Study of characteristic aroma components of baked Wujiatai green tea by HS-SPME/GC-MS combined with principal component analysis

ABSTRACT In this study, the aroma components of six Wujiatai green teas were extracted and identified using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS), and principal component analysis (PCA) was further used to reveal possible differences in these teas based on their aroma components. The results showed that, although there are some similarities in the aroma composition and content between Wujiatai green tea and regular green and black teas, the former had its own unique aroma characteristics. We suggest that the ‘baking’ procedure of Wujiatai green tea possibly causes the formation and/or increase of some aroma components, thus resulting in a more durable and prominent aroma characteristic as well as superior quality compared with the other teas investigated in this paper.


Introduction
Wujiatai green tea is a very famous tea in Xuanen City, Hubei Province, China and it has enjoyed an enviable reputation as a tribute tea from as early as the Qing dynasty (A. D.1636~1911) in ancient China due to its unique aroma and taste. Because of its high historical, scientific, and cultural value, Wujiatai green tea became a nationally registered product in 2008. With the quality of natural floral or cooked Chinese chestnut flavor and a sweet and infusionenduring taste, Wujiatai green tea is a favorite among consumers and has a good reputation both at home and abroad. China's Ministry of Agriculture awarded it the certificate, 'registration of geographical indication of agricultural product' in 2009. It has also attained the domestic good agricultural practices certification and foreign organic food certification (e.g. European Union and United States). The processing technology for Wujiatai green tea involves spreading out of fresh leaves, mechanical water removal, rolling, machine forming, and drying. Additionally, Wujiatai green tea undergoes the unique process of 'baking', which is usually applied to the production of the black tea but not to ordinary green teas. It has been shown that the 'baking' process is the most important step in improving the flavor of Wujiatai green tea (Nie & He, 2003).
Although volatile compounds represent only about 0.01% of the dry weight of tea, they play an important role in determining the flavor due to their low threshold values for high odor sensibility, thereby contributing about 40% of the quality (Rawat et al., 2007). Tea flavor is generally determined by chemical and biochemical transformation during cultivation, production, processing, and post-fermentation. Among these, processing is of vital importance in regard to the final flavor of the tea (Yang, Baldermann, & Watanabe, 2013). Differences in aroma composition and content among various teas have been attributed to differences in aroma (Lee et al., 2015). GC-MS is a powerful technique that has been widely used to isolate and identify the volatiles present in plants (Shao et al., 2014;Wang & Huang, 2015). HS-SPME is a fast, efficient, and solvent-free alternative to the conventional technique of volatile extraction (Bhattacharjee et al., 2011). SPME is mainly based on the establishment of an equilibrium between the sample matrices, the headspace above the sample, and a polymercoated fused fiber, following desorption of the analysates from the fiber and subsequent GC-MS analysis. This method has been widely used to extract volatile components in various types of food in the past few decades, such as honey (Bianchin et al., 2014), beer (da Silva et al., 2015, grapes (Sánchez-Palomo, Diaz-Maroto, & Perez-Coello, 2005), and various teas (Lin et al., 2013;Lv et al., 2015).
The final processing step of 'baking' accentuates the aroma of Wujiatai green tea in comparison to ordinary green teas, making it not dissimilar to black tea. To our knowledge, differences in the aroma components between Wujiatai green tea and other green teas or black teas belonging to the same production place (Hubei Province in China) have not yet been investigated. The 'baking' process for Wujiatai green tea that results in these chemical differences and aroma similar to black tea should be further investigated. In addition, the current evaluation of tea quality is mainly based on local and qualitative standards, and therefore a quantitative chemical standard needs to be established. It is thus important to identify the aroma-producing elements and clarify their roles in determining the individuality and quality of teas, in order to better understand what makes Wujiatai green tea so popular.
To investigate the differences in aroma determinants between Wujiatai green tea and other green teas or black teas, and further to address the problems mentioned above, these teas were subjected to fully automatic HS-SPME and GC-MS analyses. The obtained volatile profiles were further analysed using the principal component analysis (PCA) method to elucidate the characteristic differences in aroma composition and content. Our study sheds light on the chemical features responsible for aroma properties and provides a theoretical basis for the standard production and quality control of Wujiatai green tea.

Materials
The six Wujiatai green teas tested were from Xuanen City, and three ordinary green teas and three black teas were from Yichang City, Hubei Province. All were produced in 2014. The processing methods are shown in Figure 1.

HS-SPME method
Fiber coatings (65 µm polydimethylsiloxane/divinylbenzene (PDMS/DVB)) were purchased from Supelco (Bellefonte, PA, USA). The HS-SPME method was developed according to our previous study . All teas were powdered and filtered through a size 80 mesh sieve. Ground samples (2.0 g) were weighed and placed in a 20 mL sealed headspace vial, followed by an infusing of 5 mL boiling water. The HS-SPME procedures were performed using a Combi-PAL autosampler (Varian Pal Autosampler, Berne, Switzerland). Samples were stirred continuously at 250 rpm for 60 min at 80°C. After extraction, the fiber was removed from the vial and immediately inserted into the GC-MS injector for absorbance (250°C for 3.5 min) and further analyses.

GC-MS analysis
A 7890A GC system (Agilent Technologies, CA, USA) combined with a 5975C MSD (Agilent Technologies) was used for GC-MS analysis. The chromatographic column was an HP-5MS column (30 m × 0.25 mm × 0.25 µm film thickness), with high-purity helium as the gas carrier, at a flow rate of 1 mL/min. The injector temperature was 250°C and it was equipped with a splitless injector. The temperature was set initially to 50°C (held for 1 min), was increased to 210°C at 3°C min −1 (held for 3 min), then increased further to 230°C at 15°C min −1 . The MS ion source temperature was 230°C, and   Figure 1. Major processing steps for production of the three different teas.
electron energy 70 eV. The scan range was 35-500 amu and the solvent delay time was 2.8 min.

Data processing
Chromatographic peaks were recognized and identified by the NIST08.L MS data library, and retention indices (RI) were compared to previous literatures (Lin, Dai, Guo, Xu, & Wang, 2012;Shi et al., 2014). The relative content of the volatiles was obtained using the following method: Relative contentð%Þ ¼ single constituent area=total area Â 100% The RI of each compound was obtained using 1 µL n-alkane mixture (C8-C40; Sigma-Aldrich, USA) under the same GC-MS experimental conditions. Duncan's multiple range test was used to determining the significant differences among groups of teas using SPSS 16.0 software. PCA and CA were performed using SIMCA-P software (version 13.0, Umetrics, Umea, Sweden). The data were preprocessed by mean-centering and Pareto scaling prior to analysis.

Analysis and comparison of characteristic aroma components between Wujiatai green tea and other green/black teas
The volatile compounds were separated on a HP-5 column and identified by GC-MS. The identified volatile compounds and their RI are shown in Table 1. The odor description as given by panelists during gas chromatograph-olfactometry (GC-O) in the relevant literatures is listed in Table 1. The results show that a total of 94 volatile components were identified and quantified in three different types of tea: 69 in six Wujiatai green teas, 68 in three ordinary green teas, and 71 in three black teas. Alcohols and hydrocarbons were the most abundant compounds in Wujiatai and ordinary green teas, while alcohols and aldehydes were the most abundant compounds in black teas. Figure 2 shows typical GC-MS total ion chromatogram (TIC) levels extracted from three teas. A total of 12 alcohol compounds were identified in six Wujiatai green teas, representing 61.87% of the total content of the volatile components. Among identified alcohol compounds, the content of phytol (no. 19) was higher in Wujiatai green tea (37.52%) and ordinary green tea (7.39%) than in black tea (1.57%), while the content of geraniol (no. 14) was higher in black tea (29.46%) than in Wujiatai green tea (14.96%) and ordinary green tea (5.70%). The alcohols from fatty acid derivates were relatively low in all types of tea tested. Phytol, which has a typical flowery fragrance (Jumtee, Komura, Bamba, & Fukusaki, 2011), was found at highest concentrations in Wujiatai green tea whereas its content was only 7.39 and 1.57% in ordinary green tea and black tea, respectively. In Wujiatai green tea and black tea, geraniol (no. 14) and linalool (no. 8) both had a higher content than in ordinary green tea, demonstrating that these two compounds may have a major contribution to the flavor of Wujiatai green tea and black tea. These two kinds of terpene alcohol usually have a typical flowery fragrance with high aromatic value, thus possibly contributing significantly to the aroma of the tea (Guth & Grosch, 1993;Hattori, Takagaki, & Fujimori, 2003). This result also showed that the 'baking' process of Wujiatai green tea may give it similar aromatic characteristics to black tea. The content of nerolidol (no. 15) in Wujiatai green tea was significantly lower than in the other two types of tea. Note that nerolidol (no. 15) content was found to be high in half-fermented oolong teas , and therefore it seems likely that its formation is related to the particular processing technology used.
The content of hydrocarbons in Wujiatai green tea (15.82%) and ordinary green tea (20.36%) showed no significant difference, but was significantly higher than in black tea (2.36%). Usually, alkanes and polycyclic aromatic hydrocarbons, such as tridecane (no. 31), tetradecane (no. 36), pentadecane (no. 40), and anthracene (no. 49), have no effect on tea flavor while terpene compounds possibly play an important role (Alasalvar et al., 2012). These findings show that the terpene compounds may make a major contribution to the aroma of green tea. The level of D-limonene (no. 23) was 9.00% in ordinary green tea but very low in the other two types. We speculated that the low content of this compound in Wujiatai green and black tea may be associated with the processing technology of these two teas (i.e. the 'baking' and fermentation processes resulting in the loss of D-limonene). In addition, in Wujiatai green tea the contents of δ-cadinene (no. 42) and ylangene (no. 34) were higher than in the other two teas, and certain sesquiterpene components, such as α-cedrene (no. 37), β-caryophyllene (no. 38), α-elemene (no. 39), and cuparene (no. 43), were detected only in Wujiatai green tea. Therefore, it is likely that these sesquiterpene compounds make some contribution to the special fragrance of Wujiatai green tea.
Ten aldehydes were found in the six Wujiatai green teas, but with lower content compared with the black teas. These results are consistent with those of K. Wang et al., (2011), who reported that the main constituents of black tea are aldehydes. In addition, 2-phenyl-2-butenal (no. 62), (E,E)-2,4-decadienal (no. 62), 2-butyl-2-octenal (no. 64), and 5-methyl-2phenyl-2-hexenal (no. 66) were detected only in black tea. The content of aldehyde compounds may be related to the fermentation process, as noted by an obvious increase in hexanal concentration after fermentation (L.-F. Wang et al., 2008).
The ketone content was relatively low in all teas tested, but was slightly higher in ordinary green tea (10.62%) than in Wujiatai green tea (5.22%) and black tea (5.52%). The most abundant compounds among the ketones identified in Wujiatai green tea were β-ionone (no. 74), cis-jasmone (no. 70), and geranyl acetone (no. 72). No significant differences were found in regard to the contents of β-ionone (no. 74) and geranyl acetone (no. 72). Ketones have been shown to make a significant contribution to the aroma of tea due to their overall low aroma threshold values and high flavor factors (FD) value (Kanasawud & Crouzet, 1990;Schuh & Schieberle, 2006).
The content of ester compounds in Wujiatai green tea (0.63%) and ordinary green tea (2.86%) was significantly lower than in black tea (9.46%). This may be associated with differences in the processing technology between green and black tea. The content of ester compounds has been reported to be high in oolong tea (Lin et al., 2013), and we therefore speculate that the high-temperature filming of green tea may lead to the loss of some ester compounds, which therefore make little contribution to the aroma of green tea.   (Fariña et al., 2015) 0.03 ± 0.08a 0.00 ± 0.00a 0.27 ± 0.05b a RI, retention indices as determined on HP-5MS column using a homologous series of n-alkanes (C 8 -C 40 ). b The retention time (min) of identified compounds. c Compounds listed according to their structure. d MS match value of the Nist library. e Odor description: odor description as perceived by panelists according to GC-O in the relevant literature. f The content of volatile compounds is represented as mean ± standard deviation (mean ± SD), and different letters indicate significant differences in the same row (P < 0.05, ANOVA, Duncan's multiple range test). a RI, índices de retención determinados en la columna HP-5MS utilizando series homólogas de n-alkanes (C 8 -C 40 ). b El tiempo de retención (min) de los compuestos identificados. c Los compuestos se detallaron en una lista según su estructura. d El valor MS de coincidencia de la biblioteca Nist. e Descripción del olor: descripción del olor percibida por los panelistas durante GC-O en la literatura relevante. f El contenido de compuestos volátiles se representó como valor promedio ±desviación estándar (promedio ± SD) y las distintas letras indican diferencias significativas en la misma fila (P < 0,05, ANOVA, Test de Duncan de rango múltiple).
The comparison among the aroma compounds of these three different teas is shown in Figure 3. Because Wujiatai green tea and ordinary green tea are both classed as green tea, their aroma composition and content exhibited a high   degree of similarity (e.g. higher content of hydrocarbons and lower content of esters and aldehyde compounds). However, Wujiatai green tea has its own unique aroma characteristics, such as higher contents of phytol, linalool, geraniol, and certain terpenes (α-cedrene, β-caryophyllene, α-bergaptene, etc.). Although the processing technology differs between Wujiatai green tea and black tea, similar aroma characteristics were detected (e.g. high content of terpene alcohols). Since these compounds have high aromatic activity, they may be the major reason for enhanced aroma and flavour in Wujiatai green tea compared to ordinary green tea.

CA analysis
Like PCA, cluster analysis (CA) is an unsupervised data analysis method, meaning that prior knowledge of the sample under analysis is not required. CA is another method of determining differences among different teas, by dividing all samples into groups (clusters) according to similarity and finding the similarity among objects in a multidimensional space, forming clusters between the nearest objects (Zhang, Nan, Wang, Jiang, & Li, 2014). Ward's method as the amalgamation rule and the squared Euclidean distance as metric were used to establish clusters (Sun et al., 2015). The dendrogram of the CA results is shown in Figure 5; all samples were divided into three groups, which is basically the same with the results of PCA. Our results show that ordinary green tea has a flavor profile intermediate between Wujiatai and black tea. Tea aroma components can be affected by tea variety, environmental factors, cultivation conditions, processing conditions, and exogenous induction factors (Yang et al., 2013). In our study we chose tea samples from same production area as the research object in order to eliminate environmental effects and improve the accuracy of analysis. As can be seen from our results, the processing procedure of 'baking' indeed produces some unique aroma characteristics in Wujiatai green tea, based on the contents of phytol, linalool, geraniol, cis-jasmone, δ-cadinene, ylangene, αcedrene, β-caryophyllene, etc. Therefore, differences between Wujiatai and other teas may result from the process of 'baking', during which the conditions (such as temperature and time) are very important for quality control and evaluation. Meanwhile, a more comprehensive discussion of the role of the aroma-active compounds in regard to the flavour of Wujiatai green tea will be possible in the future following ongoing investigation into the association between the quantitative data obtained for the sensory attributes (e.g. GC-O, electronic nose) of several batches and their volatile compounds, using multivariate statistical methods. In addition, further investigations should be carried out with the aim of understanding the balance (variability) between the transformation and generation/ degradation of volatile compounds during the different processing stages of Wujiatai green tea.

Conclusion
In this study, the characteristic volatile profiles of Wujiatai green tea were evaluated by combining HS-SPME/GC-MS techniques with multi-statistical analysis. A total of 69 aroma compounds were identified in six Wujiatai green teas, mainly alcohols and hydrocarbons. Through PCA and CA, all tested tea samples were successfully divided into three groups, and Wujiatai green tea was found to possess a unique aroma characteristic compared with two other types. The characteristic aroma components in Wujiatai green tea were identified as including phytol, linalool, geraniol, cis-jasmone, δ-cadinene, ylangene, α-cedrene, and β-caryophyllene. Our results showed that the combination of headspace analysis and multi-statistical methods can be useful in identifying characteristic aroma components in a specific tea.

Disclosure statement
No potential conflict of interest was reported by the authors.