Ion mobility spectrometry-based method for rapidly judging authenticity of Tongzhou green tea

Abstract

The invention discloses an ion mobility spectrometry-based method for rapidly judging the authenticity of Tongzhou green tea, and belongs to the technical field of food quality detection. The method comprises the following steps: 1) establishing an ion mobility spectrometry detection method for aroma components of the Tongzhou green tea extracted by hot water; 2) collecting the aroma fingerprint spectrum of the Tongzhou tea leaves under different drying modes and extraction conditions; the method comprises the following steps of 1, establishing a fingerprint spectrum database of the characteristic aroma types of the Tongzhou tea leaves under different drying modes and extraction conditions, 2, distinguishing and identifying the Tongzhou green tea processed in different drying modes, 3, establishing a fingerprint spectrum database of the characteristic aroma types of the Tongzhou tea leaves under different drying modes and extraction conditions, and 4, distinguishing and identifying the Tongzhou green tea processed in different drying modes.

Description

Technical Field

[0001] The present invention belongs to the technical field of food quality detection, and particularly relates to a rapid method for discriminating false Chizhou green tea based on ion mobility spectrometry. Background Art

[0002] Tea is one of the three major non-alcoholic beverages in the world and has various health benefits, such as antioxidant, anti-inflammatory, anti-aging, anti-cancer, prevention of hypertension and cardiovascular diseases, and so on. Green tea is one of the most produced and consumed tea varieties in China and has attracted great interest due to its unique aroma, flavor, color, clarity of tea soup and other quality characteristics. The main processes of green tea production include steps such as fixation, rolling, shaping and drying. Drying, as an important part of the processing process, plays a crucial role in promoting flavor formation during the processing.

[0003] The aroma of tea is an important factor in measuring the quality of tea. The rich and pleasant aroma of tea plays a key decisive role in the quality, grade and price of tea, and is also an important basis for consumers to choose. The pleasant aroma of tea is composed of thousands of volatile components. The ever-changing combinations of these components and their interaction effects are the material basis for forming various fragrance types. Different drying methods have a decisive impact on the aroma and flavor of tea. Consumers have a special preference for freeze-dried tea that can maintain the fresh aroma of tea. There are often cases where tea processed by other drying methods is passed off as freeze-dried tea in the market, and it is difficult for ordinary consumers and dealers to distinguish.

[0004] Traditional sensory evaluation technology is the mainstream method for evaluating the quality of tea. In terms of aroma evaluation, tea tasters evaluate the type, concentration, purity and persistence of tea aroma through their sense of smell. The results of sensory evaluation are easily affected by the professional qualities and sensory differences of tea tasters, and it is difficult to eliminate the subjective components of humans. Moreover, affected by external objective factors, it is difficult to completely objectively and accurately evaluate the aroma quality and fragrance type of tea, and it is difficult to complete the evaluation of a large number of tea samples in a short time. With the rapid development of modern instrumental analysis technology, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) have become important means for detecting tea aroma substances. These means usually require complex aroma extraction pretreatment, such as solid-phase microextraction (SPME), distillation extraction, etc. These not only take a long time, but also have a certain degree of damage to the samples. At the same time, due to the low sensitivity of these means, it is difficult to distinguish the subtle differences caused by different processing techniques for the same tea variety. The limitations of the above technologies are difficult to meet a series of important requirements such as the rapid discrimination of tea aroma fragrance types and the on-line monitoring of aroma during tea processing or storage.

[0005] Ion mobility spectrometry (IMS) is a rapid separation and detection technology for gas-phase ions that has been popular since the 1970s. Its principle is that under atmospheric pressure, volatile and semi-volatile compounds are ionized into gas-phase ions and then move in an external electric field. Different types of ions have different electrophoretic velocities due to differences in characteristic parameters such as mass, charge number, collision cross section, and spatial structure of the molecule, resulting in different times required for them to reach the ion detector, thus achieving the purpose of separation and detection. Ion mobility spectrometry combines high separation capacity gas chromatography (GC) with fast response ion mobility spectrometry (IMS). It has the advantages of high sensitivity, fast analysis speed, low cost, and easy portability. It has been widely used in the rapid on-site screening of drugs and explosives. In recent years, domestic and foreign research has gradually expanded the application of ion mobility spectrometry to other fields, including quality inspection and flavor analysis of agricultural products, online monitoring of environmental pollutants, non-destructive detection of freshness of meat products, identification of adulteration of edible oils, and process monitoring of coffee roasting. Similarly, the advantages of ion mobility spectrometry, such as short analysis time (single spectrum scanning time is in milliseconds) and no sample pretreatment, can also meet the needs of rapid and non-destructive detection of tea aroma. In recent years, there have been reports on the use of ion mobility spectrometry for tea aroma analysis, but there are relatively few practical applications. By establishing a fingerprint library of tea aroma types of Chizhou green tea with different drying methods and combining it with chemical informatics tools, rapid determination of Chizhou green tea with different drying methods can be achieved. Summary of the invention

[0006] The ion mobility spectrometry technology used in the present invention shows many unique advantages in the application of Chizhou green tea counterfeiting. On the one hand, in the sample processing link, compared with traditional detection methods, the hot water extraction combined with headspace sampling greatly simplifies the process. It is only necessary to place an appropriate amount of tea powder in the headspace bottle, incubate it at a specific temperature, and then use a gas-tight syringe to extract the headspace gas and inject it into the inlet, avoiding the cumbersome, complicated and time-consuming operation such as solid phase microextraction, retaining the original state of the aroma components of tea to the greatest extent, reducing the risk of component loss due to pretreatment, and laying the foundation for subsequent accurate detection. On the other hand, the fine setting of the instrument operating parameters ensures the accuracy and efficiency of the detection. With high-purity nitrogen as the carrier gas, the flow rate is accurately controlled according to the detection process. The low-speed stable airflow in the early stage ensures the smooth introduction of the initial state of the sample. The rapid increase in the flow rate in the later stage helps the volatile components to pass through the detection area quickly, and with the appropriate chromatographic column and migration tube temperature and electric field strength, the ions of different aroma components can be separated in order according to their characteristics and quickly detected. Under this synergistic effect, not only is the scanning time of a single spectrum as short as milliseconds, able to capture the subtle characteristics of the tea aroma in an instant, but the entire detection process is smooth and coherent, without the need for long waiting times, thus meeting the timeliness requirements of on-site rapid detection.

[0007] During the fingerprint construction phase, we fully considered two key factors: different drying methods and extraction conditions. Drying methods include common hot air drying, microwave drying, vacuum freeze drying, and vacuum freeze + microwave combined drying. Each method will cause unique changes in aroma components during tea processing. By collecting IMS fingerprints for tea samples with typical characteristic aromas evaluated by experts according to the established detection process, we accurately locked the characteristic fingerprints of unique aromas under different drying methods, including key identifiers such as the number of peaks, peak positions, and relative intensities. These characteristic fingerprints are like the "identity codes" of tea leaves, corresponding to tea leaves produced by different drying processes one by one, providing an accurate comparison basis for the subsequent identification of massive tea samples. Extraction conditions should not be ignored either. From incubation temperature, stirring speed to injection volume and other details, any slight changes may affect the release and detection results of aroma components. Therefore, based on a large number of experiments, we determined the best combination of extraction conditions and applied it throughout the entire research process. In this way, the established fingerprint library not only comprehensively covers the characteristic information of different drying methods, but also internalizes the influencing factors of the extraction conditions, making the reference value of the library more accurate and reliable.

[0008] When faced with tea samples to be tested, the established standard procedures are strictly followed. Each tea sample is sampled and tested multiple times to obtain technical repeated data. After professional data preprocessing, noise interference, calibration signal deviation, etc. are removed to ensure the purity of the data. Then, advanced multivariate statistical analysis methods such as cluster analysis or discriminant analysis, especially orthogonal partial least squares discriminant analysis (OPLS-DA) software tools, are used to deeply explore the information hidden behind the data. With volatile flavor substances as the core variables, drying methods and extraction conditions as guiding variables, the differences between different tea samples can be clearly and intuitively presented. Through real-time comparison with the fingerprint library, the processing method of the tea to be tested can be quickly locked. Whether it is the common low-grade hot air dried green tea in the market pretending to be high-end vacuum freeze-dried green tea, or other confusing counterfeiting situations, they can be accurately identified in a short time, which effectively defends the market reputation of Chizhou green tea and protects the rights and interests of consumers. At the same time, it also provides strong technical support for tea production and processing companies to control product quality and regulate market order.

[0009] In the future, we plan to further expand the application boundaries of this technology, and deeply explore the deep connection between the aroma components of tea and the quality and taste of tea. It will not only be limited to authenticity detection, but also provide forward-looking guidance for the development of new tea products and quality improvement, and promote the entire Chizhou green tea industry to develop in a direction with higher quality and more innovative vitality. DETAILED DESCRIPTION

[0010] The present invention is based on ion mobility spectrometry technology for anti-counterfeiting identification of Chizhou green tea. Green tea samples processed by different drying methods are collected as samples. Different green tea samples are subjected to ion mobility spectrometry analysis under different extraction conditions, and the data is analyzed by special software to form characteristic spectra, establish a fingerprint database, and realize the anti-counterfeiting identification of Chizhou green tea. The specific implementation methods are as follows: S1 Experimental material preparation S1.1 Tea sample collection Collect green tea samples processed by different batches and different drying methods from multiple well-known tea gardens and tea processing factories in Chizhou. It covers tea leaves processed by traditional manual frying combined with hot air drying, which have a strong fried aroma; there are also tea leaves prepared by using advanced vacuum freeze-drying technology, retaining a fresher and more natural fresh aroma; some tea leaves processed by microwave drying and a small amount of tea leaves processed by vacuum freeze-drying combined with microwave drying. Ensure that the samples have wide representativeness and can reflect the diversity of Chizhou green tea in actual production.

[0011] For each batch of tea leaves, according to the standard sampling process, multi-point sampling is carried out from different parts and different packaging levels of the tea leaves. After mixing evenly, sub-samples for subsequent experiments are further divided to ensure the uniformity and authenticity of the sampled tea samples.

[0012] S1.2 Instrument and reagent preparation Ion mobility spectrometer: Select a commercial model with a high-precision temperature control module, a stable electric field generation device, and a high-sensitivity ion detector. Before the experiment, according to the calibration process provided by the instrument manufacturer, the instrument is fully calibrated with a standard gas mixture to ensure the measurement accuracy of key parameters such as the temperature of the migration tube and the electric field strength, and the error is controlled within a very small range.

[0013] Hermetic syringe: Purchase a syringe with high temperature resistance, high pressure resistance, and excellent airtightness, with a specification suitable for 85°C and a capacity of 500 μL, to ensure that there is no leakage or sudden temperature change when extracting headspace gas, which may affect the gas composition.

[0014] Headspace vial: Use a 20 mL borosilicate glass headspace vial, which has good chemical stability and sealing performance, can withstand an 80°C high-temperature incubation environment, and prevent chemical reactions with the aroma components of tea leaves or gas leakage during the experiment.

[0015] Nitrogen: Select high-purity nitrogen with a purity of up to 99.99% as the carrier gas, provided by a professional gas supplier, and equipped with a high-precision gas flow controller to accurately regulate the flow rate of nitrogen at different detection stages.

[0016] S2 Refinement of hot water extraction and ion mobility spectrometry detection process S2.1 Sample treatment steps Accurately weigh 2 grams of tea powder using an electronic balance with a precision of 0.001 grams to ensure the accuracy of each weighing. Carefully pour the weighed tea powder into a 20 mL headspace vial, and add 10 mL of ultrapure water, then mix well.

[0017] Quickly place the headspace vial containing the tea powder into a thermostatic water bath preheated to 80 °C, and at the same time, turn on the magnetic stirring device and set the stirring speed to 500 rpm. Under this temperature and stirring rate, the aroma components in the tea can be efficiently and stably released into the headspace gas. The incubation process lasts for 15 min. During this period, strictly control the time through a timer to ensure that the incubation time of each sample is consistent and reduce experimental errors.

[0018] After the incubation is completed, immediately use an airtight syringe preheated to 85 °C to slowly and steadily extract 500 μL of the headspace gas. During the extraction process, pay attention to keeping the syringe vertical to avoid gas mixing with impurities or backflow. Then quickly inject the extracted gas into the inlet of the ion mobility spectrometer.

[0019] S2.2 Instrument Operating Parameter Setting Gas Flow Rate Regulation: At the initial stage of starting the ion mobility spectrometer, first introduce nitrogen carrier gas into the system at a flow rate of 2 mL / min and maintain this flow rate for 2 min to make the internal environment of the instrument reach a preliminary stable state, ensuring that the sample gas will not be disturbed by the gas flow impact when entering. Then, through programming to control the gas flow controller, linearly increase the nitrogen flow rate to 100 mL / min within 10 min and maintain this flow rate for 10 - 20 min to quickly push the volatile aroma components through the chromatographic column and migration tube to achieve efficient separation and detection.

[0020] Temperature Control: Accurately set the temperature of the chromatographic column to 60 °C, and use the temperature control module to monitor and feedback-adjust in real time to ensure that the stationary phase in the chromatographic column can provide a suitable separation environment for the aroma components. The temperature of the migration tube is maintained at 45 °C. This temperature can not only promote the ionization process to proceed smoothly but also avoid the ion structure being unstable or unnecessary chemical reactions occurring due to too high a temperature. At the same time, set the electric field strength to 365 V cm - 1 to provide a stable and differentiated driving force for ions with different masses and charge numbers, enabling them to migrate to the ion detector orderly according to their characteristics.

[0021] S3 Fingerprint Map Construction and Database Improvement S3.1 Characteristic Fingerprint Map Acquisition Select tea samples with clear and typical characteristic fragrance through sensory evaluation by senior tea experts as benchmark samples. For example, for vacuum freeze-dried tea, select samples with distinct fresh floral fragrance, slightly fruity fragrance and long-lasting aroma; for hot-air dried tea, select samples with strong stir-fried fragrance and accompanied by baking aroma.

[0022] According to the above hot water extraction and ion mobility spectrometry detection process, these benchmark samples are repeatedly detected under different extraction conditions. The changes in extraction conditions include adjusting the incubation temperature at intervals of 2°C between 75°C and 85°C, changing the stirring speed in the range of 400 rpm - 600 rpm in steps of 50 rpm, and increasing or decreasing the injection volume by 50 μL between 400 μL and 600 μL. By comprehensively covering these variable combinations, a rich variety of IMS fingerprint spectra are collected to accurately capture the subtle changes in the aroma components of tea under different drying methods and extraction conditions ( Figure 1 )

[0023] For each collected fingerprint spectrum, using professional image processing software and chemical analysis algorithms, accurately identify and label the number of peaks, peak positions and relative intensities of the common peaks. These key identifications constitute the characteristic fingerprint of tea aroma and are stored in the local database as the basic template for subsequent comparison.

[0024] S3.2 Fingerprint Spectrum Database Management and Update Establish a fingerprint spectrum database architecture based on a relational database management system (such as MySQL). Store the information of different drying methods, extraction conditions and corresponding tea characteristic fingerprints in different data tables respectively, and associate them through unique identifiers for quick query and comparison.

[0025] With the collection of new tea samples and the generation of new detection data, regularly update and maintain the database. Adopt an automated data import script combined with a manual review mechanism to ensure the accuracy and integrity of the new data. At the same time, set up a data backup strategy to prevent data loss due to hardware failures, human errors, etc., and ensure the long-term stable operation of the database.

[0026] S4 Detection and Anti-Counterfeiting Practical Application of Tea Samples to be Tested S4.1 Sample Pretreatment and Detection When receiving a tea sample to be tested, it is also processed according to the standard process of weighing 2 grams of tea powder, incubating at 80°C for 15 minutes, and extracting 500 μL of headspace gas with an airtight syringe at 85°C. Conduct three independent samplings and detections on the tea sample to be tested to ensure sufficient technical replicate data is obtained to improve the reliability of the detection results.

[0027] During the detection process of the ion mobility spectrometer, the operating status of the instrument is monitored in real time, including the stability of gas flow rate, temperature fluctuations, and the signal intensity of the ion detector, etc. Once an anomaly is detected, the detection is immediately paused, the cause of the fault is investigated and repaired to avoid the production of incorrect data due to instrument failure.

[0028] S4.2 Data Analysis and Forgery Judgment After collecting the IMS fingerprint data of the tea sample to be tested, operations such as noise reduction, baseline correction, and peak identification are first performed using data preprocessing software. Remove the spurious peaks generated by factors such as instrument electronic noise and environmental interference, and calibrate the peak position deviation caused by signal drift to ensure the purity and accuracy of the data.

[0029] The preprocessed data is imported into SIMCA 14.1 software for orthogonal partial least squares discriminant analysis (OPLS-DA). Using 43 volatile flavor substances already existing in the database as the dependent variables and the drying method and extraction conditions as the independent variables, a discriminant model is constructed. Through model calculation, the coordinate position of the tea sample to be tested in the multi-dimensional space will be compared with the characteristic regions of tea leaves with different drying methods in the database ( Figure 2 ). If the data point of the tea sample to be tested falls within the concentrated area of tea leaves with a certain specific drying method, it is determined that its processing method is consistent with it; if it deviates from the known area, it is determined that there may be suspicion of forgery, and further verification is carried out in combination with manual sensory evaluation. Finally, a detailed test report is issued according to the forgery judgment result, clarifying the authenticity of the tea sample to be tested, and providing a strong basis for market supervision and enterprise quality control. Description of the Drawings

[0030] Figure 1 IMS Fingerprint of Volatile Components in Tea Leaves under Different Drying Methods and Extraction Conditions Figure 2 OPLS-DA Analysis of Volatile Components in Tea Leaves under Different Drying Methods and Extraction Conditions.

Claims

1. A rapid method for detecting the authenticity of Chizhou green tea based on ion mobility spectrometry, characterized by the following steps: 1) Establishment of an ion mobility spectrometry detection method for the aroma components of Chizhou green tea extracted with hot water; 2) Collection of aroma fingerprint spectra of Chizhou tea under different drying methods and extraction conditions; 3) Establishment of a fingerprint spectrum library for the characteristic fragrance types of Chizhou tea under different drying methods and extraction conditions; 4) Distinguishing and identifying Chizhou green tea processed by different drying methods.

2. The rapid method for falsification judgment of Chizhou green tea based on ion mobility spectrometry according to claim 1, wherein The specific steps of step 1) include the following steps: A) Weigh 2 g of tea powder into a 20 mL headspace vial, add 10 mL of ultrapure water, directly incubate at 80 °C with a stirring speed of 500 rpm for 15 min to obtain an extraction sample. Subsequently, inject 500 μL of headspace gas into the inlet with an airtight syringe at 85 °C. B) Gas: Use nitrogen (purity 99.99%) as the carrier gas, with a flow rate of 2 mL / min maintained for 2 min, and then linearly increased to 100 mL / min within 10 min and maintained for 10 - 20 min. C) Ion mobility spectrometry (IMS) detection: The column temperature is 60 °C, the drift tube temperature is 45 °C, the electric field strength is 365 V cm-1. Put in the tea sample, and continuous acquisition can be triggered after starting purge injection. The acquisition frequency is 1 Hz.

3. The rapid method for detecting counterfeit Chizhou green tea based on ion mobility spectrometry as claimed in claim 1, characterized in that The specific steps of step 2) include the following steps: Take typical tea samples that have been sensory evaluated by experts and have the characteristic fragrance types unique to this processing method, and collect IMS fingerprint spectra under different drying methods and extraction conditions according to the conditions described in step 1) and step 2). Determine the characteristic fingerprints of the fragrance types unique to this processing method, including the number of peaks, peak positions, and relative intensities of the common peaks, and establish a fingerprint spectrum library.

4. The rapid method for verifying the authenticity of Chizhou green tea based on ion mobility spectrometry according to claim 1, wherein The specific steps of step 3) include the following steps: Collect IMS fingerprint spectra of the tea samples to be tested under different drying methods and extraction conditions according to the conditions described in step 1) and step 2). Each tea sample is sampled three times for detection to obtain three technical replicates. After data preprocessing, use multivariate statistical analysis methods such as cluster analysis or discriminant analysis to distinguish and identify the unique fragrance types. 1) All experimental samples are analyzed in 3 replicates. Use SIMCA 14.1 software for orthogonal partial least squares discriminant analysis (OPLS-DA) to explore the differences under different drying methods and extraction conditions; 2) Using 43 volatile flavor substances as the dependent variable and drying methods and extraction conditions as the independent variables, OPLS-DA can effectively distinguish Chizhou green tea from different drying methods. The OPLS-DA model shows obvious differences among four common drying methods and extraction conditions, and each drying method and extraction condition shows a relatively concentrated distribution.

5. The rapid method for detecting the falsification of Chizhou green tea based on ion mobility spectrometry according to claim 1, characterized in that The specific steps of step 4) include the following steps: The IMS fingerprint spectra of the samples to be tested under different drying methods and extraction conditions were collected according to the conditions described in step 1) and step 2). Each tea sample was sampled three times for testing to obtain three technical replicates. After data preprocessing, the data were compared with the fingerprint library of characteristic aroma types of Chizhou tea under different drying methods and extraction conditions to determine the processing method of the tea to be tested, and to quickly identify the counterfeiting of low-grade tea such as hot-air dried green tea that is common in the market and is posing as vacuum freeze-dried green tea.

Images