Characteristic Flavor Substances of Guizhou Black Tea and the Environmental Factors Influencing Their Formation Using Stable Isotopes and Headspace Gas Chromatography-Ion Mobility Spectrometry

Ge, Yonghui; Huang, Yongji; Wang, Ling; Jia, Luyu; Wang, Jinhua

doi:https://doi.org/10.1155/2024/9019953

Journal of Food Biochemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusion and Discussion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 9019953 | https://doi.org/10.1155/2024/9019953

Characteristic Flavor Substances of Guizhou Black Tea and the Environmental Factors Influencing Their Formation Using Stable Isotopes and Headspace Gas Chromatography-Ion Mobility Spectrometry

Yonghui Ge,¹Yongji Huang,¹Ling Wang,¹Luyu Jia,²and Jinhua Wang¹

Academic Editor: Hongxun Tao

Received05 Sept 2023

Revised19 Mar 2024

Accepted01 Apr 2024

Published17 Apr 2024

Abstract

Guizhou is one of the most important black tea producing areas in China. The purpose of this study was to explore the characteristic flavor compounds of Guizhou black tea and investigate the influence of environmental factors on the formation of black tea flavor components. In this study, HS-GC-IMS was used to identify 143 compounds from black tea samples. OPLS-DA was employed to analyze the data, effectively distinguishing black tea from different origins. A total of 83 significant flavor compounds were selected as potential markers using the VIP variable selection method and OAV screening. Cluster analysis was used to identify the characteristic flavor compounds in black tea samples from different origins. In addition, by investigating the climate environment of various production regions and analyzing the volatile components along with stable C and N isotope ratios of samples, it was discovered that the development of volatile components in black tea could be significantly influenced by ambient temperature and light. In regions with higher temperatures, the concentration of volatile components with floral, fruity, and sweet aromas is higher, resulting in a more intense aroma in black tea. In regions with low ambient temperatures but strong sunlight, black tea contains higher levels of components that give it a fresh and nutty aroma. As a result, the aroma of black tea is relatively elegant and fresh. At the same time, it was found that the stable isotope ratios of C and N in black tea were also significantly affected by ambient temperature and were positively correlated. This study introduces a fresh perspective for the comprehensive examination of Guizhou black tea, offering theoretical guidance for optimizing planting conditions and enhancing product quality. Its positive influence on promoting the development of the Guizhou black tea industry is noteworthy.

1. Introduction

Tea is among the most consumed beverages worldwide [1]. Tea contains many healthy active ingredients, such as polyphenols, caffeine, theanine, and theaflavins [2, 3]. According to the Food and Agriculture Organization of the United Nations statistics (FAOSTAT) database (https://www.fao.org/faostat/), China is one of the world’s largest tea producers and consumers, with a large production and market share of black tea. Guizhou Province is also among the major black tea-producing areas in China. In 2022, tea plantations in Guizhou covered an area of more than 4,666 square kilometers, with an output value of 57.09 billion Chinese yuan.

Tea’s flavor is the most important determinant of its quality. Flavor refers to tea taste and aroma, with volatile organic compounds (VOCs) being significant contributors to its characteristic flavor. The aroma of tea is predominantly made up of volatile flavor compounds [4]. Plant volatiles are mainly divided into terpenoids (with floral, fruity aroma), fatty acid metabolic compounds (alcohols, ketones, aldehydes, etc., with sweet aroma), and phenylalanine/aromatic cyclic metabolic compounds (including volatile phenylpropanoids and aromatic compounds with floral, nutty aroma) [1, 5]. The terpenoids are synthesized primarily through the methylerythritol-4-phosphate (MEP) or mevalonate (MVA) pathways. Fatty acid metabolic compounds are primarily synthesized through the degradation of the unsaturated C18 fatty acids linolenic and linoleic acid in the lipoxygenase (LOX) pathway [6]. Phenylalanine/aromatic cyclic metabolites are primarily synthesized through the shikimic acid pathway [7]. At present, it has been found that the synthesis pathways of the above volatile components will be affected by external environmental factors.

Ion mobility spectroscopy (IMS) is a highly effective and sensitive analytical technique based on ion mobility differences in the gas phase under a constant electric field. In addition, headspace-gas chromatography ion mobility spectrometry (HS-GC-IMS) combines the advantages of high resolution provided by gas chromatography and high sensitivity provided by ion transfer spectrometry [8–11] to rapidly detect trace VOCs in samples without requiring a sample pretreatment. Currently, HS-GC-IMS has been widely used in the detection of volatile components such as fruit juices [9], peppers [4], huajiao [12], green tea [13], and other foods.

In recent years, stable isotope technology has emerged as an excellent novel method for origin identification and has been widely utilized in various fields such as soil science, medicine, agriculture, biology, ecology, and environmental studies. The isotopic fractionation effect is influenced by various factors, including altitude, soil composition, water sources, topography, and atmospheric composition. These factors lead to variations in isotope ratios among organisms in different regions, contributing to distinct regional profiles in isotope ratios. Therefore, the detection of the isotope composition of organisms can reflect their growth environment and their characteristics as a result of adaptation to environmental changes. Moreover, since the stable isotope composition in living organisms is an intrinsic property that is not affected during processing or storage [14], stable isotope technology has also been used to identify tea-producing areas [15–18].

Numerous studies have been conducted on the identification and characterization of volatile compounds in tea to explore the mechanism of pleasurable aroma formation and reveal the relationship between tea metabolite composition and aroma production [10, 19–21]. However, there are few studies on the relationship between the volatile components of tea and environmental factors. In this study, HS-GC-IMS was used to analyze the characteristic flavor components of black tea and to investigate the key flavor components of black tea from various regions. According to the characteristics that the synthesis of volatile components and isotopic composition of plants are affected by natural factors in the plant environment, the influence of environmental factors on the unique flavor components of Guizhou black tea and the relationship between the isotopic composition and environmental factors were innovatively determined by the ratio of C and N stable isotopes in black tea samples. It is hoped that a correlation model can be established between the formation of Guizhou black tea flavor and environmental factors. This would provide a new research direction for the comprehensive study of Guizhou black tea and offer theoretical guidance for the future development of the black tea industry.

2. Materials and Methods

2.1. Samples

All black tea samples used in this experiment were collected in the spring of 2022 from four main black tea-producing areas in Guizhou and two main black tea-producing areas outside of Guizhou in China. The samples were collected by picking one bud, one leaf, one bud, and two leaves, and they were processed aseptically to make black tea using Chen’s methods [19]. The information on sampling locations is shown in Table 1 and Figure 1.

2.2. Headspace Gas Chromatography-Ion Mobility Spectrometry (HS-GC-IMS)

2.2.1. Instruments and Equipment

The FA2204 Electronic analytical balance was purchased from China, and the Flavor Spec® HS-GC-IMS was purchased from G.A.S. Germany.

2.2.2. HS-GC-IMS Determination Method

Prior to the experiment, the tea samples were processed as described previously with minor modifications [13, 22]. Accordingly, an HS-GC-IMS FlavourSpec® (GAS mbH, Dortmund, Germany) instrument was utilized for the study. About 1 g of samples was injected into a 20 mL headspace vial and incubated for 20 min at 85°C. Subsequently, 500 μL of headspace gas was automatically collected with a heated syringe (85°C) and was injected into the HS-GC-IMS for analysis. The samples were then carried into the capillary column (FS-SE-54-CB-1, 15 m × 0.53 mm, 60°C) by nitrogen (purity ≥99.999%) using the following flow procedure: initial flow rate was maintained at 2 mL/min for 2 min, increased to 10 mL/min at 10 min, then 100 mL/min at 20 min, and finally 150 mL/min at 30 min. After preseparation, analytes were ionized in positive ion mode using a 3H ionization source located in the ion mobility spectrometer ionization chamber and then transferred to the drift tube at 45°C under the drift gas (nitrogen, purity ≥99.999%) at 150 mL/min. The drift tube length was 98 mm, and its linear voltage was 500 V/cm. Volatile compounds were identified by comparing retention indices (RIs) and were then compared with the data obtained from the NIST17 mass spectral library and the IMS database (GAS mbH, Dortmund, Germany).

2.2.3. HS-GC-IMS Result Analysis

The VOC analysis software was used to inspect the analytical spectrogram to determine the retention and migration time for qualitative analysis. The internal standard method was used to calculate the substance content, and the content calculation formula is as follows:where is the concentration of volatile substances, is the concentration of standard substances, is the peak area of volatile substances, and is the peak area of standard substances.

In addition, the HS-GC-IMS Library Search software was compared with the NIST and IMS databases for qualitative analysis of characteristic flavor compounds. The 3D difference map, 2D overhead difference map, and fingerprint map of VOCs were constructed using Reporter and Gallery Plot. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) and Partial Least Squares Discriminant Analysis (PLS-DA) were used to analyze the differences between the sample groups. The key flavor substances were selected by calculating the volatile component VIP value and odor activity value (OAV) value.

2.3. Stable Isotope Analysis

2.3.1. Instruments and Equipment

The XP6 electronic balance was purchased from Mettler-Toledo International Inc in Switzerland; the DI/CF-MAT253 gas isotope ratio mass spectrometer was purchased from Thermo Fisher Scientific; and the YXQM-0.4L planetary ball mill was purchased from MITR in China.

2.3.2. Measurements of C and N Element Content and Their Stable Isotope Ratios

Tea samples were crushed with a ball mill and sieved through a 100-mesh screen prior to the experiment. Approximately 1 mg from each tea sample was weighed and transferred to a 3.5 × 5 mm pressed tin capsule. The samples were placed sequentially into the automatic solid sample tray of the element analyzer. After reduction resulting from combustion, the carbon and nitrogen elements in the samples were converted into pure CO₂ and N₂ gas. Then, the isotope ratio mass spectrometer was used for the detection of isotope composition.

The δ-value (‰) was determined by comparing the isotope value of the examined sample to internationally accepted standard materials. The isotopic values were expressed according to the following formula:where and are the isotopic ratios of the sample and the standard materials, respectively. Isotope standards IAEA-CH-3 and IAEA-N1 were used for the and calibration.

2.3.3. Meteorological Information Collection

GPS was used to locate and record the sampling locations’ longitude, latitude, and altitude. Moreover, the China Meteorological Science Data Center was queried for the average annual precipitation, average high temperature, average low temperature, and number of sunny days.

2.3.4. Data Analysis

The correlation model between the carbon and nitrogen isotope ratios and environmental factors was established by the partial least squares (PLS) method. The least squares method is a commonly implemented method in unitary linear regression analysis. It minimizes the sum of the squared errors between the observed and predicted values, generating a best-fitting line to obtain the functional relationship between the independent variable and the dependent variable. A quantitative prediction model was established to assess the relationship between carbon and nitrogen isotope ratios and environmental factors, using 8 environmental factors as independent variables and each stable isotope ratio as dependent variables. The model included 18 samples, of which 16 were randomly selected and used as the training set and 2 as the prediction sets. The model results were expressed in terms of coefficient of determination (R), root mean square error (RMSE), and relative prediction deviation (RPD) for all samples (where RPD = DS/RMSE).

The box diagrams and bar diagrams were created using the Origin 2021 software. OPLS-DA was conducted on the SIMCA 14.1 program, and VIP value, partial least squares, and PLS analysis were performed on XLSTAT.

3. Results and Discussion

3.1. Black Tea Volatile Flavor Compounds

3.1.1. Analysis of Volatile Compounds of Black Tea Samples from Different Regions by HS-GC-IMS

The volatile flavor compounds in 4 black tea samples from different regions in Guizhou and 2 black tea samples from regions outside of Guizhou were determined by GC-IMS. Using the Reporter plug-in of the included VOC analysis software, the volatile compounds of black tea from different regions were compared, and the results are shown in Figure 2.

(a)

(b)

(c)

The GC-IMS 3D spectra of black tea samples from 6 regions are shown in Figure 2(a). The variation in volatile compounds across different samples is clearly visible in the figure. The top view of the 2D GC-IMS diagram of black tea samples from 6 regions is shown in Figure 2(b). The background of the image is blue, with the vertical axis representing the retention time (s) of gas chromatography, and the horizontal axis representing the ion migration time. The red vertical line at 1.0 on the horizontal axis indicates the reaction ion peak (RIP). Both the migration time and RIP were normalized. Each peak on the left and right sides of the RIP corresponds to a volatile organic compound, and the color indicates the compound concentration: white indicates a lower concentration, red is a higher concentration, and a darker color corresponds to a greater concentration. As shown in Figure 2(b), most volatile compound signals of the black tea samples from 6 different Guizhou regions were in a retention time range of 0∼1750 s and drift time range of 1.0∼1.7 s, indicating that the volatile compounds of the black tea samples from different regions were highly similar. However, their content in the different regions was different. For example, the two GZZY samples were similar to WY and BS, while the GZAS, GZPA, and GZDY samples shared a higher similarity. The WY sample was taken as a reference to accentuate the differences, and the signal peak in the reference spectrum was deducted to obtain the differential spectral map between different samples (Figure 2(c)). The background was white after deduction, and red indicated that the corresponding compound content was higher than WY, while a darker color indicated greater differences and a blue color indicated that the corresponding compound content was lower than WY. As shown in Figure 2(c), the volatile compound contents of GZAS, GZPA, and GZDY were significantly different from those of WY and BS (red box in the figure). In contrast, the volatile compound contents of BS, GZZY, and WY were relatively low, with BS and WY still exhibiting some differences (green box in the figure). Finally, the volatile compounds of GZZY and WY samples were very similar. Therefore, overall, the differences between Guizhou tea samples and non-Guizhou tea samples are small (purple box in figure) and cannot be clearly distinguished.

3.1.2. Qualitative and Quantitative Analysis of Volatile Flavor Compounds

To more intuitively assess the changes in the content of volatile compounds in different samples, the retention time and migration time of volatile flavor compounds from samples from different regions were compared, and the HS-GC-IMS migration time data were compared with the NIST database to perform qualitative analysis of volatile compounds. Quantitative analysis was performed using the content calculation formula, and the results are shown in Table 2. A total of 184 volatile compounds were detected in the black tea samples, and 143 volatile compounds (including dimers and monomers) were identification through the database. As shown in Figure 3, there were 30 aldehydes (accounting for 21%∼27% of the total volatile compounds), 30 alcohols (16%∼24% of the total volatile compounds), 26 ketones (21%∼28% of the total volatile compounds), and 21 heterocyclic classes (7%∼14% of the total volatile compounds). There were 18 esters (4%–7% of the total volatile compounds), 9 terpenes (1% of the total volatile compounds), 5 acids (11%–21% of the total volatile compounds), and 4 sulfides (2%-3% of the total volatile compounds). The total volatile compound content in the GZZY, BS, and WY samples was higher than that in the other three samples, and the volatile compounds of the WY and GZZY samples were highly similar.

The Gallery Plot plug-in was used to plot the fingerprint spectrum of volatile compounds (Figure 4). Each row in the figure represents all the signal peaks identified in a sample, and each column represents the signal peaks of the same volatile organic compound in different samples, followed by M and D, which correspond to the monomers and dimers of the same compounds, and the number indicates the unidentified peaks.

The detailed volatile organic compound information for each sample and the differences in volatile content between samples are shown in Figure 4. Volatile compounds that were present in high amounts (in mg/kg) across all samples were as follows: Acetic acid (15.37∼30.05 mg/kg), Linalool (2.44∼15.53 mg/kg), 2-Octanone (0.59∼12.60 mg/kg), (E)-2-Hexenal (6.57∼10.57 mg/kg), Furfural (0.79∼9.83 mg/kg), (Z)-3-Hexenol (2.90∼7.46 mg/kg), 1-Hydroxy-2-propanone (2.19∼7.28 mg/kg), Acetone (3.97∼6.97 mg/kg), Hexanal (5.29∼6.61 mg/kg), 4-Methyl-3-penten-2-one (2.37∼6.42 mg/kg), Linalool oxide (0.64∼6.13 mg/kg), 1-Penten-3-ol (4.91∼5.93 mg/kg), 2-Butanone (4.10∼5.71 mg/kg), Dimethyl sulfide (3.57∼5.11 mg/kg), 1-Pentanol (2.44 to 4.60 mg/kg), and Propanal (3.48∼3.93 mg/kg). Among these compounds, Linalool, (E)-2-Hexenal, (Z)-3-Hexenol, Hexanal, Linalool oxide, and 1-Pentanol have been shown to be the main aroma compounds in black tea [19, 20, 25–30].

The types of volatile flavor compounds identified in the black tea from different regions or varieties were similar. However, they differed in relative abundance in the different samples (i.e., compounds that were only present in high amounts in a specific sample but very low or no amounts in other samples). For example, the characteristic compounds in the WY sample are 22, Dimethyl disulfide, Myrcene, 5-Methyl-3-heptanone, 6-Methyl-5-hepten-2-one, 2-Propanol, and 1-Hexano1-D. The distinct compounds in the BS samples were 33, 34, 35, and 36 and included Pyrrolidine, beta-Pinene, Diacetyl, 1-Penten-3-one-D, cis-4-Heptenal, Benzaldehyde, and Phenylacetaldehyde. The compounds characteristic of the GZZY sample are 12, Linalool oxide, 4-Methyl-2-pentanone, 3-Methyl-2-pentanone, 4-Methyl-3-penten-2-one, 2-Hexanone, Ethyl pentanoate-D, 1-Propanethiol, and 4-Methy1-1 pentanol. The characteristic compounds in the GZAS sample were Ethyl isobutyrate, 1-Octen-3-one, Camphene, Heptanal-D, and Octanal. The characteristic compounds in the GZPA samples are 1, 4, 26, 27, Styrene, o-Xylene, 2-acetylfuran, 2-Methylpyrazine, 2-acety1-1 pyrroline, 2, 3-dimethylpyrazine, Pyridine, 3-Ethylpyridine, 5-Methyl-2-furfural, Propanoic acid, 2-Methylpropanoic acid, 2-Cyclohexen-1-one, 5-Methyl-2 (3H)-furanone, Methyl acetate, (Z)-3-Hexenyl propionate, and 3-Methyl-3-buten-1-ol. The distinct compounds in the GZDY sample were 10, 39, 40, and Ethyl acetate.

3.1.3. Analysis of Volatile Aroma Compounds in Black Tea with OPLS-DA

OPLS-DA is a supervised discriminant analysis statistical method that can establish a relationship model between the samples and their volatile compound abundance to categorize them. The model evaluation parameters were the independent variable fit index (R2X), dependent variable fit index (R2Y), and model prediction index (Q2). R2X and R2Y represent the interpretation rate of the built model for X and Y matrices, respectively, and Q2 represents the prediction ability of the model. The model is considered highly reliable when all three indices approach 1. Q2 > 0.5 indicates a reliable model, and Q2 > 0.9 indicates a highly reliable predictive model.

The OPLS-DA model was established according to the volatile compounds identified in black tea samples from 6 different regions. The results are shown in Figure 5(a). The six samples could be distinguished effectively, and the model indices R2X = 0.983, R2Y = 0.997, and Q2 = 0.993 indicate a very high and reliable prediction ability. Thus, the model can be used to distinguish black tea samples from different regions. In addition, to prevent the model from overfitting the data, a replacement test (n = 200) was also carried out on the model. The results are shown in Figure 5(b). All the green R2 values and blue Q2 values on the left are lower than the original point on the right, and the intercept between the regression line and the vertical axis (on the left) of the Q2 point is negative, indicating that the model does not overfit and has good prediction ability. Thus, it can be used for discriminant analysis of aroma compounds from black tea samples of different origins.

(a)

(b)

3.1.4. Analysis of Volatile Aroma Compounds of Black Tea with PLS-DA

The 143 compounds identified in Table 2 were assessed with PLS-DA. PLS-DA is a supervised discriminant analysis method, a multivariate statistical analysis method, which can determine the classification of research objects according to the observed or measured values of several variables. PLS-DA maximizes the difference between groups according to a predefined classification (Y variable), achieving better separation results than principal component analysis.

According to the PLS-DA score plots (Figure 6), WY and GZZY were in close proximity, indicating that the two samples have similar aroma types. In comparison, the other four samples were relatively dispersed and independent, indicating that the model can distinguish different black tea origins.

WY black tea is known for its floral, fruity, and dried longan aroma, while GZZY black tea is known for its fruity and sweet aroma. As is shown in Figure 6, Terpinolene, Limonene141, Butanal, 2-Hexenal, 3-Methyl-1-butanol, 4-Methyl-1-pentanol, 2-Propanol, Diethyl acetate, gamma-Terpinene, 1-Pentanol-D, 2-Methyl-2-pentenal, Hexyl propionate, Ethyl crotonate, 2-Heptanone, and Butyl acetate were significantly correlated with the aromas of WY and GZZY samples, which exhibited strong fruity and floral aroma, in line with the common aroma characteristics of WY and GZZY samples. The BS sample was highly correlated with Diacetyl, Methyl hexanoate, Heptanal, beta-Pinene, Nonanal, etc., which are associated with sweetness and woody fragrance. The PA sample was correlated 2-cyclohexen-1-one, 5-Methyl-2 (3H)-furanone, 2, 3-dimethylpyrazine, 3-Ethylpyridine, 2-Methylpyrazine, and Propanoic acid, which give a floral and sweet aroma. The GZDY sample was highly correlated with 1-Penten-3-ol, 2-Methyl-1-propanol, 2-Methylpropanal, Acetic acid, Dimethyl sulfide, and Ethyl acetate. These compounds potentially give GZDY black tea a subtle floral and fruity aroma.

3.1.5. Analysis of Key Flavor Compounds of Black Tea from Different Guizhou Regions

In order to further analyze the contribution rates of key flavor compounds of black tea products from 4 Guizhou regions and 2 regions outside of Guizhou, a variable importance (VIP) >1 and a predicted value <0.05 were used to identify the key volatile compounds. A total of 50 flavor compounds were screened and are listed in Figure 7. They include 13 aldehydes, 10 alcohols, 7 ketones, 6 terpenes, 5 esters, 5 heterocycles, 3 sulfur-containing compounds, and 1 acid.

The OAV is the ratio of the concentration of each compound to its detection threshold. The odor activity value can determine the contribution of each volatile compound in the sample to the overall aroma. When OAV ≥1, the aroma compound can theoretically be considered to contribute to the overall aroma. Moreover, the higher the OAV, the larger the contribution to aroma. Using the threshold values of related compounds provided by references [31, 32], the OAV of all volatile compounds was calculated. Finally, 83 compounds with OAV ≥1 in at least one sample were obtained, including 1 acid, 13 alcohols, 22 aldehydes, 11 esters, 11 heterocyclic compounds, 16 ketones, 3 sulfides, and 6 terpenes. When a volatile compound has an OAV ≥100 in the sample, it can be considered to have a greater contribution to the overall aroma. Therefore, all the volatile compounds with VIP greater than 1 or OAV ≥100 in the sample were considered as the main aroma compounds of black tea (68 in total), and cluster analysis was conducted accordingly (Figure 3). A stronger correlation between the compound and the overall aroma of the sample is indicated by a lighter color, while weaker correlations are indicated with darker colors.

As shown in Figure 8, the cluster analysis showed that GZZY and WY samples had a high similarity. The GZDY and GZPA samples were also similar, while GZAS samples exhibited some overlap with the GZZY, WY, GZDY, and GZPA samples, while the BS samples were distinct.

At the same time, although no significant difference in volatile compounds was found between Guizhou black tea samples and non-Guizhou black tea samples, it can be seen from Figure 8 that although there was a certain overlap between samples from the same production area, each sample had distinct volatile compounds that were highly correlated with that particular sample, and these patterns had a high similarity with the PLS-DA results. In accordance with these criteria, the distinct compounds present in each sample could be the ones that give the characteristic aroma of each sample. For example, the compounds with high correlation with the WY sample were Dimethyl disulfide, 1-Octen-3-one, 2-Octanone, 2-Methylisoborneol, Butyl acetate and Linalool, 1-Octen-3-ol, 2, 3-diethyl-6-methylpyrazine, 2-propanol, 1-Hexanol, alpha-Terpinene, 2, 5-dimethylpyrazine, and 5-Methyl-3-heptanone. Most of these compounds have a floral and fruity aroma, while some have a nutty aroma and sour taste and may be the source of the dry longan aroma in WY black tea. Among them, Dimethyl disulfide, 2-Propanol, 1-Hexanol, alpha-Terpinene, 2, 5-dimethylpyrazine, and 5-Methyl-3-heptanone had the strongest correlation with the WY sample. These compounds may be the distinct flavor compounds in WY samples. Compounds with high correlation with the BS sample included Cyclopentanone, Benzaldehyde, cis-4-Heptenal, 1-Penten-3-one, Diacetyl, beta-Pinene, Phenylacetaldehyde, (E, E)-2, 4-Hexadienal, 2-Octanone (2-Methylisoborneol, Butyl) acetate, (E)-2-Hexenal, 1-Octen-3-ol, (E)-2-Octenal, Butanal, 3-Methyl-1-butanol, 2-Methylpropanal, Pentanal, and Heptanal. The aroma of these compounds may serve as the source of BS black tea’s rich, sweet, fruity, and fragrant aroma. cis-4-Heptenal, 1-Penten-3-one-D, Diacetyl, beta-Pinene, Phenylacetaldehyde, and (E, E)-2, 4-Hexadienal had the highest correlation with the BS samples. Therefore, these compounds may be the distinct flavor compounds in BS samples. The compounds with high correlation with the GZZY samples were 2-octanone, 2-methylisoborneol, Linalool, 2, 3-diethyl-6-methylpyrazine, (E)-2-Octenal, Limonene, 4-Methyl-3-penten-2-one, 2-Pentylfuran, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, Ethyl pentanoate, Butanal1-Pentanol, Acetophenone, cis-2-Penten-1-ol, Dimethyl sulfide, Acrolein, 1-Penten-3-ol, Nonanal, and 5-Methyl-3-heptanone. Most exhibit floral and fruit aromas and may be the source of the intensely floral and fruitarian aromas of GZZY samples. The correlation between Limonene, 4-Methyl-3-penten-2-one, 2-Pentylfuran, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, Ethyl pentanoate, and GZZY samples was the strongest, which suggested that these compounds might be the compounds giving the characteristic aroma in GZZY tea samples. The compounds with high correlation with GZAS samples were 1-Octen-3-one, Cyclopentanone, Benzaldehyde, Cism-4-Heptenal, 3-Methyl-1-butanol, 2-Butylfuran, 1-Butanol, 1-Pentanol, 3-Methyl-2-butenal, Pentanal, Heptanal, Propanal, cis-2-Penten-1-ol, Methional, 1-Penten-3-ol, Thiophene, Octanal, (E)-2-Pentenal, Ethyl isobutyrate, and Camphene, most of which confer a grassy aroma alongside with floral and fruity notes and potentially contribute to the fresh smell of the GZAS samples. Among them, Limonene, 4-Methyl-3-penten-2-one, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, and Ethyl pentanoate had the strongest correlation with the GZAS samples. Thus, they might be the key contributing volatiles to the characteristic aroma of the GZAS samples. The compounds with high correlation with the GZPA samples were 2-Methylpropanoic acid, (E)-2-Nonenal, o-Xylene, Methyl acetate, Hexanal, 1-Propanol, 2-Butanone, 3-Methyl-1-butanol, 3-Methyl-2-butenal, 2-Methylpropanal, 2-Acetyl-1-pyrroline, Pyridine, and 3-Hydroxy-2-butanone, which contribute to the sweet smell of the PA black tea. 2-Methylpropanoic acid, (E)-2-Nonenal, o-Xylene, Methyl acetate, Hexanal, 1-Propanol, and 2-Acetyl-1-pyrroline exhibited the strongest correlation with the GZPA samples. Thus, they might be the key contributing volatile compounds to the aroma of the GZPA tea samples. The compounds with high correlation with the GZDY sample were 3-Methylbutanal, 2-Methylpropanal, Pentanal, 3-Hydroxy-2-butanone, Acetophenone, 3-Carene, Ethyl acetate, CS-2-penten-1-OL, Methional, Dimethyl sulfide, and Nonanal which can bring various flavors to GZDY black tea, including floral, fruity, nutty, and sweet flavors. Among them, 3-Methylbutanal, 3-Carene, and Ethyl acetate strongly correlated with the characteristics of the GZDY sample, suggesting that these compounds might be the distinct aroma compounds in the GZDY sample.

3.1.6. Summary of the Volatile Compound Profiles in Different Black Tea Samples

In summary, GC-IMS technology was implemented to determine volatile compounds from 4 black tea samples from Guizhou and 2 black tea samples from regions outside of Guizhou. A total of 184 volatile compounds were detected, and 143 compounds were annotated through database comparisons. Among them, 30 aldehydes (accounting for 21%∼27% of total volatile compounds), 30 alcohols (16%∼24% of total volatile compounds), 26 ketones (21%∼28% of total volatile compounds), 21 heterocyclic compounds (7%∼14% of total volatile compounds), and 18 esters (4%∼7% of total volatile compounds), 9 terpenes (1% of total volatile compounds), 5 acids (11%∼21% of total volatile compounds), and 4 sulfides (2%∼3% of total volatile compounds) were analyzed by OPLS-DA. The results showed that OPLS-DA could effectively distinguish the black tea samples from different regions. By calculating the VIP and OAV values of the volatile compounds in the samples, a total of 83 important volatile aroma compounds were selected. After analysis by PLS-DA and cluster analysis, the distinctive aroma compounds of black tea samples from different regions in Guizhou were identified. The analysis of the main aroma compounds in the black tea samples from different regions revealed that three samples, GZAS, GZPA, and GZDY, had high similarity. Similarly, the GZZY and WY samples had a high similarity, consistent with the OPLS-DA and PLS-DA results. At the same time, although no significant difference in volatile compounds between Guizhou black tea samples and non-Guizhou black tea samples was found, distinct flavor compounds were identified in black tea samples from different regions. The distinctive aroma compounds in the WY sample were Dimethyl disulfide, 2-Propanol, 1-Hexanol, alpha-Terpinene, 2, 5-dimethylpyrazine, and 5-Methyl-3-heptanone. The distinctive aroma compounds in BS samples were cis-4-Heptenal, 1-Penten-3-one-D, Diacetyl, beta-Pinene, Phenylacetaldehyde, and (E, E)-2, 4-Hexadienal. The distinctive aroma compounds in the GZZY sample were Limonene, 4-Methyl-3-penten-2-one, 2-Pentylfuran, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, and Ethyl pentanoate. The characteristic aroma compounds in the GZAS sample were Limonene, 4-Methyl-3-penten-2-one, 2-Pentylfuran, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, and Ethyl pentanoate. In GZPA samples, the distinctive aroma compounds were 2-Methylpropanoic acid, (E)-2-Nonenal, o-Xylene, Methyl acetate, Hexanal, 1-Propanol, and 2-Acetyl-1-pyrroline, while in the GZDY sample, the characteristic aroma compounds were 3-Methylbutanal, 3-Carene, and Ethyl acetate.

3.2. Effects of Environmental and Meteorological Factors on the Formation of the Characteristic Flavor of Black Tea

Through HS-GC-IMS analysis, the distinctive flavor components of black tea samples from various origins were identified. However, it was also observed that the similarity in volatile components between the GZZY sample, a black tea produced in Guizhou Province, and the WY sample, a black tea produced in Fujian Province, was higher than that of other black teas from Guizhou Province. To investigate this phenomenon further, the researchers conducted the following study.

3.2.1. Effects of Environmental Factors in Different Black Tea Regions on Volatile Components of Black Tea

Through the weather website, we checked the annual climate conditions of each producing area. It was found that the sample producing areas with similar volatile components had similar climate conditions. Therefore, it was speculated that environmental factors may be one of the reasons for the difference in volatile components of black tea.

At present, studies have shown that the synthesis of volatile components in plants is affected by environmental factors. For example, high temperature can promote the synthesis and subsequent release of floral and fruity terpene compounds by inducing and increasing the activity of enzymes related to MEP and MVA pathways. It can also significantly express LOX in plants, promoting the accumulation of related metabolites (alcohols, ketones, aldehydes, etc.), most of which can bring a sweet odor [33]. The average temperature of the WY region and GZZY region is higher than that of the other four regions. Therefore, the content of related volatile components in the WY sample and GZZY sample is significantly higher than that of the other four samples, making their floral, fruity, and sweet fragrance stronger. The shikimic acid pathway in plants is inhibited under long-term high-temperature conditions but can be activated under intense light and low-temperature conditions, thus promoting the synthesis of phenylpropane and aromatic compounds with sweet, floral, and nutty aromas in plants [34]. GZAS, GZPA, and GZDY, thanks to their high altitude, low average temperatures, and abundant sunlight, produce black teas that are fresh and light, with some nutty notes. This provides theoretical guidance for the study of the flavor of black tea produced in Guizhou. However, as GC-IMS technology is an emerging technology, its database is not complete and refined, and there are still many compounds that cannot be annotated, so it is impossible to know whether these compounds are the key flavor compounds in black tea. We anticipate that GC-IMS results will become more precise as technology advances and GC-IMS data become more and more refined.

3.2.2. Relationship between C and N Stable Isotope Composition and Environmental Factors in Black Tea

Environmental and meteorological factors not only affect the synthesis of volatile aroma compounds but also have a great influence on the fractionation of stable isotopes in plants. Different environmental meteorological factors can alter the ratio of stable isotopes in plants. For example, the carbon isotope fractionation of carbon assimilated in organisms is affected by multiple environmental factors such as temperature, precipitation, light, and atmospheric pressure [35]. Specifically, in arid areas with low air humidity and strong evaporation, plants reduce stomatal conductance, decreasing intercellular CO₂ concentration and increasing the value of carbon assimilated by photosynthates. During respiration, temperature also affects the CO₂ produced by the leaves and the values of the main organic compounds of the leaves [35]. Nitrogen is an important biological element and one of the key components of amino acids, significantly impacting organisms’ growth and development [36]. Nitrogen isotope fractionation in plant tissues is mainly affected by soil properties, fertilization, altitude, temperature, and other factors [37, 38]. For example, when the ambient temperature increases, the biological activity of nitrifying bacteria and ammoniating bacteria in the soil is enhanced. This accelerates the rate of soil mineralization and nitrification, leading to an increase in the content of soil ¹⁵N, consequently raising of plants [39–41].

Based on the aforementioned as a preliminary reference, it is hypothesized that there might exist a potential correlation between the volatile components of black tea and its internal isotopic compositions. By measuring the and values of black tea samples from six distinct regions, in conjunction with local environmental factors, we assessed the relationship between environmental factors and isotope fractionation across different regions, with the aim of establishing a relational model capable of reflecting both the isotope ratios and volatile composition of black tea.

The stable isotope ratios of black tea samples from 4 Guizhou regions and 2 non-Guizhou regions were obtained, as shown in Table 3. In addition, the values of the samples from Guizhou province ranged from −28.71‰ to −25.37‰, while the values ranged from 0.75‰ to 3.31‰. The values of the WY samples ranged from −26.11‰ to −25.52‰, while the values ranged from 2.77‰ to 2.99‰. Furthermore, the and values of BS samples ranged between −27.53‰ and −26.44‰ and between 0.02‰ and 0.22‰, respectively.

In order to investigate the impact of environmental and meteorological factors on the ratio of stable isotopes of carbon and nitrogen in plants, a quantitative prediction model was developed using PLS analysis of the environmental meteorological factors and the values in each sample region (Figure 9). The quantitative prediction model results are shown in Table 4. As and RPD increase, the RMSE decreases, and the model accuracy improves. The values of the and models were 0.09 and 0.63, respectively. The RMSE values were 0.94 and 0.64, respectively, and the RPD values were 0.94 and 1.06, respectively. This indicates that the prediction model based on eight environmental and meteorological factors is more accurate than the prediction model.

(a)

(b)

(c)

(d)

Further analysis of the importance of the model variables shows that if the VIP value of the environmental factor is all greater than 1, it indicates that the environmental factor has a significant impact on the ratio of carbon and nitrogen isotopes. The results are depicted in Figure 9. Variables with VIP values of carbon isotope ratios greater than 1 include annual rainfall, mean low temperature, mean high temperature, and altitude, indicating that temperature, rainfall, and altitude have significant effects on values. In addition, the positive correlation coefficients of the four factors showed that the value of black tea was positively correlated with the temperature and rainfall of the producing area and negatively correlated with the altitude of the producing area. Regarding the values, four environmental factors, total rainfall, number of sunny days, number of cloudy days, and mean low temperature, had VIP values greater than 1. Among the four factors, only the normalized correlation coefficients of number of sunny days were negative, while those of the other three factors were positive. This result shows that the value is affected by several factors, such as precipitation temperature and sunshine.

At present, a large number of studies show that when the environmental precipitation decreases or the soil moisture decreases, the water stress increases. In order to reduce water transpiration loss in the body, plants often close part of their stomata, reducing stomatal conductance and intercellular CO₂ concentration, resulting in an increase in plant [42]. However, when the precipitation is too high or the soil moisture is too high, the soil microbial activity decreases, the respiration rate decreases sharply, the soil nitrification is inhibited, and the availability of soil inorganic nitrogen decreases, thus making the soil ¹⁵N poor and resulting in the decrease of plant [43]. According to Wang et al. [44], as the altitude increases, the temperature decreases, the atmospheric CO₂ partial pressure decreases, and the amount of CO₂ available to plants decreases. Plants, due to insufficient CO2 supply, cause carboxylase enzymes to be unable to fractionate 13C, leading to a direct synthesis of organic compounds, thereby resulting in an increase in plant .

In this study, the and values of black tea are positively correlated with the temperature of the producing area, which is consistent with the results of Julien et al. [35]. However, the correlation results of and values of black tea with environmental elevation and rainfall are different from the current mainstream research results. Water et al. [45] believe that the positive ratio of value to environmental elevation may be due to the influence of regional water use efficiency and drought stress elimination. In addition, Schulze [46], Heaton [47], and Codron [48] also found that the and values in plants were positively correlated with regional rainfall under some special circumstances. To sum up, the effects of other factors on the stable isotope ratio of black tea may be superimposed, making the situation more complicated. Therefore, the mechanism of how various environmental factors affect the stable isotope ratio of black tea still needs to be further studied.

3.3. Summary of the Effects of Environmental Factors on the Formation of Volatile Components in Black Tea

According to the environmental and meteorological conditions of six black tea samples and the variation in volatile components in each sample, it was observed that the volatile components of black tea were significantly influenced by temperature, sunlight, and other factors. The levels of terpenoids, alcohols, ketones, and aldehydes in black tea samples were found to be higher in regions with elevated temperatures. These compounds contribute to the stronger floral, fruity, and sweet flavors characteristic of teas produced in these regions. In regions with slightly lower average temperatures and abundant sunshine, the plants contain higher levels of substances with fragrant, floral, and nutty aromas. This results in tea produced in these areas having a lighter, sweeter smell, a fresher aroma, and a hint of nuttiness.

In addition to the volatile components, external environmental factors also influence the internal isotopic composition of tea. The and values of black tea samples from six regions were detected, and a PLS-VIP regression model was used for analysis. The results showed that the value of black tea samples was mainly affected by the temperature of origin, altitude, and rainfall. It was positively correlated with temperature and rainfall but negatively correlated with altitude. The value is mainly affected by precipitation, temperature, and sunshine. It is positively correlated with precipitation and temperature but negatively correlated with sunshine.

Combined with the analysis of the relationship between the two and the environmental factors in the producing area, it was found that the content of volatile components in black tea samples was positively correlated with and due to the environmental temperature in the producing area, indicating that the stable isotope ratio in black tea samples may reflect the content of volatile components. However, due to the small sample size, this conclusion needs to be further studied after expanding the sample size.

4. Conclusion and Discussion

In this study, GC-IMS technology was implemented to determine volatile compounds from 4 black tea samples from Guizhou and 2 black tea samples from regions outside of Guizhou. A total of 184 volatile compounds were detected, and 143 compounds were annotated through database comparisons. The data were analyzed by OPLS-DA, and the results showed that the HS-GC-IMS could effectively distinguish black tea samples from different regions. By calculating the VIP and OAV values of the volatile compounds in the samples, a total of 83 important volatile aroma compounds were selected. After analysis by PLS-DA and cluster analysis, the distinctive aroma compounds of black tea samples from different regions in Guizhou were identified. The distinctive aroma compounds in the GZZY sample were Limonene, 4-Methyl-3-penten-2-one, 2-Pentylfuran, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, and Ethyl pentanoate. These substances give GZZY samples strong floral and fruity aromas. The distinctive aroma compounds in the GZAS sample were Limonene, 4-Methyl-3-penten-2-one, 2-Pentylfuran, 1-Propanethiol, Ethyl butanoate, gamma-Terpinene, and Ethyl pentanoate. These substances give the GZAS sample a fresh grassy, floral, and fruity aroma. The characteristic aroma compounds in GZPA samples were 2-Methylpropanoic acid, (E)-2-Nonenal, o-Xylene, Methyl acetate, Hexanal, 1-Propanol, and 2-Acetyl-1-pyrroline. These substances have a strong sweet fragrance, which makes the GZPA sample smell with some honey odor; and in the GZDY sample 3-Methylbutanal, 3-Carene and Ethyl acetate. In addition, among the black tea samples from the other two regions, the characteristic flavor substances in WY black tea are Dimethyl disulfide, 2-Propanol, 1-Hexanol, alpha-Terpinene, 2, 5-Dimethylpyrazine, and 5-Methyl-3-heptanone. These ingredients give WY sample a special dry longan aroma. The characteristic flavor substances in BS black tea samples were cis-4-Heptenal, 1-Penten-3-one-D, Diacetyl, beta-Pinene, Phenylacetaldehyde, and (E, E)-2, 4-Hexadienal which gave BS black tea samples a strong fruity flavor.

In addition, through the investigation of the climate environment of different producing areas, combined with the analysis of the volatile component content and stable C and N isotope ratios in each sample, it was found that the formation of volatile components in black tea was greatly affected by ambient temperature and light. In the regions with higher temperature, the volatile components with floral, fruity, and sweet aroma were higher, and the black tea had a stronger aroma. In the regions where the temperature is low but the light is strong, the fragrant and nutty components in black tea are higher, and the aroma of black tea is relatively elegant and fresh. At the same time, it was found that the stable C and N isotope ratios in black tea were also significantly affected by ambient temperature and were positively correlated.

This study examined how characteristic flavor compounds and environmental factors affect the flavor of Guizhou black tea. It offers a theoretical framework for optimizing planting conditions and enhancing product quality. Additionally, it sets a new direction for in-depth research on Guizhou black tea and contributes positively to the industry’s development.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Yonghui Ge was responsible for project administration, supervision, and review and editing. Yongji Huang was responsible for investigation, review and editing, visualization, software, and manuscript revision. Ling Wang was responsible for conceptualization, methodology, software, validation, formal analysis, original draft preparation, and visualization. Luyu Jia was responsible for investigation, resources, and validation. All authors have read and approved the manuscript.

Acknowledgments

We express our gratitude to Dr. Jing Tian from the Institute of Geochemistry, Chinese Academy of Sciences, for the technical support provided to this research. This work was supported by the Project of Development of Guizhou Functional Sauce-Flavor Baijiu Formulated and By-Products, the Project Funded by Guizhou Mingguan Biotechnology Co., LTD; and the Guizhou Forest and Grass Development Co., LTD. “Research and Application of microbial organic Fertilizer of Paulownia Meal against Solanaceae Plant Pathogens” project; (GZ-LFGS-HZ135-009) and the PhD start-up grant (GYU-ZRD (2018)-009), the Project Funded by Guiyang University; and the Guizhou Province Biological and Pharmaceutical Engineering Research Center (Grant No. QJHKY2019051), the Discipline and Master’s Site Construction Project of Guiyang University by Guiyang City Financial Support Guiyang University (Grant No. SY-2020), Guiyang University Idesia Polycarpa Maxim Processing Engineering Technology Research Center.

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Copyright © 2024 Yonghui Ge et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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