Discrimination Based on Volatile Compounds and Differential Analysis of Chinese Dark Tea

Simultaneous distillion and extraction (SDE)/gas chromatographymass spectrometry (Gc-Ms) detection coupled with partial least squares-discriminate analysis (PLS-DA) were used to discriminate Chinese dark teas (CDTs). A total of 84 volatile compounds were taken to conduct the cluster analysis. 9 kinds of CDTs could be divided into 3 groups in consistent with the manufacturing processes. Besides, ingredients that were related with the quality of CDT, such as free amino acid, soluble sugar and catechines were analyzed. Differential trends were disclosed between Pu-Er raw tea and Pu-Er ripe tea as well as raw dark green tea (RDGT).


Introduction
Chinese dark tea (CDT) (Zhang et al., 2013), which was characterized with the post-fermentation, was popular among those who take tea as their favorite beverage.Pharmaceutical research pronounced Chinese dark tea therapeutic benefits in the treatment with hyperlipemia (Li et al., 2012), cardiovascular disease (CVD) (Xu et al., 2010), diabetes (Zhang et al., 2009;Li et al., 2014) and etc (Lv et al., 2007;Hou et al., 2010;Xiong et al., 2012;Liu et al., 2014).Generally, manufacture of CDTs was beginning with the fixation, rolling, pile-fermentation and drying sequentially.Then the raw dark green tea (RDGT) was obtained (Xia, 2016).After which, with steaming and pressing, the RDGT was compressed into brick, cake or other types.However, CDTs were consisted of different categories of teas, such as RDGT, Pu-Er raw tea, Pu-Er ripe tea, and so on.Each of them was prepared through respective process, so the connotation of chemical compositions would be distinguished and the subsequent derivation of them would be disparate as well (Lv et al., 2013).Distinguished with Pu-Er raw tea, there was pile fermentation during the manufacture process of Pu-Er ripe tea as well as RDGT.Moreover, loose tea distinguished from compressed tea in autoclaved typing.
Discrimination of diverse dark teas was the pursuit of researchers as well as consumers.However, several precise means could be employed.Liquid chromatography-mass spectrometry was employed to investigate the metabolic changes of Pu-Er tea during the pile-fermentation.And discrimination between the raw material group and pile-fermentation process groups or the final product group were obtained (Chen et al., 2013).Using electronic nose and ultrasound-assisted extraction-dispersive liquid-liquid microextraction-gas chromatographymass spectrometry, Ye and co-workers discriminated raw Pu-Er tea from ripened Pu-Er tea (Ye et al., 2016).Combined with chemometrics methods, GC-MS identification of volatile compounds is fast and ideal for discrimination of Pu-Er teas with different processing technologies and storage times (Lv et al., 2015a).Taking 77 volatile compounds into PCA, Pu-Er green tea could be clearly distinguished from regular green tea.Meanwhile, GC-MS fingerprints were used to demonstrate the production sites of Pu-Er green tea in Yunnan (Lv et al., 2015b).At the other hand, aged Pu-Er tea were clustered away from ripened Pu-Er tea using principal component analysis (PCA) and hierarchical cluster analysis (HCA) based on comparison of ten major constituents (Yi et al., 2015).
Herein, comprehensive analyses were taken up to elucidate the different succession of quality-related constituents between RDGT in bulk, Pu-Er raw tea and Pu-Er ripe tea in cake type.These results highlight our current understanding of the exact objective and accurate analytical method to discriminate CDTs varieties.

Material and Methods
All the tea samples were commercially obtainable from Colors of Yunnan Tea CO.LTD.All the solvents and chemicals were commercially available.RDGT in bulk aged for 3, 2, 1 years, Pu-Er raw tea, which was in chitsu pingcha aged for 7, 5, 3 years and Pu-Er ripe tea which was in chitsu pingcha aged for 8, 7, 6 years were chosen to conduct the research.Listed in Table I, each of them was numbered in order to simplify the experiment (Table 1).All of the tea samples were ground to pass through 30-60 mesh and sealed for future use.Tea samples were weighted on analytical balance (Metiler Toledo, ME-104E).UV absorption spectrum was obtained from Shimadzu UV-3600.According to GB/T 8314-2013, free amino acids were detected at the wavelength of 570 nm.
Anthrone colorimetry method was adopted to determine the content of soluble total sugar.
GC-MS data were collected from Agilent 7890A-5975C.Capillary column of Agilent DB-5Ms (30 m × 0.25 mm inner diameter, 0.25 μm film thickness) was chosen to conduct the detection.The stepped temperature program was set as below: An initial temperature of 50 °C was held for 2 min and then increased to 85 °C at 5 °C min -1 ; this temperature was held for 2 min and then increased to 110 °C at 2 °C min -1 ; and then increased to 130 °C at 7 °C min -1 ; heating up to 230 °C at 5 °C min -1 , this temperature was maintained for 8 min.The injector temperature was 230 °C.Helium (percentage purity > 99.999%) was used as the carrier gas.The column flow rate was 1 mL min -1 and the split ratio was 1:1.The mass spectrometer was used under the following conditions: ionisation voltage, 70 eV; ion source temperature, 230 °C; interface temperature, 230 °C.The mass scan range was 30-400 atomic mass units (amu).Mass spectral identification was achieved by comparing spectra with the commercial mass spectral databases NIST-14.Components were tentatively identified by agreement of their retention times and mass spectra with published data and, if available, with those of authentic compounds.

Experimental
Simultaneous distillion and extraction was used to absorb the volatile compounds.10 g of the tea sample were sealed in the tube, and 300 ml boiling purity water were added.After balancing for 10 min, 50 ml distilled ether was added into the tube on the other side to extract the volatile compounds.Refluxing for 50 mins, then the heater was removed.After separation of the water, organic phase was dried with Na 2 SO 4 .Then filtrated and concentrated to 1-2 ml for GC-MS detection.The temperature of the injection port was set at 230 °C.The data was collected and analyzed on the Agilent ChemStation.

Difference in the Constituent of Volatile Compounds
Listed in Table 2, ingredients of the volatile compounds in RDGT, Pu-Er raw tea and Pu-Er ripe tea were distinguished.84 compounds were isolated, among which there were 13 aldehydes, 19 ketones, 6 esters, 16 alcohols, 8 alkoxides and 12 alkanes.Furthermore, 9 alkenyl aldehydes and 14 ketenes could be isolated.
Particularly worth mentioning is that linalool was not detected in Pu-Er ripe tea in spite of the highest account in RDGT and Pu-Er raw tea.Also, alcohols in RDGT and Pu-Er raw tea were dominate while alkoxides in Pu-Er ripe tea were the maximum compounds.Remarkably, naphthalene and naphthalene were also detected in Pu-Er ripe tea as well as Pu-Er raw tea, but none in RDGT.Interestingly, alkoxides in RDGT aged for 3 years was 5.93%, about 60 times more than RDGT aged for 1 year.Taking these 84 volatile compounds as variables, partial least squares-discriminate analysis was conducted to predict the category of the dark tea.Shown as Figure 1, Pu-Er raw tea and Pu-Er ripe tea as well as RDGT could be distinguished faultlessly in accordance with the traditional classification of CDT.And the correlation analysis jas.ccsenet.
It is well known that the material for CDT was coarse and old, so the content of cellulose in CDT was much higher than the other teas.When it turned to soluble total sugar, the tendency was increasing which benefited from the hydrolysis of cellulose consisted in the old leaves.In respect to the attenuation of soluble total sugar in Pu-Er ripe tea, consumption by microorganism was much stronger than the hydrolysis of cellulose (B1, B2 and B3, Figure 3).

Table 1 .
Tea sample of CDTs

Table 5 .
At the same time, sensory evaluation was conducted according to GB/T 14487-2008 as well as GB/T 23776-2009 and the results were disclosed as below in Table5.Overall, both Pu-Er raw tea and Pu-Er ripe tea obtained higher score than RDGT.Sensory evaluation of CDTs GC in Pu-Er content of gall th time.It is w an in Pu-Er rip . Metabolic Changes during the Pu-erh Tea Pile-Fermentation Revealed by a Liquid Chromatography Tandem Mass-Spectrometry-Based Metabolomics Approach.J.Food Sci., 78(11), 1665-1672.http://dx.doi.org/10.1111/1750-3841.12288