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.

RÉSUMÉ

En este estudio, se extrajeron e identificaron los componentes aromáticos de seis tés verdes Wujiatai utilizando microextracción en fase sólida del espacio de cabeza (HS-SPME) juntamente con cromatografía de gases y espectrometría de masas (GC-MS), además de análisis de componentes principales (PCA) para revelar las posibles diferencias entre estos tés en base a sus componentes aromáticos. Los resultados mostraron que a pesar de que se encontraron similitudes en la composición aromática y el contenido del té verde Wujiatai, el té verde normal y el té negro normal, el primero obtuvo unas características aromáticas únicas. Nosotros sugerimos que el proceso de ‘horneado’ del té verde Wujiatai posiblemente cause la formación y/o el aumento de algunos de sus componentes aromáticos, lo cual resulta en unas características aromáticas más prominentes y con mayor duración, además de tener una calidad superior en comparación con otros tés que se investigaron en este estudio.

KEYWORDS:

• Wujiatai green tea
• aroma component
• headspace solid-phase microextraction
• gas chromatography-mass spectrometry
• principal component analysis

PALABRAS CLAVE:

• Té verde Wujiatai
• componente aromático
• microextracción en fase sólida sin espacios vacíos
• cromatografía de gases y espectrometría de masas
• análisis de componentes principales

1. 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 infusion-enduring 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 polymer-coated 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.

2. Materials and methods

2.1. 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.

Figure 1. Major processing steps for production of the three different teas.

Figura 1. Los principales pasos del proceso de producción de los tres tés diferentes.

2.2. 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 (Lv et al., 2014). 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.

2.3. 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 electron energy 70 eV. The scan range was 35–500 amu and the solvent delay time was 2.8 min.

2.4. 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:

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.

3. Results and discussions

3.1. 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.

Table 1. Volatile compounds and their relative contents in three different teas.

Tabla 1. Compuestos volátiles y su contenido en tres tés diferentes.

NO.	RI^a	Retention time ^b	Compound^c	MS match value^d	Odor description^e	Relative percentage content [%]^f
						Wujiatai green teas	Ordinary green teas	Black teas
			Alcohols
1	843	5.142	(Z)-3-Hexen-1-ol	90	Fresh, fruity green (L.-F. Wang et al., 2008)	0.02 ± 0.06a	0.00 ± 0.00a	0.27 ± 0.15b
2	859	5.528	(E)-2-Hexen-1-ol	88		0.00 ± 0.00a	0.00 ± 0.00a	0.07 ± 0.06b
3	861	5.600	1-Hexanol	94	Newly cut grass (Fariña et al., 2015)	0.00 ± 0.00a	0.00 ± 0.00a	0.21 ± 0.10b
4	979	10.328	1-Octen-3-ol	91	Potato, mushroom, tomato (Du, Song, Baldwin, & Rouseff, 2015)	0.15 ± 0.06a	0.08 ± 0.07a	0.07 ± 0.03a
5	1034	12.935	Benzyl alcohol	95	Mildly sweet, roasted (L.-F. Wang et al., 2008)	0.00 ± 0.00a	0.00 ± 0.00a	0.38 ± 0.15b
6	1072	14.185	Linalool oxide I (cis-furan)	90	Sweetly floral, green, fruity (Du et al., 2015)	0.12 ± 0.12a	0.78 ± 0.37a	3.70 ± 4.81b
7	1087	15.517	Linalool oxide II (trans-furan)	90	Sweet floral, green, fruity (Du et al., 2015)	0.18 ± 0.15a	0.40 ± 0.07a	2.19 ± 0.35b
8	1098	16.160	Linalool	97	Floral, fruity, rose-like (Du et al., 2015)	6.13 ± 2.54a	3.06 ± 0.92b	6.77 ± 1.84a
9	1110	16.755	Phenylethyl alcohol	96	Pleasantly floral, mild rose-like (Jumtee et al., 2011)	0.98 ± 1.55a	0.36 ± 0.34a	3.13 ± 3.02b
10	1169	19.448	Linalool oxide III (cis-pyran)	90	Sweetly floral, green, fruity(Du et al., 2015)	0.00 ± 0.00a	0.02 ± 0.04a	0.21 ± 0.05b
11	1175	19.748	Linalool oxide IV (trans-pyran)	90	Sweetly floral, green, fruity (Du et al., 2015)	0.40 ± 0.45a	0.90 ± 0.76ab	1.38 ± 0.14b
12	1188	20.437	α-Terpineol	98	Mint(Lv et al., 2012)	0.18 ± 0.18a	0.09 ± 0.16a	0.31 ± 0.14a
13	1228	22.300	Nerol	95	Rose-like, floral (Lv et al., 2012)	0.00 ± 0.00a	0.00 ± 0.00a	0.70 ± 0.47b
14	1256	23.589	Geraniol	96	Floral (ros-like), sweet (L.-F. Wang et al., 2008)	14.96 ± 17.07a	5.70 ± 3.30b	29.46 ± 12.88a
15	1554	36.538	Nerolidol	92	Floral (rose-like), apple, green (L.-F. Wang et al., 2008)	1.15 ± 1.19a	4.22 ± 1.06b	4.42 ± 4.48b
16	1595	37.758	Cedrol	90	Woody (Lv et al., 2012)	0.00 ± 0.00a	0.00 ± 0.00a	0.20 ± 0.34b
17	1653	39.852	α-Cadinol	91	Pine nut (Chen, Sheu, & Wu, 2006)	0.00 ± 0.00a	0.00 ± 0.00a	0.13 ± 0.22b
18	1949	50.017	Isophytol	98		0.08 ± 0.05a	0.09 ± 0.08a	0.00 ± 0.00a
19	2122	55.126	Phytol	94	Floral (Jumtee et al., 2011)	37.52 ± 15.34a	7.39 ± 1.54b	1.57 ± 0.80b
			Hydrocarbons and aromatic hydrocarbons
20	910	8.054	α-Pinene	95	Pine, turpentine (Cheng et al., 2015)	0.38 ± 0.94a	0.00 ± 0.00a	0.00 ± 0.00a
21	1010	11.925	α-Terpinene	96	Grassy, plastic, smokey (Moon, Cliff, & Li-Chan, 2006)	0.00 ± 0.00a	0.06 ± 0.06b	0.00 ± 0.00a
22	1022	12.336	1-Methyl-2-(1-methylethyl)-benzene	95		0.08 ± 0.13a	0.14 ± 0.14a	0.00 ± 0.00b
23	1026	12.512	D-Limonene	94	Lemon, citrus, mint (Cheng et al., 2015)	0.56 ± 1.03a	9.00 ± 5.75b	0.81 ± 0.20a
24	1037	13.120	(E)-3,7-Dimethyl-1,3,6-octatriene	97		0.02 ± 0.05a	0.39 ± 0.24b	0.00 ± 0.00a
25	1051	13.603	Ocimene	98		0.31 ± 0.50a	0.59 ± 0.31a	0.00 ± 0.00b
26	1056	14.023	γ-Terpinene	96	Citrus (L.-F. Wang et al., 2008)	0.06 ± 0.14a	0.44 ± 0.09b	0.11 ± 0.11a
27	1178	19.821	Naphthalene	93	Floral/fruity (Zepka, Garruti, Sampaio, Mercadante, & Da Silva, 2014)	1.30 ± 1.30a	0.44 ± 0.39a	0.00 ± 0.00b
28	1200	20.990	Dodecane	95		0.28 ± 0.17a	0.40 ± 0.07a	0.00 ± 0.00b
29	1224	22.176	3-Carene	95	Fruity (Boonbumrung et al., 2001)	0.00 ± 0.00a	0.00 ± 0.00a	0.08 ± 0.14b
30	1287	24.934	2-Methyl-naphthalene	97		0.94 ± 0.70ab	1.12 ± 0.67b	0.00 ± 0.00c
31	1300	25.584	Tridecane	98		0.00 ± 0.00a	0.10 ± 0.10b	0.02 ± 0.03a
32	1302	25.670	1-Methyl-naphthalene	97		0.36 ± 0.26a	0.00 ± 0.00b	0.00 ± 0.00b
33	1334	27.653	α-Cubebene	90	Herby (Qiao et al., 2008)	0.65 ± 0.55a	0.37 ± 0.38a	0.00 ± 0.00b
34	1346	28.543	Ylangene	95		0.70 ± 0.86a	0.07 ± 0.12b	0.00 ± 0.00b
35	1385	29.489	β-Guaiene	90	Woody (Lv et al., 2012)	0.06 ± 0.13a	0.00 ± 0.00a	0.00 ± 0.00a
36	1400	29.943	Tetradecane	95		0.80 ± 0.65a	1.01 ± 0.34a	0.00 ± 0.00b
37	1409	30.218	α-Cedrene	90	Woody (Lv et al., 2012)	0.66 ± 1.62a	0.00 ± 0.00b	0.00 ± 0.00b
38	1417	30.556	β-Caryophyllene	96	Floral (Lv et al., 2012)	0.76 ± 1.04a	0.00 ± 0.00b	0.00 ± 0.00b
39	1496	33.669	α-Eelemene	88		0.86 ± 1.07a	0.00 ± 0.00b	0.00 ± 0.00b
40	1500	34.046	Pentadecane	95		0.72 ± 0.94a	0.59 ± 0.31a	0.06 ± 0.11b
41	1508	34.375	α-Farnesene	94	Floral, green plants (Kesen, Kelebek, & Selli, 2014)	0.00 ± 0.00a	0.00 ± 0.00a	0.79 ± 1.37b
42	1520	34.859	δ-Cadinene	90	Spicy, woody (L.-F. Wang et al., 2008)	2.42 ± 3.38a	0.55 ± 0.25b	0.21 ± 0.15b
43	1532	35.467	Cuparene	93		0.40 ± 0.99a	0.00 ± 0.00b	0.00 ± 0.00b
44	1572	36.786	Fluorene	95		0.08 ± 0.15a	0.99 ± 0.24b	0.00 ± 0.00a
45	1598	37.921	γ-Cadinene	91	Spicy, woody (L.-F. Wang et al., 2008)	0.22 ± 0.53a	0.44 ± 0.76a	0.00 ± 0.00b
46	1600	37.938	Hexadecane	96		1.24 ± 1.10a	0.43 ± 0.38b	0.17 ± 0.30b
47	1700	41.616	Heptadecane	98		0.61 ± 0.57a	0.49 ± 0.11a	0.00 ± 0.00b
48	1706	41.816	2,6,10,14-Tetramethyl-pentadecane	95		0.35 ± 0.42a	1.46 ± 0.85b	0.00 ± 0.00c
49	1765	43.856	Anthracene	94		0.69 ± 1.06a	0.22 ± 0.39a	0.00 ± 0.00b
50	1800	45.140	Octadecane	96		0.27 ± 0.25a	0.49 ± 0.21a	0.06 ± 0.10b
51	1809	45.457	2,6,10,14-Tetramethyl-hexadecane	95		0.04 ± 0.09a	0.57 ± 0.24b	0.05 ± 0.09a
			Aldehydes
52	818	3.588	Hexanal	90	Fruity, green, grassy (Du et al., 2015)	0.11 ± 0.11a	0.07 ± 0.13a	0.71 ± 0.46b
53	840	5.027	(E)-2-Hexenal	92	Fragrant, sweet, fruity (L.-F. Wang et al., 2008)	0.00 ± 0.00a	0.00 ± 0.00a	0.28 ± 0.26b
54	880	6.791	Heptanal	97	Fatty, rancid (Cheng et al., 2015)	0.13 ± 0.07a	0.13 ± 0.03a	0.09 ± 0.09a
55	957	9.321	Benzaldehyde	95	Almondy, moldy (Du et al., 2015)	0.07 ± 0.09a	0.15 ± 0.05a	0.37 ± 0.06b
56	1042	13.252	Phenyl acetaldehyde	97	Flowery, gingery, rose-like (Du et al., 2015)	0.07 ± 0.06a	0.06 ± 0.11a	1.51 ± 0.71b
57	1048	13.586	1-Ethyl-1H-pyrrole-2-carbaldehyde	90		0.03 ± 0.07a	0.00 ± 0.00a	0.50 ± 0.42b
58	1100	16.361	Nonanal	90	Grassy, tea, vegetables, (Moon et al., 2006)	1.19 ± 0.83a	2.21 ± 0.33a	1.07 ± 0.42a
59	1196	20.810	Safranal	98	Herbaceous (L.-F. Wang et al., 2008)	0.02 ± 0.06a	0.28 ± 0.09b	0.08 ± 0.14a
60	1205	21.255	Decanal	93	Herbal, fatty (Lv et al., 2012)	0.05 ± 0.08a	0.71 ± 0.09b	0.42 ± 0.20b
61	1218	21.799	β-Cyclocitral	98	Mild green, minty, fruity (L.-F. Wang et al., 2008)	0.50 ± 0.46a	0.78 ± 0.27a	0.43 ± 0.18a
62	1273	24.248	2-Phenyl-2-butenal	93		0.00 ± 0.00a	0.00 ± 0.00a	0.72 ± 0.38b
63	1313	26.265	(E,E)-2,4-Decadienal	94	Fatty, green, vegetable-like (Du et al., 2015)	0.00 ± 0.00a	0.00 ± 0.00a	0.25 ± 0.26b
64	1371	28.766	2-Butyl-2-octenal	91	Cured ham, meaty (Bueno et al., 2011)	0.00 ± 0.00a	0.00 ± 0.00a	0.48 ± 0.43b
65	1405	30.303	Dodecanal	93	Beefy, citrus (Lin & Rouseff, 2001)	0.84 ± 1.05a	3.13 ± 0.37b	2.40 ± 0.67b
66	1490	33.574	5-Methyl-2-phenyl-2-hexenal	96	Wax, cured meat, roasted (Moon et al., 2006)	0.00 ± 0.00a	0.00 ± 0.00a	0.89 ± 0.87b
			Ketones
67	982	10.546	2,3-Octadione	89		0.12 ± 0.08a	0.06 ± 0.02a	0.01 ± 0.02a
68	985	10.657	6-Methyl-5-hepten-2-one	90	Sweet, nutty, raspberry (Du et al., 2015)	0.11 ± 0.06a	0.12 ± 0.13a	0.19 ± 0.09a
69	1030	12.777	2,2,6-Trimethylcyclohexan-1-one	92		0.14 ± 0.17a	0.11 ± 0.19a	0.06 ± 0.10a
70	1397	29.819	cis-Jasmone	96	Floral (L.-F. Wang et al., 2008)	1.03 ± 1.00a	1.60 ± 1.00a	0.10 ± 0.11b
71	1428	31.022	α-Ionone	98	Woody, berryies, floral, nutty (L.-F. Wang et al., 2008)	0.18 ± 0.38a	0.46 ± 0.40a	0.34 ± 0.20a
72	1455	32.131	Geranyl acetone	95	Sweet, herbal, woody (Du et al., 2015)	0.73 ± 0.62a	1.09 ± 0.13a	0.64 ± 0.32a
73	1483	33.300	4-(2,6,6-Trimethylcyclohexa-1,3- dienyl)-but-3-en-2-one	93		0.10 ± 0.09a	0.00 ± 0.00b	0.23 ± 0.09a
74	1486	33.412	β-Ionone	96	Woody, floral (rose-like, violets) (L.-F. Wang et al., 2008)	2.63 ± 3.86a	4.75 ± 2.38a	3.37 ± 2.08a
75	1918	49.020	Farnesyl acetone	92		0.18 ± 0.11a	2.43 ± 0.60b	0.58 ± 0.09a
			Esters
76	1190	20.549	Methyl salicylate	95	Sweet, spicy, minty (L.-F. Wang et al., 2008)	0.26 ± 0.27a	0.39 ± 0.04a	3.28 ± 0.81b
77	1238	22.540	cis-3-Hexenyl isovalerate	90		0.00 ± 0.00a	0.00 ± 0.00a	0.34 ± 0.16b
78	1245	22.724	cis-3-Hexenyl valerate	90		0.00 ± 0.00a	0.00 ± 0.00a	0.17 ± 0.17b
79	1325	26.668	Methyl geraniate	87		0.00 ± 0.00a	0.00 ± 0.00a	0.68 ± 0.46b
80	1383	29.190	(Z)-3-Hexenyl hexanoate	90	Green, fruity, woody (Du et al., 2015)	0.05 ± 0.07a	0.92 ± 0.45ab	1.54 ± 1.66b
81	1387	29.408	Hexyl hexanoate	91		0.00 ± 0.00a	0.19 ± 0.20b	0.39 ± 0.41b
82	1391	29.558	(E)-2-Hexenyl Hexanoate	94	Green, leafy, apples (Du et al., 2015)	0.00 ± 0.00a	0.00 ± 0.00a	0.45 ± 0.62b
83	1566	36.752	cis-3-Hexenyl benzoate	95		0.15 ± 0.36a	0.15 ± 0.27a	1.07 ± 0.61b
84	1578	37.026	Hexyl benzoate	93		0.00 ± 0.00a	0.00 ± 0.00a	0.23 ± 0.20b
85	1927	49.358	Hexadecanoic acid methyl ester	99		0.17 ± 0.11a	0.76 ± 0.23ab	0.95 ± 0.74b
86	2093	54.543	Methyl linoleate	99		0.00 ± 0.00a	0.16 ± 0.07b	0.14 ± 0.18b
87	2099	54.736	Methyl linolenate	99		0.00 ± 0.00a	0.29 ± 0.13b	0.22 ± 0.25b
			Furans
88	989	10.824	2-Pentyl-furan	90	Geraniums, cooked spices (Bueno et al., 2011)	0.47 ± 0.37a	1.78 ± 0.95b	0.56 ± 0.09a
89	998	11.330	cis-2-(2-Pentenyl)furan	88		0.00 ± 0.00a	0.05 ± 0.05b	0.08 ± 0.01b
			Nitrogen compounds
90	1291	25.203	Indole	96	Nutty, floral (L.-F. Wang et al., 2008)	0.42 ± 0.76a	1.85 ± 1.21b	0.00 ± 0.00c
91	1840	46.622	Caffeine	97		5.42 ± 1.57a	24.34 ± 3.97b	10.38 ± 1.35c
			Acids
92	1975	50.608	Hexadecanoic acid	99		0.39 ± 0.18a	0.71 ± 0.71a	0.66 ± 1.15a
			Lactones
93	1528	34.983	Dihydroactinidiolide	96	Woody (Lv et al., 2012)	3.52 ± 2.35a	2.67 ± 0.62a	1.29 ± 0.87a
			Phenols
94	1351	27.691	2,6-Dimethoxyphenol	90	Nutty, smokey (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 (C₈–C₄₀). ^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 (C₈–C₄₀). ^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).

Figure 2. TICs of three representative tea samples by GC-MS (a, Wujiatai green tea; b, ordinary green tea; c, black tea).

Figura 2. TIC de tres muestras representativas de té mediante GC-MS (a, Té verde Wujiatai; b, té verde normal; c, té negro).

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 (Ma et al., 2014), 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-2-phenyl-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.

Caffeine (no. 91), which makes little contribution to the aroma of tea (Lv, Wu, Jiang, & Meng, 2014), showed a higher content in ordinary green tea (24.34%) than in Wujiatai green tea (5.42%) and black tea (10.38%). Indole (no. 90), which has a typical flowery fragrance and may contribute to overall green tea odor (Hattori et al., 2003), was detected only in green tea. Two furans – 2-pentylfuran (no. 88) and cis-2-(2-pentenyl) furan (no.89) – identified in various types of tea (Rawat et al., 2007; K. Wang et al., 2011) were found in the present study (Table 1). The content of 2-pentylfuran (no. 88) in ordinary green tea (1.78%) was higher than in the other two types.

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.

Figure 3. Comparison among volatile compounds in three kinds of tea.

Figura 3. Comparación entre los compuestos volátiles de tres tipos de té.

3.2. PCA of GC-MS data

PCA is a non-targeted statistical method (Cai et al., 2013; Song, Xia, & Tomasino, 2015; Wu, Meyer, Whitaker, Litt, & Kennelly, 2013), and was used in this study to determine whether there were differences in levels of volatile components among these three teas. All identified 94 aroma components in Table 1 were analysed by PCA based on their relative percentages. The score and loading plots of triplicate results are shown in Figure 4. The serial numbers of variables (see Figure 4b) were consistent with the code of aroma components given in Table 1. All tea samples were successfully divided into three groups in the score plot, with no overlap (see Figure 4a). This indicates that each group’s tea possessed an aroma characteristic. For variables closer to the corresponding samples the greater the probability contribution of these samples. As can be seen from Figure 4b, these variables, which include V42 (δ-cadinene), V27 (naphthalene), V46(hexadecane), V39 (α-elemene), V34 (ylangene), V43 (cubebene), V35 (β-guaiene), V67 (2,3-octadione), V49 (anthracene), V19 (phytol), and V4 (1-octen-3-ol), made a major contribution to Wujiatai green tea and some were only identified in Wujiatai green tea; V90 (indole), V58 (nonanal), V48 (2,6,10,14-tetramethyl-pentadecane), V21 (α-terpinene), V88 (2-pentyl-furan), V24 ((E)-3,7-dimethyl-1,3,6-octatriene), V51 (2,6,10,14-tetramethyl-hexadecane), V23 (D-limonene) etc. made a major contribution to ordinary green tea; and V7 (linalool oxide II), V10 (linalool oxide III), V84 (hexyl benzoate), V52 (hexanal), V77 (cis-3-hexenyl isovalerate), V76 (methyl salicylate), V1((Z)-3-hexen-1-ol), V79 (methyl geraniate), V56 (phenyl acetaldehyde), and V13 (nerol) made a major contribution to black tea. Nevertheless, the complete separation of all tea samples indicated that each class has its own unique aroma profile.

Figure 4. PCA biplot of score values and major loading values (a, score plot; b, score and loading plots of triplicate results). O, ordinary green tea; B, black tea; W, Wujiatai green tea.

Figura 4. Diagrama de dispersión biespacial PCA de los resultados y los principales valores de carga (a, gráfico de resultados; b, resultados y gráficos de carga de los resultados triplicados), O representa el té verde normal, B representa el té negro normal y W representa el té verde Wujiatai.

3.3. 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.

Figure 5. CA dendrogram of all 12 teas. O, ordinary green tea; B, black tea; W, Wujiatai green tea.

Figura 5. Dendrograma CA de los 12 tés, O representa el té verde normal, B representa el té negro normal y W representa el té verde Wujiatai.

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, α-cedrene, 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.

4. 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.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [No.31460228] and scientific research funds from Yunnan Province Department of Education [No.2014Y089].