Characterization of white tea metabolome: Comparison against green and black tea by a nontargeted metabolomics approach

Accepted Manuscript Characterization of white tea metabolome: Comparison against green and black tea by a nontargeted metabolomics approach

Weidong Dai, Dongchao Xie, Meiling Lu, Pengliang Li, Haipeng Lv, Chen Yang, Qunhua Peng, Yin Zhu, Li Guo, Yue Zhang, Junfeng Tan, Zhi Lin PII: DOI: Reference:

S0963-9969(17)30102-3 doi: 10.1016/j.foodres.2017.03.028 FRIN 6635

To appear in:

Food Research International

Received date: Revised date: Accepted date:

2 August 2016 14 February 2017 10 March 2017

Please cite this article as: Weidong Dai, Dongchao Xie, Meiling Lu, Pengliang Li, Haipeng Lv, Chen Yang, Qunhua Peng, Yin Zhu, Li Guo, Yue Zhang, Junfeng Tan, Zhi Lin , Characterization of white tea metabolome: Comparison against green and black tea by a nontargeted metabolomics approach. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/j.foodres.2017.03.028

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Characterization of white tea metabolome: comparison against green and black tea by a nontargeted metabolomics approach

Weidong Dai1, Dongchao Xie1, Meiling Lu2, Pengliang Li1, Haipeng Lv1, Chen Yang1,

1

SC

RI

PT

Qunhua Peng1, Yin Zhu1, Li Guo1, Yue Zhang1, Junfeng Tan1, *, Zhi Lin1, **

Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture,

NU

Tea Research Institute, Chinese Academy of Agricultural Sciences, 9 Meiling South

2

MA

Road, Hangzhou, Zhejiang 310008, PR China

Agilent Technologies (China) Limited, No. 3 Wangjing North Road, Chaoyang Distr.,

*

Corresponding

PT E

D

Beijing, 100102, P. R. China

author:

Tel.:

+86

571

86653154;

E-mail

addresses:

E-mail

addresses:

Corresponding

AC

**

CE

[email protected]

[email protected]

author:

Tel.:

+86

571

86650617;

ACCEPTED MANUSCRIPT Abstract White tea is considered the least processed form of tea and is reported to have a series of potent bioactivities, such as antioxidant, anti-inflammatory, anti-mutagenic, and anti-cancer activities. However, the chemical composition of white tea and the

PT

dynamic changes of the metabolites during the manufacturing process are far from

RI

clear. In this study, we applied a nontargeted metabolomics approach based on

SC

ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) to comprehensively profile the characteristic

NU

metabolites of white tea. There were significant differences in the content of amino

MA

acids, catechins, dimeric catechins, flavonol and flavone glycosides, and aroma precursors in white tea compared with green and black teas that were manufactured

D

from the same fresh tea leaves. Furthermore, the dynamic changes of the metabolites

PT E

in the tea samples with various withering durations of 0, 4, 8, 12, 16, 20, 24, 28, and 36 h were also profiled. This study offers a comprehensive characterization of the

CE

metabolites and their changes in white tea.

AC

Keywords: white tea, withering, metabolomics, LC-MS, dynamic change

Chemical compounds studied in this article Theanine (PubChem, CID: 439378); Glutamic acid (PubChem, CID: 33032); Quercetin (PubChem, CID: 5280343); Kaempferol (PubChem, CID: 5280863); Myricetin Theaflavin

(PubChem, (PubChem,

CID: 5281672);

Apigenin

CID: 114777);

(PubChem,

Epigallocatechin 2

CID: 5280443);

gallate

(PubChem,

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

CID: 65064); Catechin (PubChem, CID: 73160); Caffeine (PubChem, CID: 2519)

3

ACCEPTED MANUSCRIPT 1 Introduction Tea is the most consumed beverage after water in the world because of its health benefits and satisfactory sensory experience (Ho, Zheng, & Li, 2015; Namita, Mukesh, & Vijay, 2012; Sharangi, 2009). Compared with green tea and black tea, the two most

PT

popular teas, white tea is a rare form of tea that undergoes the least amount of

RI

processing. Only a prolonged withering process and drying process are included in the

SC

manufacturing of white tea. In the prolonged withering process, a slight fermentation (oxidation) occurs, which is catalyzed by endogenous polyphenol oxidase (PPO) and

NU

peroxidase (POD) in tea leaves, and these enzymes produce the unique aroma and

MA

taste of white tea (Hashimoto, Goto, Sakakibara, Oi, Okamoto, & Kanazawa, 2007; K. Wang, Liu, Liu, Huang, Xu, Li, et al., 2011; Yang, Baldermann, & Watanabe, 2013).

D

Recent studies have revealed that white teas have a series of potent bioactivities,

PT E

including antioxidant (T. Dias, Tomás, Teixeira, Alves, Oliveira, & Silva, 2013; , Socorro, Silva, & Oliveira, 2014), anti-inflammatory (Thring,

CE

Hili, & Naughton, 2011; L. Zhao, La, & Grenier, 2013), anti-mutagenic (Santana-Rios,

AC

Orner, Amantana, Provost, Wu, & Dashwood, 2001), anti-cancer (Hajiaghaalipour, Kanthimathi, Sanusi, & Rajarajeswaran, 2015; Mao, Nie, Tsu, Jin, Rao, Lu, et al., 2010; Shukla, 2007), and neuroprotective activities (Almajano, Vila, & Ginés, 2011; López & Calvo, 2011). These bioactivities depend on the unique metabolite phenotype of white tea. There are some studies that have been carried out to investigate the major compositions of white tea and have compared white tea with other teas. Alcazar et al. (Alcazar, 4

ACCEPTED MANUSCRIPT Ballesteros, Jurado, Pablos, Martín, Vilches, et al., 2007) reported that white teas have the highest contents of alanine, arginine, asparagine, histidine, isoleucine, leucine, phenylalanine, serine, and theanine compared with green, black, oolong, and pu-erh teas, and the teas can be classified using the free amino acid profile combined with a

PT

chemometric method. Santana-Rios et al. (Santana-Rios, Orner, Amantana, Provost,

RI

Wu, & Dashwood, 2001) reported that white tea has higher contents of gallic acid,

SC

theobromine, epigallocatechin (EGC), caffeine, and epicatechin gallate (ECG) and lower contents of theophylline, catechin (C), and epicatechin (EC) compared with

NU

green tea. Zhao et al. (Y. Zhao, Chen, Lin, Harnly, Yu, & Li, 2011) tentatively

MA

identified 68 compounds in green pu-erh, green, and white teas using the UPLC combined with diode array detector (DAD) and MS detection, and found that gallic

D

acid, 1,2,6-trigalloylglucose, and caffeine concentrations were significantly higher in

PT E

white tea than in the other two teas.

However, the chemical constituent investigations in these studies were carried out

CE

using tea samples purchased from markets, which differ in variety, region,

AC

manufacturing procedure, and storage. The initial metabolite contents in fresh tea leaves are also different in these teas. Therefore, these tea samples might be not appropriate for the investigations of the impact of the white tea manufacturing process on tea metabolites (Unachukwu, Ahmed, Kavalier, Lyles, & Kennelly, 2010). In addition, only a small number of major constituents, such as catechins, caffeine, theobromine, and free amino acids were compared in these studies. A comprehensive survey of the metabolome of white tea and its comparison to those of green tea and 5

ACCEPTED MANUSCRIPT black tea are urgently needed. Furthermore, the dynamic changes of the metabolome during the manufacturing process have been less studied. The in-depth mapping of the dynamic changes of the characteristic metabolites will be helpful for the manufacturing of high-quality white teas.

PT

Metabolomics, which is emerging as an important part of system biology, detects

RI

dozen, even hundreds, of endogenous metabolites simultaneously. It provides a global

SC

view of the metabolome and has been widely applied in food and tea studies (W. Dai, Qi, Yang, Lv, Guo, Zhang, et al., 2015; Lee, Lee, Chung, Shin, Lee, Lee, et al., 2011;

NU

Tan, Dai, Lu, Lv, Guo, Zhang, et al., 2016; Xu, Hu, Wang, Wan, & Bao, 2015). In

MA

this work, we manufactured white, green, and black tea from the same fresh tea leaves and their metabolite profiles were compared by using an UHPLC-QTOF/MS-based

D

nontargeted metabolomics approach to discover the chemical characteristics of white

PT E

tea. In addition, the dynamic changes of characteristic metabolites during the

CE

manufacturing process of white tea were also profiled.

AC

2 Experimental 2.1 Chemicals

Deionized water was produced by a Milli-Q water purification system (Millipore, Billerica, Massachusetts). Methanol of LC–MS grade was purchased from Merck (Darmstadt, Germany). Formic acid, EGC, C, epigallocatechin gallate (EGCG), ECG, EC,

kaempferol

3-O-glucoside,

kaempferol

3-O-galactoside,

kaempferol

3-O-rutinoside, vitexin, luteolin-8-C-glucoside, isovitexin, quercetin 3-O-glucoside 6

ACCEPTED MANUSCRIPT (isoquercitrin), tryptophan, glutamic acid, proline, glutamine, aspartic acid, asparagine, leucine, isoleucine, threonine, lysine, histidine, valine, arginine, γ-aminobutyric acid (GABA), myricitrin, kaempferol 3-O-arabinoside, guanosine, quinic acid, chlorogenic acid, sulfacetamide, sulfafurazole, flumequine, and adenosine were from Sigma (St.

theaflavin,

theaflavins-3-gallate,

and

RI

apigenin-6,8-C-diglucoside,

PT

Louis, MO). Myricetin 3-galactoside, procyanidin B1, procyanidin B2, theogallin,

SC

theaflavins-3,3’-digallate were purchased from Chemfaces (Wuhan, China). Theanine, quercetin 3-rhamnoglucoside (rutin), quercetin 3-O-galactoside, theobromine,

NU

gallocatechin (GC), tyrosine, and phenylalanine were obtained from J&K Scientific

MA

Ltd. (Beijing, China). Epiafzelechin was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Caffeine was purchased from Enzo Life Sciences Inc.

PT E

2.2 Tea sample treatment

D

(Farmingdale, NY).

Clonal tea leaves of the “Fuding Dabaicha”

r ty (one bud with three leaves)

CE

were collected, and divided into 3 portions for the manufacturing of white, green, and

AC

black teas (Figure 1). The manufacturing process of white tea was as follows: 40 kg fresh tea leaves were withered at 30 ºC and a relative humidity of 47% for 36 h, and then dried at 120 ºC for 20 min and 80 ºC for 20-30 min to a moisture content of approximately 5%. The manufacturing process of green tea was modified from a previous work (W. Dai, et al., 2015). Briefly, after withering for 2 h, the tea leaves were fixed using a rotary continuous fixation machine to terminate the activities of the endogenous enzymes. Then, the leaves were rolled for 1 h before they were dried at 7

ACCEPTED MANUSCRIPT 120 °C for 20 min and then at 80 °C for 20−30 min to a moisture content of approximately 5%. The manufacturing process of black tea was modified from another previous work (Tan, et al., 2016). After 12 h of withering at 30 ºC and a relative humidity of 47%, a roller was used to roll the tea leaves for 80 min. Then, the

PT

tea leaves were fermented during 4 h in an environment control cabinet at 30°C and

RI

90% of relative humidity, and then were immediately dried to a moisture content of

SC

approximately 5% to terminate the fermentation using a hot air dryer at 120 °C for 20 min and then at 80 °C for 20−30 min. For the sampling, one part of the tea sample

NU

was collected. The sampling was repeated six times for the manufactured teas (green,

MA

white, and black teas) and was repeated three times for the tea samples with various withering times (0, 4, 8, 12, 16, 20, 24, and 28 h). 50 milliliters of hot deionized water

D

(100 °C) was added to 0.3 g ground tea powder (< 0.15 mm) and held at 100 °C for 5

PT E

min to extract tea metabolites. Then, 1.6 mL of the solution was centrifuged at 10,000 g (Centrifuge 5810R, Eppendorf) for 10 min. The supernatants were filtered through a

CE

0.22 μm membrane and were then analyzed by LC–MS. Three internal standards of

AC

sulfacetamide, sulfafurazole, and flumequine (0.2 μg/mL) were spiked into the tea samples to evaluate the stability during the LC–MS process. In addition, quality control (QC) samples were prepared by mixing equal amount of each tea sample and were also used to evaluate the metabolomics process. 2.3 Nontargeted metabolomics analysis The metabolomics measurements were carried out following procedures we developed previously (Tan, et al., 2016). Briefly, an UHPLC system (UHPLC Infinity 8

ACCEPTED MANUSCRIPT 1290, Agilent Tech., Santa Clara, CA) coupled to a Q-TOF mass spectrometer (Q-TOF 6540, Agilent Tech., Santa Clara, CA) was used for the initial LC-MS data acquisition. The chromatographic separation of the tea metabolome was conducted on a Zorbax Eclipse Plus C18 column (100 × 2.1 mm, 1.8 μm, Agilent Tech., Littlefalls,

PT

DE). The column was maintained at a constant temperature of 40 °C. Binary mobile

RI

phases were used for elution at a flow rate of 0.4 mL/min, where solvent A was an

SC

aqueous solution containing 0.1% (v/v) formic acid and solvent B was pure methanol. The linear gradient elution profile was as follows: 0 min, 10% B; 4 min, 15% B; 7

NU

min, 25% B; 9 min, 32% B; 16 min, 40% B; 22 min, 55% B; 28 min, 95% B; and 30

MA

min, 95% B. The total analysis time for one sample injection was 30 min. 4 min was allowed for column equilibration between two consecutive injections. The injection

D

volume was 3 μL. The effluent from the column was detected using a Q-TOF mass

PT E

spectrometer with a dual jet stream electrospray ionization (ESI) source and operated in the positive mode. The major MS parameters were the same as previously reported

CE

(Tan, et al., 2016). A mass scan range of 100–1100 m/z was applied for the full scan

AC

analysis. The Q-TOF/MS was calibrated daily following the manufacturer's procedure, and the reference ions with an m/z of 121.0509 (purine) and 922.0098 (hexakis phosphazine) were continuously infused via the reference sprayer during data acquisition for online calibration to ensure the MS accuracy. The metabolites were identified according to authentic standards, accurate mass, MS2 spectra, metabolomics databases (Metlin and Human Metabolome Database), and previous work (W. Dai, et al., 2015; Lv, Zhu, Tan, Guo, Dai, & Lin, 2015; Tan, et al., 2016). Raw LC-MS files 9

ACCEPTED MANUSCRIPT are available at MetaboLights with accession number MTBLS403. 2.4 Data processing The raw data files acquired by LC-MS analysis were first processed by the DA Reprocessor software (Agilent Tech., Santa Clara, CA) to extract the metabolite

PT

feature ions, and then, the data were imported into the Mass Profiler Professional

RI

software (Version 13.0, Agilent Tech., Santa Clara, CA) for peak alignment. The

SC

tolerances of retention time shift and mass shift for peak alignment were set as 0.15 min and 2 mDa, respectively. Ions with a relative standard deviation (RSD) less than

NU

30% in the QC sample analyses were used for further univariate and multivariate

MA

statistics (W. Dai, Yin, Zeng, Kong, Tong, Xu, et al., 2014; W. D. Dai, Wei, Kong, Jia, Han, Zhang, et al., 2011). Principal component analysis (PCA) was performed using t

r

tr

, Sweden) after weight normalization

D

the Simca-P

PT E

and Pareto scaling to investigate the overall tea metabolome variation among the white, green, and black teas and to determine the characteristic metabolites in white

CE

tea. Heat-map analysis and clustering analysis were performed using the

AC

MultiExperiment Viewer (version 4.8.1) to illustrate the dynamic changes of characteristic metabolites in white tea after the data transformation into the fold change of the individual mass intensity to average mass intensity. The significance of the difference of the metabolite between groups was tested using a non-parametric Kruskal-Wallis H test or a Tukey s-b(K) test with the PASWstat software (Version 18.0, USA).

3 Results and discussion 10

ACCEPTED MANUSCRIPT 3.1 Analysis of the tea metabolome by UHPLC Q-TOF/MS As shown in Figure S1-A, the typical total ion current (TIC) chromatograms of white tea, green tea, and black tea showed visually distinct differences. After peak alignment, 2137, 1802, and 1719 metabolite ion features were detected in white, green,

PT

and black tea, respectively. Among them, 101 metabolites were structurally identified

RI

(Table S2), and 85 out 101 metabolites were found in three teas simultaneously

SC

(Figure S1-B in Supplementary Material). To evaluate the performance of the LC-MS analysis, PCA analysis of QC samples and relative standard deviation (RSD)

NU

calculation of three internal standards of sulfacetamide, sulfafurazole, and flumequine

MA

were applied. The QC samples were crowded in the center of the PCA score plot (Figure S2). The RSD of the retention times, m/z, and mass intensities of the three

D

internal standards were ranged from 0.06–0.18%, 4.7×10-5–8.1×10-5%, and

PT E

4.24–5.71%, respectively (Table S1). These results indicated good reproducibility of the LC-MS analyses.

CE

3.2 Characteristics of white tea metabolome

AC

The differences among the metabolomes of white, green, and black tea that were manufactured from the same batch of tea leaves were investigated using PCA analysis (Figure 2). The principal components 1 and 2 explained 56.2% and 26.2% of total variances, respectively, and distinct differences were observed among the three groups of teas in the PCA score plot (Figure 2-A). The PCA loading plot illustrated the main differential metabolites in white tea (Figure 2-B). 3.2.1 Amino acids 11

ACCEPTED MANUSCRIPT Several previous studies have reported that white tea contains a higher amino acid content than green and black teas (Alcazar, et al., 2007; Chen, Chen, Zhang, & Wan, 2009). In this study, the free amino acid contents showed drastic variations among three teas (Table S3). Most of the amino acids, including valine, phenylalanine,

PT

proline, leucine, isoleucine, tryptophan, threonine, lysine, histidine, arginine, and

RI

tyrosine exhibited the highest contents in white tea and the lowest contents in green

SC

tea. The high levels of these amino acids in white tea might be due to the protein breakdown during the prolonged withering process (Y

Lu J

g C

’ r y

NU

Singanusong, et al., 2006). By contrast, theanine, glutamic acid, glutamine, and

MA

aspartic acid, which are recognized as main contributors for the umami taste of tea infusion, showed the lowest contents in white tea and the highest contents in green

D

tea.

PT E

3.2.2 Catechins and dimeric catechins Catechins are considered the main compounds that account for the antioxidant

CE

activity of tea. They can be converted to dimeric, oligomeric, and polymeric

AC

compounds, such as theaflavins, theacitrins, theasinensins, theanaphthoquinones, and thearubigins in the presence of polyphenol oxidase (PPO) and peroxidase (POD) during the tea fermentation process (Stodt, Blauth, Niemann, Stark, Pawar, Jayaraman, et al., 2014). Catechins, including EGCG, EGC, EC, ECG, GC, C, and GCG, were at lower levels in white tea and had the lowest levels in black tea when compared with those in green tea (Table S3). This result is consistent with the fermentation degree in that black, white, and green tea is fully fermented, slightly fermented, and 12

ACCEPTED MANUSCRIPT non-fermented, respectively. However, these results are different from Santana-

’s

findings, who reported that white tea had higher contents of EGC and ECG compared with green tea (Santana-Rios, Orner, Amantana, Provost, Wu, & Dashwood, 2001). This contrast might be due to the inclusion of different varieties of white tea and green ’s work, whereas the same batch of tea leaves was included in this

PT

tea in Santana-

RI

study.

SC

Methylated catechins are naturally present in tea leaves and were recently reported to have a stronger antiallergic (Fujimura, Umeda, Yano, Maeda-Yamamoto, Yamada,

NU

& Tachibana, 2007; Maeda-Yamamoto, Ema, Monobe, Tokuda, & Tachibana, 2012)

MA

and antihypertensive effects (Kurita, Maeda-Yamamoto, Tachibana, & Kamei, 2010) than catechins. In this study, interestingly, methylated catechins, such as EC

D

3-O-(3-O-methylgallate) and EGC 3-methylgallate, exhibited the highest contents in

PT E

white tea compared with those in green tea and black tea. Because methylated catechins are consumed during the fermentation process (Tan, et al., 2016) (EC

CE

3-O-(3-O-methylgallate) and EGC 3-methylgallate were at a significantly low level in

AC

black tea), it is assumed that the methylation of catechins may occur during the prolonged withering process of white tea. However,

theaflavins,

including

theaflavin,

theaflavins-3-gallate,

and

theaflavins-3,3’-digallate, were at significantly high concentrations in white tea because of the slight fermentation, but the levels were much lower than those in black tea (Figure S3). Theasinesins and procyanidins are also important dimeric catechins in teas, however, the variations in their levels after different manufacturing processes are 13

ACCEPTED MANUSCRIPT rarely surveyed. In a previous work on black tea fermentation, the levels of theasinensins increased sharply within the early period of fermentation and then slowly decreased, whereas the level of procyanidins reached the maximum within the first hour of fermentation and then decreased rapidly within 1–6 h to low contents

PT

(Tan, et al., 2016). In this study, theasinesins (theasinesin A and B) had significantly

RI

higher contents in white tea and the highest in black tea compared with those in green

SC

tea (Figure S3), indicating that the fermentation process could be responsible of theasinesins formation. By contrast, procyanidins (procyanidin B1, B2, B3, B5, and

NU

C1) showed an opposite trend: they had the lowest contents in black tea and lower

MA

contents in white tea compared with those in green tea (Figure S3), indicating that procyanidins consumption occurs during the withering and fermentation processes.

D

3.2.3 Flavonol glycosides and flavone glycosides

PT E

Flavonol glycosides and flavone glycosides are also major phenolic constituents in tea and have strong antioxidative bioactivity and potential benefits for the

CE

cardiovascular system (Wu, Xu, Héritier, & Andlauer, 2012). In this study, flavonol

AC

glycosides and flavone glycosides had different profiles according to their aglycones and conjugated sugars (Table S3). Kaempferol glycosides, including kaempferol 3-O-glucoside, kaempferol 3-O-galactoside, kaempferol 3-O-rutinoside, kaempferol 3-glucosylrutinoside, kaempferol 3-galactosyrutinoside, kaempferol 3,7-dirhamnoside, kaempferol 3-O-arabinoside, and kaempferol 3-(6’’-galloylglucoside), showed the highest levels in white tea compared with those in green tea and black tea. The quercetin

glycosides

showed interesting tendencies. Glucosylated quercetin 14

ACCEPTED MANUSCRIPT compounds,

including

quercetin

3-O-glucoside

(isoquercitrin),

quercetin

3-rhamnoglucoside (rutin), quercetin 3-O-glucosylrutinoside, quercetin diglucoside, and quercetin triglucoside, presented slightly lower contents in white tea than in green tea, whereas galactosylated quercetin compounds (quercetin 3-O-galactoside and

PT

quercetin 3-O-galactosylrutinoside) had relatively high contents in white tea.

RI

Myricetin glycosides also showed similar tendencies. In white tea, myricetin

SC

3-glucoside showed a lower content, while myricetin 3-galactoside showed a slightly higher content. Apigenin-C-monoglycosides, such as vitexin and isovitexin, presented

apigenin-6,8-C-diglucoside,

and

MA

apigenin-6-C-glucosyl-8-C-arabinoside,

NU

the highest contents in green tea, whereas apigenin-C-diglycosides, including

apigenin-6-C-arabinoside-8-C-glucoside, showed the highest contents in white tea.

D

3.2.4 Aroma precursors

PT E

Aroma primeverosides and glucosides are regarded as aroma precursors, which release aroma compounds during the tea manufacturing and brewing processes. In this

CE

study, phenylethyl primeveroside, benzyl primeveroside, linalool primeveroside, and

AC

linalool oxide primeveroside were found in low contents in white tea and were nearly consumed completely in black tea (Figure S4). The extremely low contents of aroma precursors in black tea were consistent with the results obtained by Wang et al., who observed that phenylethyl primeveroside, benzyl primeveroside, and linalool oxide primeveroside were almost disappeared after the fermentation stage of black tea (D. M. Wang, Kurasawa, Yamaguchi, Kubota, & Kobayashi, 2001). 3.3 Dynamic changes of metabolites during the manufacturing process of white 15

ACCEPTED MANUSCRIPT tea Long-time withering is the key process for the manufacturing of white tea. In this study, the dynamic changes of the characteristic metabolites during the withering process (0, 4, 8, 12, 16, 20, 24, 28, and 36 h) were mapped. The PCA analysis showed

PT

obvious stepwise alterations of the tea metabolome during the withering process from

RI

0 to 36 h (Figure 3). The amino acid contents changed dramatically (Figure 4 and

SC

Table S4). The content of tryptophan, histidine, isoleucine, lysine, phenylalanine, proline, leucine, valine, and tyrosine increased significantly during the withering

NU

process. The fold changes of the levels of tyrosine, valine, leucine, phenylalanine,

MA

lysine, proline, and isoleucine reached up to > 5. The level of glutamic acid and aspartic acid decreased significantly during the withering process, particularly during

D

the first 20 h. Nearly half of their contents were depleted after 36 h of withering.

PT E

Other characteristic metabolites, such as catechins, theasinensins, procyanidins, theaflavins, flavonol glycosides, flavone glycosides, and aroma precursors, were

CE

clustered into 3 groups in the heat-map (Table S4). The levels of the metabolites in

AC

group I, including TF, TF 3-gallate, TF digallate, theasinensin A, and theasinensin B, increased significantly during the withering process. These metabolites are dimeric catechins and their fold changes reached up to >15. Metabolites in group II were mainly flavonol-O-glycosides and flavone-C-glycosides, and they did not change drastically. The level of the methylated catechins of EGC 3-methylgallate and EC 3-O-(3-O-methylgallate) increased during the first 28 h and then slightly decreased during the period of 28-36 h. The amounts of methylated catechins in white tea were 16

ACCEPTED MANUSCRIPT more than 1.7-fold than those in fresh leaves (0 h). This finding will be useful for manufacturing methylated catechin-rich teas. The following compounds decreased significantly during the withering process: group III metabolites, including catechins of GC, GCG, C, EGC, and EC; the aroma precursors of benzyl primeveroside,

PT

linalool primeveroside, and linalool oxide primeveroside; and procyanidins. Less than

RI

50% of the content of GC, GCG, and C were retained in the final white tea product

SC

compared with the initial content in fresh leaves. Approximately 59-90% of the contents of aroma primeverosides were retained in the tea leaves after the 36 h of the

NU

withering process.

MA

4 Conclusion

In this study, white, green, and black teas were manufactured from the same fresh leaves,

and

their

chemical

constituents

were

compared

using

a

D

tea

PT E

UHPLC-QTOF/MS-based non-targeted metabolomics approach. Multivariate and univariate statisticcl analyses suggested great variations in the tea metabolome among

CE

white, green, and black teas. Amino acids, catechins, dimeric catechins, and aroma

AC

precursors were the metabolites with most variation among the three types of tea. Their dynamic changes during the white tea withering process (0, 4, 8, 12, 16, 20, 24, 28, and 36 h) were also mapped. The levels of the amino acids,

tyrosine, valine,

leucine, phenylalanine, lysine, proline, and isoleucine and the dimeric catechins of TF, TF 3-gallate, TF digallate, theasinensin A, and theasinensin B increased more than 5-fold, whereas the level of the catechins of GC, GCG, and C were reduced by > 50%.

17

ACCEPTED MANUSCRIPT Acknowledgements The authors appreciate the funding support from the National Natural Science Foundation of China (No. 31500561), the Earmarked Fund for China Agricultural Research System (No. CARS-23), and the Science and Technology Innovation Project

AC

CE

PT E

D

MA

NU

SC

RI

PT

of Chinese Academy of Agricultural Sciences (No. CAAS-ASTIP-2014-TRICAAS).

18

ACCEPTED MANUSCRIPT References Alcazar, A., Ballesteros, O., Jurado, J., Pablos, F., Martín, M., Vilches, J., & Navalon, A. (2007). Differentiation of green, white, black, Oolong, and Pu-erh teas according to their free amino acids content. Journal of Agricultural and Food

PT

Chemistry, 55(15), 5960-5965.

RI

Almajano, M., Vila, I., & Ginés, S. (2011). Neuroprotective effects of white tea

SC

against oxidative stress-induced toxicity in striatal cells. Neurotoxicity Research, 20(4), 372-378.

of

free

amino

acids

content

in

tea

infusions

with

MA

determination

NU

Chen, L., Chen, Q., Zhang, Z., & Wan, X. (2009). A novel colorimetric

2,4-dinitrofluorobenzene. Journal of Food Composition and Analysis, 22(2), 137-141.

D

Dai, W., Qi, D., Yang, T., Lv, H., Guo, L., Zhang, Y., Zhu, Y., Peng, Q., Xie, D., Tan,

PT E

J., & Lin, Z. (2015). Nontargeted analysis using ultraperformance liquid chromatography–quadrupole time-of-flight mass spectrometry uncovers the effects of

CE

harvest season on the metabolites and taste quality of tea (Camellia sinensis L.).

AC

Journal of Agricultural and Food Chemistry, 63(44), 9869-9878. Dai, W., Yin, P., Zeng, Z., Kong, H., Tong, H., Xu, Z., Lu, X., Lehmann, R., & Xu, G. (2014). Nontargeted modification-specific metabolomics study based on liquid chromatography–high-resolution mass spectrometry. Analytical Chemistry, 86, 9146-9153. Dai, W. D., Wei, C., Kong, H. W., Jia, Z. H., Han, J. K., Zhang, F. X., Wu, Z. M., Gu, Y., Chen, S. L., Gu, Q., Lu, X., Wu, Y. L., & Xu, G. W. (2011). Effect of the 19

ACCEPTED MANUSCRIPT traditional Chinese medicine tongxinluo on endothelial dysfunction rats studied by using urinary metabonomics based on liquid chromatography-mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 56(1), 86-92. Dias, T., Tomás, G., Teixeira, N., Alves, M., Oliveira, P., & Silva, B. (2013). White

PT

tea (Camellia sinensis (L.)): antioxidant properties and beneficial health effects.

RI

International Journal of Food Sciences and Nutrition, 2(2), 19-26.

SC

, G. a. D., Socorro, S., Silva, B. M., & Oliveira, P. F. (2014). White tea as a promising antioxidant medium additive for sperm storage

NU

at room temperature: a comparative study with green tea. Journal of Agricultural and

MA

Food Chemistry, 62(3), 608-617.

Fujimura, Y., Umeda, D., Yano, S., Maeda-Yamamoto, M., Yamada, K., &

D

Tachibana, H. (2007). The 67kDa laminin receptor as a primary determinant of

PT E

anti-allergic effects of O-methylated EGCG. Biochemical and Biophysical Research Communications, 364(1), 79-85.

CE

Hajiaghaalipour, F., Kanthimathi, M., Sanusi, J., & Rajarajeswaran, J. (2015).

AC

White tea (Camellia sinensis) inhibits proliferation of the colon cancer cell line, HT-29, activates caspases and protects DNA of normal cells against oxidative damage. Food Chemistry, 169, 401-410. Hashimoto, T., Goto, M., Sakakibara, H., Oi, N., Okamoto, M., & Kanazawa, K. (2007). Yellow tea is more potent than other types of tea in suppressing liver toxicity induced by carbon tetrachloride in rats. Phytotherapy Research, 21(7), 668-670. Ho, C.-T., Zheng, X., & Li, S. (2015). Tea aroma formation. Food Science and 20

ACCEPTED MANUSCRIPT Human Wellness, 4(1), 9-27. Kurita, I., Maeda-Yamamoto, M., Tachibana, H., & Kamei, M. (2010). Antihypertensive effect of Benifuuki tea containing O-methylated EGCG. Journal of Agricultural and Food Chemistry, 58(3), 1903-1908.

PT

López, V., & Calvo, M. I. (2011). White tea (Camellia sinensis Kuntze) exerts

RI

neuroprotection against hydrogen peroxide-induced toxicity in PC12 cells. Plant

SC

Foods for Human Nutrition, 66(1), 22-26.

Lee, J.-E., Lee, B.-J., Chung, J.-O., Shin, H.-J., Lee, S.-J., Lee, C.-H., & Hong, Y.-S.

NU

(2011). 1 H NMR-based metabolomic characterization during green tea (Camellia

MA

sinensis) fermentation. Food Research International, 44(2), 597-604. Lv, H.-P., Zhu, Y., Tan, J.-F., Guo, L., Dai, W.-D., & Lin, Z. (2015). Bioactive

PT E

Foods, 19, 194-203.

D

compounds from Pu-erh tea with therapy for hyperlipidaemia. Journal of Functional

Maeda-Yamamoto, M., Ema, K., Monobe, M., Tokuda, Y., & Tachibana, H. (2012).

CE

Epicatechin-3-O- 3″-O-methyl)-gallate content in various tea cultivars (Camellia

AC

sinensis L.) and its in vitro inhibitory effect on histamine release. Journal of Agricultural and Food Chemistry, 60(9), 2165-2170. Mao, J. T., Nie, W.-X., Tsu, I.-H., Jin, Y.-S., Rao, J. Y., Lu, Q.-Y., Zhang, Z.-F., Go, V. L. W., & Serio, K. J. (2010). White tea extract induces apoptosis in non–small cell lung cancer cells: the role of peroxisome proliferator-activated receptor-γ and 15-lipoxygenases. Cancer Prevention Research, 3(9), 1132-1140. Namita, P., Mukesh, R., & Vijay, K. J. (2012). Camellia Sinensis (green tea): a 21

ACCEPTED MANUSCRIPT review. Global Journal of Pharmacology, 6(2), 52-59. Santana-Rios, G., Orner, G. A., Amantana, A., Provost, C., Wu, S.-Y., & Dashwood, R. H. (2001). Potent antimutagenic activity of white tea in comparison with green tea in the Salmonella assay. Mutation Research/Genetic Toxicology and Environmental

PT

Mutagenesis, 495(1), 61-74.

RI

Sharangi, A. (2009). Medicinal and therapeutic potentialities of tea (Camellia

SC

sinensis L.)–A review. Food Research International, 42(5), 529-535. Shukla, Y. (2007). Tea and cancer chemoprevention: a comprehensive review. Asian

NU

Pacific Journal of Cancer Prevention, 8(2), 155-166.

MA

Stodt, U. W., Blauth, N., Niemann, S., Stark, J., Pawar, V., Jayaraman, S., Koek, J., & Engelhardt, U. H. (2014). Investigation of processes in black tea manufacture

D

through model fermentation (oxidation) experiments. Journal of Agricultural and

PT E

Food Chemistry, 62(31), 7854-7861.

Tan, J., Dai, W., Lu, M., Lv, H., Guo, L., Zhang, Y., Zhu, Y., Peng, Q., & Lin, Z.

CE

(2016). Study of the dynamic changes in the non-volatile chemical constituents of

AC

black tea during fermentation processing by a non-targeted metabolomics approach. Food Research International, 79, 106-113. Thring, T. S., Hili, P., & Naughton, D. P. (2011). Antioxidant and potential anti-inflammatory activity of extracts and formulations of white tea, rose, and witch hazel on primary human dermal fibroblast cells. Journal of Inflammation, 8(1), 27. Unachukwu, U. J., Ahmed, S., Kavalier, A., Lyles, J. T., & Kennelly, E. J. (2010). White and green teas (Camellia sinensis var. sinensis): variation in phenolic, 22

ACCEPTED MANUSCRIPT methylxanthine, and antioxidant profiles. Journal of Food Science, 75(6), C541-C548. Wang, D. M., Kurasawa, E., Yamaguchi, Y., Kubota, K., & Kobayashi, A. (2001). Analysis of glycosidically bound aroma precursors in tea leaves. 2. Changes in glycoside contents and glycosidase activities in tea leaves during the black tea

PT

manufacturing process. Journal of Agricultural and Food Chemistry, 49(4),

RI

1900-1903.

SC

Wang, K., Liu, F., Liu, Z., Huang, J., Xu, Z., Li, Y., Chen, J., Gong, Y., & Yang, X. (2011). Comparison of catechins and volatile compounds among different types of tea

NU

using high performance liquid chromatograph and gas chromatograph mass

MA

spectrometer. International Journal of Food Science & Technology, 46(7), 1406-1412. Wu, C., Xu, H., Héritier, J., & Andlauer, W. (2012). Determination of catechins and

D

flavonol glycosides in Chinese tea varieties. Food Chemistry, 132(1), 144-149.

PT E

Xu, J., Hu, F.-L., Wang, W., Wan, X.-C., & Bao, G.-H. (2015). Investigation on biochemical compositional changes during the microbial fermentation process of Fu

CE

brick tea by LC–MS based metabolomics. Food Chemistry, 186, 176-184.

AC

Yang, Z., Baldermann, S., & Watanabe, N. (2013). Recent studies of the volatile compounds in tea. Food Research International, 53(2), 585-599. Yao, L. L u X J

g Y C

N

’ r y

S g u

g

tt N & Xu

Y. (2006). Compositional analysis of teas from Australian supermarkets. Food Chemistry, 94(1), 115-122. Zhao, L., La, V. D., & Grenier, D. (2013). Antibacterial, antiadherence, antiprotease, and anti-inflammatory activities of various tea extracts: potential benefits for 23

ACCEPTED MANUSCRIPT periodontal diseases. Journal of Medicinal Food, 16(5), 428-436. Zhao, Y., Chen, P., Lin, L., Harnly, J. M., Yu, L., & Li, Z. (2011). Tentative identification, quantitation, and principal component analysis of green pu-erh, green,

AC

CE

PT E

D

MA

NU

SC

RI

PT

and white teas using UPLC/DAD/MS. Food Chemistry, 126(3), 1269-1277.

24

ACCEPTED MANUSCRIPT Figure captions Figure 1. The manufacturing processes of white, green, and black teas. Figure 2. Metabolomics analysis of the differences of the tea metabolome of white, green, and black teas (n = 6): (A) a score plot of the PCA demonstrating distinct

PT

metabolomes for three types of teas. R2X = 0.824, Q2 = 0.780; (B) a loading plot of

RI

the PCA (black triangles with red frames indicate characteristic metabolite features in

SC

white tea).

Figure 3. PCA score plot of tea samples with various withering durations from 0 to 36

NU

h. R2X = 0.821, Q2 = 0.686.

MA

Figure 4. Dynamic changes of amino acid contents during the manufacturing process of white tea; data are shown as mean ± SD (n = 3). The mass intensity was detected

AC

CE

PT E

D

with 0.3 g tea powder.

25

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 1

26

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 2

27

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 3

28

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Figure 4

29

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract

30

ACCEPTED MANUSCRIPT Highlights 

White, green, and black teas were manufactured from the same fresh tea leaves.



The three tea metabolomes were compared by a LC-MS based metabolomics approach.



Amino acids, catechins, dimeric catechins, and aroma precursors are the most

Dynamic changes of metabolites during white tea withering process were

CE

PT E

D

MA

NU

SC

RI

profiled.

AC



PT

changeable metabolites.

31