Shidong Lvab,
Yuanshuang Wua,
Jifu Weic,
Ming Liana,
Chen Wanga,
Xuemei Gaoa and
Qingxiong Meng*a
aFaculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, People’s Republic of China. E-mail: qxmeng@scbg.ac.cn; Fax: +86-871-65920570; Tel: +86-871-65920541
bKunming Grain & Oil and Feed Product Quality Inspection Center, Kunming 650118, Yunnan, People’s Republic of China
cResearch Division of Clinical Pharmacology, The First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, People’s Republic of China
First published on 7th October 2015
Volatile changes and the post-fermentation ageing process of tea remain largely unknown. Additionally, the understanding of ageing and storage processes of tea mostly rely on sensory experience and lack the support of scientific and accurate data. In this paper, a method was developed based on head-space solid phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) combined with multivariate statistical methods to assess volatile profiles in different types of Pu-erh teas, including raw, ripe and aged Pu-erh teas. A total of 122 aroma components were identified in 57 Pu-erh teas. Differences in the manufacturing method and years in storage of Pu-erh teas resulted in different compositions and contents of volatile components. The characteristic volatiles in aged teas were hexadecanoic acid, dihydroactinidiolide, caffeine, linalool, 6,10,14-trimethyl-2-pentadecanone, β-ionone, cedrol, and phytol; the characteristic volatiles in raw teas were linalool, tridecane, caffeine, dihydroactinidiolide, β-ionone, 6,10,14-trimethyl-2-pentadecanone, dodecane, etc.; and the characteristic volatiles in ripe teas were 1,2,3-trimethoxybenzene, hexadecanoic acid, 1,2,4-trimethoxybenzene, dihydroactinidiolide, 6,10,14-trimethyl-2-pentadecanon, caffeine, and 1,2,3-trimethoxy-5-methyl-benzene. Through principal component analysis (PCA), clustering analysis (CA) and orthogonal projection to latent structures-discriminate analysis (OPLS-DA), three different kinds of Pu-erh teas were classified successfully. Additionally, aged Pu-erh teas showed similar volatile constituents as ripe teas. This study suggested that HS-SPME/GC-MS combined with chemometrics methods is accurate, sensitive, fast, universal and ideal for rapid routine analysis and discrimination of Pu-erh teas with different processing technologies and storage times.
The ageing of tea is common and drinking aged tea seems likely to become a trend in some countries. Many teas, such as Anji white tea, Fuzhuan tea, High Mountain Oolong tea, Sichuan dark tea, and Pu-erh tea, have a better quality (taste and flavour) and better health efficacy if they are subjected to long-term ageing. Pu-erh tea has been recognized since the Tang dynasty (AD 618–906) in China, and the taste has been shown to improve with longer preservation times.5 Thus, Pu-erh aged tea is generally more expensive than newly produced Pu-erh tea. Tea consumers and merchants are often willing to pay higher prices to obtain older Pu-erh tea. With the increasing awareness of the importance of healthy living, the consumption demand of high quality Pu-erh aged tea products has been increasing significantly. To obtain higher profits, some manufacturers are misleading customers by labelling inferior or relatively new Pu-erh tea products as older Pu-erh tea. Sensory evaluation is currently a common method used to differentiate various teas that have undergone different processing methods.6 However, this approach cannot always result in an objective judgement because it is easily influenced by physical and mental conditions.7 Moreover, this approach is unable to reflect the chemical difference of various ages of tea and usually lacks a comprehensive view of the chemical composition of teas. Thus, developing a method for discriminating the natural ageing process and identifying the processing methods (raw tea and ripe tea) is urgently required not only for consumers to protect their interests but also for tea producers to apply quality control. Additionally, analysing the chemical composition changes from different production years using modern instrument analysis methods can help us to understand the dynamic changes of chemical compounds during the ageing process while tea is stored .
The rapid development of modern instrument analysis technology, such as LC (liquid chromatography), near infrared reflection (NIR), inductively coupled plasma mass spectrometry (ICP-MS), nuclear magnetic resonance (NMR), GC-MS, etc., make it possible to obtain more information (variables) of large samples (objects) in a relatively shorter time.8–10 Consequently, making use of different multivariate statistical methods to maximize the extraction of useful information from large data sets is of great importance. These methods mainly include principal component analysis (PCA), cluster analysis (CA), soft independent modelling of class analogy (SIMCA), orthogonal partial least squares discriminate analysis (OPLS-DA), etc.11–13 Currently, instrumental analysis technologies combined with these chemometrics methods have been applied with increasing success in the analysis of samples of different tea types. For instance, using GC-MS and the fingerprinting technique combined with global volatile profiling, we have revealed that green teas have the feature to produce area-dependent chemical components.14 Lin et al.15 successfully used HS-SPME combined with PCA, CA and linear discriminate analysis (LDA) to discriminate five Oolong tea (Camellia sinensis) varieties; Ye et al.16 applied similar methods to distinguish between Hubei green teas and Henan green teas in China. Other analysis technologies, such as NMR,17 ICP-MS,18 NIR,19 and LC-MS20 combined with multivariate statistical methods also obtained satisfactory results when applied to identify geographic origins and processing types of various tea samples. Therefore, multivariate statistical methods combined with HS-SPME/GC-MS could be an effective and convenient tool for comprehensive analysis of different tea volatiles in order to assess tea quality and to investigate the behaviour of volatiles during long-term tea storage.
To our knowledge, the study of chemical composition associated with processing methods and production length of Pu-erh tea is very limited. Ku et al.21 have used LC-MS and chemometrics methods to analyse the processing type and change of water-soluble components in Pu-erh tea with different post-fermentation lengths, demonstrating that a chemometrics method was an effective tool for identifying processing types and the post-fermentation length of Pu-erh tea. Xu et al.22 used NIR spectroscopy combined with chemometrics methods to discriminate the type (raw tea or ripe tea) and predict the age of Chinese tuo tea. Nevertheless, there is no study on volatile profiling using GC-MS and multivariate statistical method analyses from different production years and processing types of Pu-erh tea, especially Pu-erh aged tea. Additionally, the study of ageing processes and the similarities and differences between Pu-erh aged tea and ripe tea are essentially unknown.
Our previous study14,23 used the HS-SPME/GC-MS methodology to investigate the fingerprint profile of Dayi Pu-erh ripe tea and Pu-erh green tea; however, it mainly involved the fingerprint similarity analysis method, such as the correlation coefficient of similarity and overlapping chromatographic peaks (ORP), and did not involve the OPLS-DA method; additionally, only a single processed type of Pu-erh tea was involved. In the present study, the GC-MS method combined with PCA, CA and OPLS-DA techniques was adopted to probe potential differences in aroma characteristics among three types of Pu-erh teas (raw, ripe and aged). Changes in the content of volatile components in relation to the ages and production process of Pu-erh teas were also explored. Our study might provide a reference point for a fast and simple method for identifying Pu-erh teas that have been produced with different ages and processing methods.
No. | Sample ID | Type | Production year | Production period | Amount |
---|---|---|---|---|---|
1 | O1 | Aged tea | 1914 | 100 years | 1 |
2 | O2 | Aged tea | 1984 | 30 years | 1 |
3 | O3 | Aged tea | 1989 | 25 years | 1 |
4 | O4 | Aged tea | 1991 | 23 years | 1 |
5 | O5 | Aged tea | 1994 | 20 years | 1 |
6 | O6 | Aged tea | 1997 | 17 years | 1 |
7 | O7 | Aged tea | 1998 | 16 years | 1 |
8 | O8 | Aged tea | 1999 | 15 years | 2 |
9 | O9 | Aged tea | 2000 | 11 years | 1 |
10 | R1 | Raw tea | 2004 | 10 years | 1 |
11 | R2 | Raw tea | 2005 | 9 years | 1 |
12 | R3 | Raw tea | 2006 | 8 years | 1 |
13 | R4 | Raw tea | 2007 | 7 years | 1 |
14 | R5 | Raw tea | 2008 | 6 years | 1 |
15 | R6 | Raw tea | 2009 | 5 years | 3 |
16 | R7 | Raw tea | 2010 | 4 years | 3 |
17 | R8 | Raw tea | 2011 | 3 years | 2 |
18 | R9 | Raw tea | 2012 | 2 years | 2 |
19 | R10 | Raw tea | 2013 | 1 year | 8 |
20 | P1 | Ripe tea | 1996 | 18 years | 1 |
21 | P2 | Ripe tea | 2000 | 14 years | 1 |
22 | P3 | Ripe tea | 2001 | 13 years | 1 |
23 | P4 | Ripe tea | 2002 | 12 years | 1 |
24 | P5 | Ripe tea | 2003 | 11 years | 1 |
25 | P6 | Ripe tea | 2004 | 10 years | 2 |
26 | P7 | Ripe tea | 2005 | 9 years | 2 |
27 | P8 | Ripe tea | 2006 | 8 years | 2 |
28 | P9 | Ripe tea | 2007 | 7 years | 1 |
29 | P10 | Ripe tea | 2008 | 6 years | 1 |
30 | P11 | Ripe tea | 2009 | 5 years | 1 |
31 | P12 | Ripe tea | 2010 | 4 years | 2 |
32 | P13 | Ripe tea | 2011 | 3 years | 2 |
33 | P14 | Ripe tea | 2012 | 2 years | 3 |
34 | P15 | Ripe tea | 2013 | 1 year | 3 |
No. | RIa | Compoundb | Relative percentage contentc [% (range)] | ||
---|---|---|---|---|---|
Aged tea (n = 10) | Raw tea (n = 23) | Ripe tea (n = 24) | |||
a RIs, retention indices as determined on a HP-5MS column using the homologous series of n-alkanes (C8–C40).b Compounds were listed in order of retention time.c The content of volatile compounds are represented as the mean value ± standard deviation (mean ± SD), different letters indicate significant differences (p < 0.05, ANOVA, Duncan’s multiple range test). | |||||
1 | 802 | 1-Pentanol | 0.00 ± 0.00a | 0.03 ± 0.09a | 0.00 ± 0.00a |
2 | 806 | Hexanal | 0.26 ± 0.29a | 0.00 ± 0.00b | 0.07 ± 0.12b |
3 | 843 | cis-3-Hexenol | 0.03 ± 0.07a | 0.00 ± 0.00b | 0.00 ± 0.00b |
4 | 861 | 1-Hexyl alcohol | 0.02 ± 0.04a | 0.00 ± 0.00b | 0.00 ± 0.00b |
5 | 884 | 2-Heptanone | 0.10 ± 0.09a | 0.03 ± 0.05b | 0.02 ± 0.04b |
6 | 894 | 2-Heptanol | 0.11 ± 0.17a | 0.02 ± 0.04b | 0.01 ± 0.05b |
7 | 957 | Benzaldehyde | 0.29 ± 0.17a | 0.20 ± 0.06b | 0.14 ± 0.08b |
8 | 979 | 1-Octen-3-ol | 0.36 ± 0.26a | 0.90 ± 0.99b | 0.03 ± 0.05a |
9 | 982 | 2,3-Octadione | 0.04 ± 0.05a | 0.06 ± 0.08b | 0.01 ± 0.02a |
10 | 985 | 6-Methyl-5-hepten-2-one | 0.45 ± 0.25a | 0.25 ± 0.11b | 0.05 ± 0.07c |
11 | 989 | 2-Pentyl-furan | 0.49 ± 0.25a | 0.91 ± 0.30b | 0.21 ± 0.13c |
12 | 998 | cis-2-(2-Pentenyl)furan | 0.09 ± 0.09a | 0.14 ± 0.09a | 0.03 ± 0.05b |
13 | 1010 | α-Terpinene | 0.04 ± 0.06a | 0.12 ± 0.06b | 0.01 ± 0.02a |
14 | 1022 | 1-Methyl-2-(1-methylethyl)-benzene | 0.27 ± 0.15a | 0.19 ± 0.18a | 0.05 ± 0.13b |
15 | 1026 | D-Limonene | 0.58 ± 0.58a | 1.20 ± 0.79b | 0.08 ± 0.09c |
16 | 1030 | 2-Ethylhexanol | 0.02 ± 0.04a | 0.00 ± 0.00a | 0.07 ± 0.18a |
17 | 1034 | Benzyl alcohol | 0.14 ± 0.16a | 0.40 ± 0.10b | 0.02 ± 0.04c |
18 | 1037 | (E)-3,7-Dimethyl-1,3,6-octatriene | 0.16 ± 0.15a | 0.27 ± 0.09b | 0.00 ± 0.00c |
19 | 1042 | Phenyl acetaldehyde | 0.22 ± 0.12a | 0.24 ± 0.10a | 0.06 ± 0.07b |
20 | 1048 | 1-Ethyl-1H-pyrrole-2-carbaldehyde | 0.02 ± 0.05a | 0.07 ± 0.11ab | 0.14 ± 0.16b |
21 | 1051 | Ocimene | 0.05 ± 0.08a | 0.45 ± 0.15b | 0.00 ± 0.00a |
22 | 1056 | γ-Terpinene | 0.30 ± 0.19a | 0.33 ± 0.11a | 0.07 ± 0.17b |
23 | 1064 | Acetophenone | 0.07 ± 0.07a | 0.03 ± 0.12a | 0.03 ± 0.06a |
24 | 1068 | (E)-2-Octen-1-ol | 0.19 ± 0.15a | 0.39 ± 0.46b | 0.00 ± 0.00a |
25 | 1072 | Linalool oxide I | 0.90 ± 0.61a | 0.97 ± 0.50a | 0.82 ± 0.53a |
26 | 1087 | Linalool oxide II | 1.48 ± 1.24a | 1.92 ± 0.63a | 1.65 ± 0.87a |
27 | 1092 | (E,E)-3,5-Octadien-2-one | 0.23 ± 0.47a | 0.06 ± 0.09b | 0.12 ± 0.16ab |
28 | 1098 | Linalool | 4.34 ± 2.71a | 14.72 ± 4.51b | 0.77 ± 0.65c |
29 | 1101 | Hotrienol | 1.20 ± 0.66a | 1.76 ± 0.48b | 0.31 ± 0.32c |
30 | 1110 | Phenylethyl alcohol | 0.25 ± 0.17a | 0.00 ± 0.00b | 0.25 ± 0.23a |
31 | 1135 | Benzene acetonitrile | 0.64 ± 0.75a | 0.00 ± 0.00b | 0.13 ± 0.32b |
32 | 1137 | 2,5-Pyrrolidinedione, 1-ethyl- | 0.12 ± 0.20a | 0.02 ± 0.08b | 0.03 ± 0.06b |
33 | 1139 | 3-Nonen-2-one | 0.05 ± 0.09a | 0.00 ± 0.00b | 0.00 ± 0.00b |
34 | 1149 | 1,2-Dimethoxybenzene | 0.47 ± 0.60a | 0.01 ± 0.03b | 1.09 ± 0.49c |
35 | 1153 | 1,4-Dimethoxy-2-methylbenzene | 0.05 ± 0.13a | 0.00 ± 0.00b | 0.00 ± 0.00c |
36 | 1159 | (E)-2-Nonenal | 0.09 ± 0.14a | 0.00 ± 0.00b | 0.06 ± 0.10ab |
37 | 1169 | Linalool oxide III | 0.19 ± 0.21a | 0.00 ± 0.00b | 0.38 ± 0.26c |
38 | 1175 | Linalool oxide IV | 1.20 ± 0.58a | 0.84 ± 0.41a | 1.31 ± 0.85a |
39 | 1178 | Naphthalene | 0.45 ± 0.40a | 0.44 ± 0.16a | 0.49 ± 0.59a |
40 | 1188 | α-Terpineol | 2.48 ± 2.09a | 2.53 ± 1.18a | 1.33 ± 0.75b |
41 | 1190 | Methyl salicylate | 0.61 ± 0.41a | 0.68 ± 0.76a | 0.40 ± 0.28a |
42 | 1196 | Safranal | 0.40 ± 0.23a | 0.37 ± 0.10a | 0.18 ± 0.13b |
43 | 1200 | Dodecane | 0.32 ± 0.85a | 3.31 ± 0.93b | 0.03 ± 0.06a |
44 | 1205 | Decanal | 0.27 ± 0.27a | 0.00 ± 0.00b | 0.26 ± 0.14a |
45 | 1218 | β-Cyclocitral | 0.42 ± 0.15a | 0.67 ± 0.18b | 0.16 ± 0.10c |
46 | 1221 | 2,3-Dihydrobenzofuran | 0.25 ± 0.65a | 0.00 ± 0.00a | 0.26 ± 0.86a |
47 | 1224 | 3-Carene | 0.09 ± 0.11a | 0.21 ± 0.08b | 0.00 ± 0.00c |
48 | 1228 | Nerol | 0.19 ± 0.20a | 0.29 ± 0.13b | 0.05 ± 0.09c |
49 | 1236 | 2-Methoxy-4-methyl-1-(1-methylethyl)-benzene | 0.20 ± 0.18a | 0.00 ± 0.00b | 0.04 ± 0.10b |
50 | 1241 | 3,4-Dimethoxytoluene | 0.85 ± 0.94a | 0.00 ± 0.00b | 0.88 ± 0.79a |
51 | 1256 | Geraniol | 1.20 ± 0.48a | 1.59 ± 0.46b | 0.44 ± 0.28c |
52 | 1259 | 7-Methoxybenzofuran | 0.06 ± 0.15a | 0.00 ± 0.00b | 0.01 ± 0.04b |
53 | 1261 | 2-Methoxybenzyl alcohol | 0.10 ± 0.11a | 0.00 ± 0.00b | 0.03 ± 0.06b |
54 | 1263 | (E)-2-Decenal | 1.07 ± 2.06a | 0.00 ± 0.00b | 0.01 ± 0.05b |
55 | 1265 | 3,5-Dimethoxytoluene | 1.24 ± 3.51a | 0.00 ± 0.00b | 0.07 ± 0.11b |
56 | 1285 | 2-Methyl-naphthalene | 0.52 ± 0.36a | 0.34 ± 0.08b | 0.37 ± 0.28ab |
57 | 1287 | Tridecene | 0.00 ± 0.00a | 0.21 ± 0.12b | 0.00 ± 0.00a |
58 | 1290 | Indole | 0.06 ± 0.11a | 0.06 ± 0.16a | 0.00 ± 0.00a |
59 | 1294 | 2-Undecanone | 0.29 ± 0.29a | 0.00 ± 0.00b | 0.20 ± 0.20a |
60 | 1300 | Tridecane | 0.01 ± 0.03a | 8.05 ± 2.06b | 0.01 ± 0.20a |
61 | 1302 | 1-Methyl-naphthalene | 0.25 ± 0.19a | 0.00 ± 0.00b | 0.22 ± 0.11a |
62 | 1316 | 1,2,3-Trimethoxybenzene | 2.94 ± 4.10a | 0.34 ± 0.19a | 11.52 ± 5.75b |
63 | 1325 | 4-Ethyl-1,2-dimethoxy-benzene | 1.43 ± 2.01a | 0.01 ± 0.04b | 2.27 ± 1.56a |
64 | 1334 | 2,6,6-Trimethyl-1-cyclohexene-1-ethanol | 0.16 ± 0.19a | 0.00 ± 0.00b | 0.14 ± 0.23a |
65 | 1351 | 2,6-Dimethoxyphenol | 0.53 ± 0.70a | 0.31 ± 0.18a | 0.29 ± 0.17a |
66 | 1362 | 2-Dodecenal | 0.08 ± 0.17a | 0.00 ± 0.00b | 0.00 ± 0.00b |
67 | 1366 | α-Ylangene | 1.51 ± 1.48a | 0.00 ± 0.00b | 0.71 ± 1.65ab |
68 | 1375 | 1,2,4-Trimethoxybenzene | 0.83 ± 1.06a | 0.00 ± 0.00a | 4.87 ± 3.37b |
69 | 1381 | β-Damascenone | 0.04 ± 0.11a | 0.02 ± 0.09a | 0.00 ± 0.00a |
70 | 1383 | 1-Methoxy-4-propenyl-benzene | 0.15 ± 0.39a | 0.00 ± 0.00b | 0.02 ± 0.08b |
71 | 1387 | α-Gurjunene | 1.70 ± 5.38a | 0.00 ± 0.00b | 0.00 ± 0.00b |
72 | 1389 | β-Guaiene | 0.00 ± 0.00a | 0.00 ± 0.00a | 0.38 ± 0.36b |
73 | 1397 | cis-Jasmone | 0.00 ± 0.00a | 0.40 ± 0.39b | 0.24 ± 0.23b |
74 | 1400 | Tetradecane | 0.63 ± 0.53a | 1.05 ± 0.19b | 0.31 ± 0.20c |
75 | 1404 | 1,2,3-Trimethoxy-5-methyl-benzene | 1.49 ± 2.67a | 0.01 ± 0.05a | 3.34 ± 3.28b |
76 | 1406 | 6,10-Dimethyl-2-undecanone | 0.12 ± 0.25a | 0.00 ± 0.00b | 0.00 ± 0.00b |
77 | 1408 | 1,2-Dimethoxy-4-n-propenyl-benzene | 0.10 ± 0.33a | 0.00 ± 0.00a | 0.33 ± 0.70a |
78 | 1411 | α-Cedrene | 0.65 ± 0.82a | 0.15 ± 0.25b | 0.49 ± 0.47a |
79 | 1417 | β-Caryophyllene | 0.21 ± 0.44a | 0.20 ± 0.25a | 0.02 ± 0.10b |
80 | 1428 | α-Ionone | 1.12 ± 0.48a | 1.22 ± 0.39a | 0.88 ± 0.37b |
81 | 1433 | 1,2-Benzopyrone | 0.53 ± 0.32a | 0.43 ± 0.19a | 0.43 ± 0.52a |
82 | 1438 | Dihydro-β-ionone | 0.13 ± 0.21ab | 0.00 ± 0.00a | 0.27 ± 0.39b |
83 | 1442 | 1-Methoxy-naphthalene | 0.07 ± 0.12a | 0.00 ± 0.00a | 0.52 ± 0.25b |
84 | 1447 | 2-Methoxy-naphthalene | 0.08 ± 0.16a | 0.00 ± 0.00a | 0.71 ± 0.56b |
85 | 1449 | 1,2,3,4-Tetramethoxybenzene | 0.71 ± 0.80a | 0.00 ± 0.00b | 1.12 ± 0.62c |
86 | 1455 | Geranyl acetone | 2.25 ± 1.22a | 2.33 ± 0.64a | 1.51 ± 0.62b |
87 | 1460 | Aromandendrene | 0.23 ± 0.38a | 0.00 ± 0.00b | 0.03 ± 0.10b |
88 | 1468 | 5-Methoxy-6,7-dimethyl-benzofuran | 0.55 ± 0.65a | 0.00 ± 0.00b | 0.09 ± 0.20b |
89 | 1483 | 4-(2,6,6-Trimethylcyclohexa-1,3-dienyl)-but-3-en-2-one | 1.44 ± 2.07ab | 0.25 ± 0.17a | 2.72 ± 2.55b |
90 | 1487 | β-Ionone | 3.28 ± 1.80a | 5.05 ± 1.23b | 3.02 ± 1.27a |
91 | 1489 | Pentadecene | 0.38 ± 0.64a | 0.89 ± 0.27b | 0.00 ± 0.00c |
92 | 1492 | 2-Tridecanone | 0.19 ± 0.39a | 0.00 ± 0.00a | 0.69 ± 1.04b |
93 | 1500 | Pentadecane | 0.77 ± 1.02a | 0.62 ± 0.24a | 0.50 ± 0.38a |
94 | 1502 | 1,2-Dimethoxy-4-(1-propenyl)benzene | 0.00 ± 0.00a | 0.00 ± 0.00a | 0.34 ± 0.35b |
95 | 1504 | Cuparene | 0.56 ± 0.93a | 0.00 ± 0.00b | 0.35 ± 0.76ab |
96 | 1506 | Dibenzofuran | 0.48 ± 0.57a | 0.68 ± 0.63a | 0.56 ± 0.30a |
97 | 1508 | α-Farnesene | 0.00 ± 0.00a | 1.40 ± 0.86b | 0.49 ± 0.50c |
98 | 1528 | Dihydroactinidiolide | 5.74 ± 3.50a | 6.16 ± 1.05a | 4.37 ± 1.38b |
99 | 1549 | 1,2,3-Trimethoxy-5-(2-allylbenzene) | 0.15 ± 0.34a | 0.00 ± 0.00b | 0.00 ± 0.00b |
100 | 1554 | Nerolidol | 0.05 ± 0.14a | 0.29 ± 0.39a | 1.37 ± 1.60b |
101 | 1572 | Fluorene | 0.88 ± 0.44a | 0.97 ± 0.32a | 0.85 ± 0.42a |
102 | 1598 | Cedrol | 3.57 ± 2.45a | 0.57 ± 0.29b | 1.73 ± 1.57c |
103 | 1600 | Hexadecane | 1.31 ± 0.80a | 1.32 ± 0.47a | 1.48 ± 0.89a |
104 | 1653 | α-Cadinol | 0.36 ± 0.48a | 0.96 ± 0.23b | 0.83 ± 0.38b |
105 | 1659 | 2,2′,5,5′-Tetramethyl-1,1′-biphenyl | 0.22 ± 0.22a | 1.05 ± 0.26b | 0.46 ± 0.36c |
106 | 1664 | 2-Methyl-hexadecane | 0.10 ± 0.22a | 0.17 ± 0.28ab | 0.35 ± 0.24b |
107 | 1700 | Heptadecane | 1.51 ± 1.30a | 0.87 ± 0.70a | 1.56 ± 0.99a |
108 | 1706 | 2,6,10,14-Tetramethyl-pentadecane | 1.77 ± 1.14a | 2.66 ± 0.91b | 1.78 ± 1.16a |
109 | 1765 | Anthracene | 1.40 ± 0.48a | 0.64 ± 0.42b | 1.54 ± 0.74a |
110 | 1800 | Octadecane | 0.94 ± 0.78a | 0.41 ± 0.45b | 1.06 ± 0.73a |
111 | 1809 | 2,6,10,14-Tetramethyl-hexadecane | 0.94 ± 0.91ab | 0.49 ± 0.42a | 1.07 ± 0.90b |
112 | 1840 | Caffeine | 4.83 ± 2.31ab | 6.13 ± 2.89b | 4.05 ± 1.90a |
113 | 1846 | 6,10,14-Trimethyl-2-pentadecanone | 4.04 ± 2.32a | 3.75 ± 1.90a | 4.21 ± 2.31a |
114 | 1900 | Nonadecane | 0.15 ± 0.24a | 0.13 ± 0.20a | 0.13 ± 0.20a |
115 | 1918 | Farnesyl acetone | 0.29 ± 0.26a | 0.38 ± 0.44a | 0.41 ± 0.31a |
116 | 1927 | Hexadecanoic acid methyl ester | 0.36 ± 0.31a | 0.51 ± 1.70a | 0.46 ± 0.33a |
117 | 1949 | Isophytol | 0.13 ± 0.15a | 0.06 ± 0.07a | 1.03 ± 0.64b |
118 | 1975 | Hexadecanoic acid | 8.44 ± 3.16a | 1.82 ± 1.49b | 10.52 ± 5.43a |
119 | 2000 | Eicosane | 0.01 ± 0.04a | 0.05 ± 0.08a | 0.18 ± 0.18b |
120 | 2093 | Methyl linoleate | 0.07 ± 0.04ab | 0.00 ± 0.00a | 0.08 ± 0.14b |
121 | 2099 | Methyl linolenate | 0.17 ± 0.13a | 0.17 ± 0.26a | 0.31 ± 0.34a |
122 | 2122 | Phytol | 3.28 ± 4.20a | 2.41 ± 1.58a | 2.12 ± 0.34a |
Alcohols | 21.95a | 30.65b | 14.69a | ||
Hydrocarbons | 18.91a | 28.19b | 15.07a | ||
Ketones | 14.66a | 14.26a | 14.81a | ||
Esters | 1.21a | 1.36a | 1.25a | ||
Aldehydes | 3.12a | 1.55b | 1.08b | ||
Nitrogen compounds | 5.65ab | 6.21b | 4.21a | ||
Lactones | 5.74a | 6.16a | 4.37b | ||
Methoxyphenolic | 11.85a | 1.05b | 27.78c | ||
Acids | 8.44a | 1.82b | 10.52a | ||
Others | 1.36a | 1.36a | 0.79a |
The volatile components identified in 57 Pu-erh teas mainly included hydrocarbons, alcohols, ketones, methoxyphenolic compounds, esters, aldehydes, furans, nitrogen compounds, lactones, and acids. As listed in Table 2, obvious differences in the volatile composition and content among these three different Pu-erh teas can be found. The main volatile components in 10 aged teas were hexadecanoic acid, dihydroactinidiolide, caffeine, linalool, 6,10,14-trimethyl-2-pentadecanone, β-ionone, cedrol, and phytol; the main volatile components in 23 raw teas were linalool, tridecane, caffeine, dihydroactinidiolide, β-ionone, 6,10,14-trimethyl-2-pentadecanone, dodecane, etc.; and the main volatile components in 24 ripe teas were 1,2,3-trimethoxybenzene, hexadecanoic acid, 1,2,4-trimethoxybenzene, dihydroactinidiolide, 6,10,14-trimethyl-2-pentadecanon, caffeine, and 1,2,3-trimethoxy-5-methyl-benzene. The Pu-erh raw tea retains the flavour and colour of the original tea because of the lack of post-fermentation in the manufacturing process, and therefore, it is not surprising that its aromatic components and taste were similar to other green teas, such as Longjing tea,25 Hubei green tea,16 Biluochun green tea,14 etc. The most obvious feature was that the content of alcohols accounted for a higher proportion (30.65%) in raw teas, especially linalool, phytol, and geraniol. These volatiles, together with some other terpene alcohols, were likely the major contributors to the herbaceous and pleasant flowery odour of the raw tea.28 In contrast, the content of these volatile compounds in aged and ripe tea were only 21.95% and 14.69%, respectively. Because aged teas were not processed by fast pile fermentation, the relative content of alcohols is obviously lower than that of raw tea. There seems to be two possible reasons for such a decrease: the first was that some alcohol compounds with lower boiling points were volatilized during the long-term storage process and the second was that some alcohol compounds were transformed into other substances by exogenous microbes and natural oxidation during long-term storage. In the case of ripe tea, the fermentation process may effectively facilitate the oxidation and decomposition of some alcohols and therefore lead to the lowest content of alcohols among the three types of teas. The content of hydrocarbons in raw tea was higher than in aged and ripe teas, especially that of tridecane and dodecane. This is possibly a result of the effective preservation of these components (that are enriched in the fresh tea leaves) in green tea without specific processing. Most of these identified hydrocarbons were saturated hydrocarbons; they usually make a minor contribution to the tea flavour.29 Unsaturated hydrocarbons play a vital role in the aroma of the tea; their content was relatively low, e.g., only 2.56% in ripe tea, and as such, may make a limited contribution to the aroma of Pu-erh tea. Ketones, with a sweet and woody flavour, exhibit a comparable content in the three types of Pu-erh tea. We speculate that processing and storage time has only a minor effect on the content of ketone compounds, and the contribution of ketone compounds to aromas of different types of Pu-erh tea is insignificant, similar to ionone. In addition, the content of methoxyphenolic compounds showed a significant difference (p < 0.001) among these three types of Pu-erh tea. The content of methoxyphenolic compounds was found to be the highest (27.78%) in ripe teas in comparison with aged teas (11.85%) and raw teas (1.05%). Methoxyphenolic compounds made a strong contribution to the stale flavour of Pu-erh ripe teas, and as such, can be used as potential markers to distinguish among these three types of Pu-erh tea.26 Some methoxyphenolic components, such as 1,2,3-trimethoxybenzene and 1,2,4-trimethoxybenzene, were commonly found in aged and ripe teas, demonstrating that these two teas share certain chemical similarities to each other. In fact, although the aged tea has not experienced a post-fermentation process, its quality characteristics were similar to ripe tea after lengthy ageing. Therefore, we suggested that methoxyphenolic compounds could be the important factor causing similar quality characteristics between aged tea and ripe tea. Similarly, through the action of microorganisms and hot and humid conditions, Pu-erh ripe tea generates many methoxyphenolic compounds within a short time. Although microorganisms have an important role in the formation of methoxyphenolic compounds, the observed high content of methoxyphenolic compounds in the aged tea suggested that the formation of these compounds did not necessarily rely on microorganisms. Consequently, the formation mechanism of methoxyphenolic compounds needs to be investigated further. The content of aldehyde compounds in aged tea (3.12%) was relatively higher than in raw tea (1.55%) and ripe tea (1.08%). Among them, the content of (E)-2-decenal was the highest in the aged tea, whereas it was almost undetectable in the other two tea types. 2-Dodecenal was only detected in the aged tea, and the content of hexanal and benzaldehyde was higher in aged tea than in the other two tea types. Therefore, these volatiles are likely to make a notable contribution to the aroma of aged tea. With regard to lactone compounds, only dihydroactinidiolide has been detected in all teas. Dihydroactinidiolide, which has a coumarin and musk flavour and has a certain contribution to the aroma of Pu-erh tea, may be a degradation product of β-carotene. As for the acid compounds, only hexadecanoic acid has been detected. Of note, its content was higher in aged tea (8.44%) and ripe tea (10.52%) than in raw tea (1.82%). It therefore seems likely that the content of hexadecanoic acid is related to the fermentation processing and storage time. In addition, the similar quality characteristics of aged and ripe tea may also be attributed to the comparable content of hexadecanoic acid in these two tea types. The main nitrogen compound in the three types of Pu-erh tea was caffeine, which was mainly related to the taste characteristics of the tea. The contents of esters, phenolics and oxygen-containing heterocyclic compounds were low in all Pu-erh teas. 2-Pentyl furan, whose formation is related to the Maillard and Strecker degradation reactions of amino acids and sugars, was detected but showed significant differences in content among the three types of Pu-erh tea.30
Pu-erh raw tea has an even richer set of chemical substances than regular green tea, including water-extractable substances and tea polyphenols, which provide a favourable material base for the transformation of chemical constituents during the post-fermentation process and the natural ageing process.2 In terms of volatile components in Pu-erh teas, after post-fermentation, the alcohol and hydrocarbon component content reduced sharply, while methoxyphenolic components increased significantly, and as a result, a great change in the aroma quality occurred. As mentioned above, it has been reported that longer ageing improves the quality of Pu-erh tea. It can be seen from our results that aged and ripe tea have some similarities based on aroma components, such as methoxyphenolic and alcohol compounds; this makes these two tea types share some similar aroma characteristics. Some water-soluble ingredients, such as tea polyphenols and flavonoid compounds, should be further compared to explore the similarities and differences of their taste characteristics. In a word, the aged tea was piled in a natural way without being processed by pile fermentation, and therefore, it is not surprising that some differences are observed in the content and composition of aroma components between aged and ripe tea. With the aid of microorganisms, ripe tea achieves similar quality characteristics as aged tea through rapid fermentation; these quality characteristics have been widely recognized by consumers.
In the process of long-term ageing, Pu-erh aged tea experiences complex chemical changes, resulting in a sharp decrease in levels of low-boiling point alcohols and hydrocarbons, and an increase in some of the high-boiling point acids, e.g., hexadecanoic acid. Because of storing the tea for several decades, some low-boiling point substances were lost via evaporation, while some ingredients were enriched because of chemical transformations from other compounds such as tea polyphenols. Du24 and Lv26 found that the formation of methoxyphenolic compounds may have a particularly close connection with the methylation of tea catechins. Overall, the mechanism of post-fermentation and long-term ageing of Pu-erh tea needs to be studied in-depth to explore the effects of various conditions on changes of tea inclusions, including polyphenols and volatile components.
However, it is extremely difficult to predict the volatile change during the storage process of Pu-erh tea. Because the tea aroma component is a very complicated system and easily influenced by many factors, it requires simultaneous characterization of large numbers of volatiles in data matrices. In subsequent work, a study of the dynamic change of the chemical composition and content associated with processing and storage length of Pu-erh tea is necessary. Additionally, not all volatile components contribute to the fragrance equally; sometimes the aromatic contribution of specific volatile ingredients on a large scale is required to be studied with more techniques in order to investigate and expose their hidden characteristics. Therefore, an electronic nose (e-nose) and gas chromatography-olfactometry (GC-O) can be used in further studies for the sensory evaluation of Pu-erh teas.
As shown in Fig. 1a and c, raw and ripe teas are well separated, and old teas are located between ripe and raw teas. However, in the UVN (Fig. 1b), ParN (Fig. 1d) and Ctr (Fig. 1e) scaling methods, the separation between samples from the same or different processing methods was not clear, and samples from different groups overlapped. Moreover, samples from one group were highly scattered and were not clearly clustered. In addition, compared with the UV and Par models, data from old teas were scattered using the UV model; the values of t[1] and t[2] were larger than in the Par model. Therefore, Par scaling is more suitable for the comprehensive analysis of Pu-erh tea volatile components.
Although PCA is an unsupervised method, it yielded sufficient results for most of the analysed types of Pu-erh teas (Fig. 1c). The best discrimination was observed between Pu-erh raw and ripe teas. The overlap observed between old and ripe teas suggests that they have some similar chemical characteristics.
The results of PCA and CA may lead to an inaccurate conclusion because of the lack of sufficient information in the original data set and the inherent flaws of these two methods, i.e., data dimension reduction and unsupervised recognition. Therefore, it is difficult to discriminate the most important variables from this loading plot in order to obtain a more accurate classification model. An alternative method, supervised OPLS-DA, was adopted to reveal the most important variables among the 122 variables but also to have better discrimination among different kinds of Pu-erh teas.
Although the number of tea samples is limited, these results showed that it is possible to discriminate and classify different processing types of Pu-erh tea based on the analysis of the volatile contents using pattern recognition techniques such as PCA, CA, and OPLS-DA. In the present study, the number of aged tea samples is relatively few because of the difficulty in obtaining reliable old tea sources. Future studies will collect more standard samples for aged tea with different ages and focus on the effect of storage time on dynamic changes in ingredients because of biochemistry and the impact of different environmental conditions on different chemical components, ultimately providing a theoretical basis for the scientific storage of Pu-erh tea. In conclusion, our study lays a foundation for improving the scientific value of Pu-erh tea and provides an understanding of the chemical composition and differences of different processing types of Pu-erh tea for consumers.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra15381f |
This journal is © The Royal Society of Chemistry 2015 |