in that... In step 1), the methanol-water solution ratio is 50-90%, the extraction temperature is 50-100℃, and the extraction time is 10-50 minutes.

6. The discrimination method as described in claim 2, characterized in that, In step 1), the high-speed centrifugation speed is 5000-10000 rpm, and the centrifugation time is 2-10 minutes.

7. The discrimination method as described in claim 2, characterized in that, In step 2), the quantitative analysis is an external standard quantitative method, specifically, the mass spectrometry response value of the target compound is calculated based on the standard working curve to obtain its content.

8. The discrimination method as described in claim 2, characterized in that, In step 3), the linear equation for determining the age of Fu brick tea is established by using the content of N-ethyl-2-pyrrolidone-substituted Fu brick tea F in the Fu brick tea sample as the x variable and the year of the Fu brick tea sample as the y variable.

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