LWT - Food Science and Technology 156 (2022) 113010

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LWT
journal homepage: www.elsevier.com/locate/lwt

Profiling of dynamic changes in non-volatile metabolites of shaken black

tea during the manufacturing process using targeted and non-targeted
metabolomics analysis
Jinjin Xue a, b, Panpan Liu c, Guiyi Guo d, Weiwei Wang a, Jianyong Zhang a, Wei Wang a,
Ting Le a, Junfeng Yin a, Dejiang Ni b, **, Heyuan Jiang a, *
a
Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou,
310008, China
b
Huazhong Agricultural University, Wuhan, 430070, China
c
Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan, 430064, China
d
Henan Key Laboratory of Tea Comprehensive Utilization in South Henan, Xinyang Agriculture and Forestry University, Xinyang, 464000, China

A R T I C L E I N F O A B S T R A C T

Keywords: Shaken black tea (SBT) is a new type of black tea that combines the traditional black tea processing technology
Shaken black tea with the shaking process of oolong tea. In this study, non-targeted metabolomics analysis based on ultra-high-
Non-targeted metabolomics pressure liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied
Dipeptides
to comprehensively analyze the non-volatile metabolites of SBT during the manufacturing process. Using
Amino acids
Metabolic pathways
multivariate statistical analysis, 93 compounds were screened as differential compounds. During the whole
process of SBT, amino acids, dipeptides, theaflavins and lipids contents showed significant increases, while that
of catechins and procyanidin C1 significantly decreased. Compared with fresh leaves, the content of proteina­
ceous amino acids (PAAs) in the dried black tea increased 1.61-fold, and total catechins decreased to 7.25%. The
results of hierarchical clustering analysis (HCA) showed that fermentation is the key process that causes the
variations of these metabolites, followed by drying and shaking. The biosynthesis metabolism of amino acids,
flavonoids and linoleic acid are abundant, mainly due to the stress response of fresh tea leaves to dehydration
and mechanical damage during shaking processing. It is beneficial to explore the quality formation during the
production of SBT through comprehensive analysis of the changes in metabolites.

1. Introduction
According to historical records, tea trees (Camellia sinensis, The­
aceae) have been cultivated and utilized in China for more than 3,000
years (Yan, Zhong, Duan, Chen, & Li, 2020). Initially, tea was discovered
as a medicinal plant and its variety of health benefits have been exten­
sively explored, including its antioxidant, anti-inflammatory, anti-mi­
crobial, and hypoglycemic properties (Carloni et al., 2013; Chen, Zhai, &
Arendrup, 2015; Hodges, Sasaki, & Bruno, 2020; Li et al., 2015).
Nowadays, tea is highly valued and widely accepted as a daily beverage
worldwide due to its unique organoleptic characteristics. The health
benefits of tea and its taste are closely related to its multitudes of
non-volatile compounds, including polyphenols, tea pigments,
alkaloids, amino acids, and organic acids (Samanta, 2020; Zhang, Cao,
Granato, Xu, & Ho, 2020). In addition to the tea cultivar, season, and
geographical environment, the processing technology is considered to
be a crucial factor that has a significant influence on the variety, pro­
portion, and content of tea components (Feng et al., 2019). In China,
fresh tea leaves are processed through six kinds of post-harvest processes
to produce green, white, yellow, oolong, black, and dark teas. According
to the degree of fermentation, they are divided into non-fermented tea
(green tea), lightly-fermented tea (white tea, yellow tea),
semi-fermented tea (oolong tea), fully-fermented tea (black tea), and
post-fermented tea (drak tea) (Jiang et al., 2019; Wang et al., 2019).
Shaking is the critical step in the manufacturing process of oolong
tea, as it reduces the astringency and enriches the characteristic taste

* Corresponding author.
** Corresponding author.
E-mail addresses: nidj@mail.hzau.edu.cn (D. Ni), jhy300@yahoo.com (H. Jiang).

https://doi.org/10.1016/j.lwt.2021.113010
Received 6 September 2021; Received in revised form 15 December 2021; Accepted 20 December 2021
Available online 25 December 2021
0023-6438/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Xue et al.
(Chen, Liu, et al., 2020). Zhang (Zhang et al., 2019) used non-targeted
metabolomics to investigate the changes of non-volatile metabolites in
three tea cultivars during the processing of oolong tea, and revealed 18
shared metabolites markers, including (− )-epigallocatechin gallate
(EGCG), L-phenylalanine (Phe), L-tryptophan (Trp), L-proline (Pro), and
hydroxy-jasmonic acid, and the content of which were dramatically
affected by the shaking process. In another study, by monitoring the
non-volatile metabolites of oolong tea during the entire process (espe­
cially several turnover and spreading), it was found that the wounding
stress applied in shaking process shifted the contents of amino acids,
flavonols, flavonol glycosides, procyanidins, and theaflavins (Chen, Liu,
et al., 2020). During shaking process, the mechanical damage caused by
the collision of fresh leaves, which promotes the oxidative polymeriza­
tion of catechins to form theaflavins and procyanidins, reducing the
astringency of tea (Chen, Liu, et al., 2020).
Black tea is one of the six traditional types of tea. In China, the
shaking technology has been applied to the production of black tea to
successfully produce a new type of black tea, known as shaken black tea
(SBT), which process comprises withering, shaking, rolling, fermenta­
tion, and drying. Different from conventional black tea, shaking tech­
nology is added before fermentation during SBT processing. Shaking
process is a combination of dynamic (turn over) and static (spreading)
process, which can be regarded as an extension of withering process. The
main purpose of shaking process is to control the change of metabolites
in tea leaves to improve tea quality through stress (dehydration and
mechanical damage) on tea leaves. Compared with traditional black tea,
SBT with a mellow taste and distinct floral odor, which was widely
accepted and enjoyed by consumers. The shaking process could decrease
the contents of (− )-epigallocatechin (EGC), (+)-catechin (C), (− )-epi­
catechin (EC), EGCG, (− )-epicatechin gallate (ECG) and total catechins,
and reduce the bitterness and astringency of tea soup, which had a
certain positive effect on the improvement of summer Keemun black tea
quality (Lei, 2017). Similarly, the effects of different shaking times (1-5
times) on the sensory of SBT were studied. The SBT made by shaking
three times was found to have a good sensory quality with sweet and
mellow taste, high contents of amino acids, theaflavins and thear­
ubigins, and low content of tea polyphenols (Zi, 2015). However, the
research on SBT mainly focused on the changes of a limited number of
metabolites or only evaluated part of the entire production process. At
present, a comprehensive analysis of the dynamic changes of
non-volatile metabolites in the processing of SBT has not been pre­
sented. What are the differential metabolites in the processing of SBT?
Which step is most critical for causing significant changes in metabo­
lites? To answer these questions, non-targeted metabolomics analysis of
non-volatile metabolites throughout the entire SBT manufacturing
process is warranted.
In the present study, a non-targeted metabolomics analysis based on
ultra-high-pressure liquid chromatography quadrupole time-of-flight
mass spectrometry (UHPLC-QTOF-MS) was used to reveal the metabo­
lite conversion during the manufacturing process of SBT. This research
could supply useful metabolomic information for the improvement of
black tea quality, and provide a firm foundation for the promotion of
SBT manufacturing process.
2. Materials and methods
2.1. Chemicals and reagents
The experimental deionized water was prepared by a Milli-Q water
purification system (Millipore, Billerica, MA, USA). The methanol, for­
mic acid, acetonitrile, ammonium acetate and ammonium hydroxide
that used for MS analysis were LC/MS-grade and purchased from
Thermo Fisher (Thermo Scientific, Waltham, MA, USA). Catechins [EC,
EGC, ECG, EGCG, C, (− )-gallocatechin (GC), (− )-catechin gallate (CG),
(− )-gallocatechin gallate (GCG)]; theaflavins [theaflavin (TF),
theaflavin-3-gallate (TF-3-G), theaflavin-3ʹ-gallate (TF-3ʹ-G) and
2
LWT 156 (2022) 113010
theaflavin-3,3ʹ-gallate (TFDG)]; amino acids [L-aspartic acid (Asp), L-
threonine (Thr), L-serine (Ser), L-glutamic acid (Glu), L-theanine (Thea),
L-alanine (Ala), L-tyrosine (Tyr), gamma-aminobutyric acid (gamma-
GABA), L-histidine (His), L-lysine (Lys), L-arginine (Arg), Pro, L-gluta­
mine (Gln), L-asparagine (Asn), L-glycine (Gly), L-leucine (Leu), L-
isoleucine (Ile), L-valine (Val), Phe, L-cystine (Cys)]; gallic acid and
caffeine (all with purity ≥98%) were purchased from the Sigma-Aldrich
Chemical Co. (St. Louis, MO, USA).
2.2. Tea processing and sample collection
Clonal tea leaves of the “Fudingdabai” variety (one bud with two
leaves) were harvested from the tea garden of Tea Research Institute,
Chinese Academy of Agricultural Sciences in September 2020. Fresh
leaves (FL) were spread out on a bamboo sieve and exposed to sunlight
at approximately 25 ◦ C for 20 min, in a process known as solar with­
ering. Afterward, the leaves were indoor-withered at 25 ◦ C for 4 h to
reach a moisture level of approximately 70% (w.b.). The leaves subse­
quently underwent a shaking process: the leaves were turned over three
times, for 5 min each time, at 1.5-h intervals (S1–S3). Subsequently, the
leaves were spread out indoors at a temperature of 25 ◦ C and a thickness
of 2–3 cm. After withering for approximately 6 h, the moisture content
of the leaves was approximately 60% (w.b.). Next, the leaves were rolled
using a 6CR-25 roller (Zhejiang Shangyang Machinery Co. Ltd., Quzhou,
China) for 60 min to rupture the tissues. The rolling process was con­
ducted as follows: without pressure for 10 min, with light pressure for
20 min, with high pressure for 20 min, and with light pressure again for
10 min. The fermentation of the leaves was carried out in a PRX-450D
environment control cabinet (Saifu Machinery Co. Ltd., Ningbo,
China) at 30 ◦ C for 3 h with a thickness of 5 cm, and the relative hu­
midity was over 95% (F). Afterward, the fermented leaves were parched
at 120 ◦ C for 10 min to inactivate the enzymes, followed by drying at 80
◦
C for 60 min in a JY-6CHZ-7B hot air-drying machine (Jiayou Ma­
chinery Co., Ltd., Quanzhou, China). Finally, we obtained dried black
tea (D) whose moisture content was less than 6%. The samples (FL, S1,
S2, S3, F) were collected and immediately stored in liquid nitrogen, then
freeze-dried to a moisture content of less than 6% in an LGJ-50C
lyophilizer (Beijing Sihuan Scientific Instrument Factory Co. Ltd., Bei­
jing, China) and stored at − 80 ◦ C until further analysis.
2.3. Non-targeted metabolomics analysis by UHPLC-QTOF-MS
Tea samples were ground to fine powder using a ball mill (MM301,
Retsch company, Haan, Germany). Ground tea leaves (20 mg) were
weighted into 2.0-mL tubes, then 500 μL of extract solution (methanol:
acetonitrile: water = 2: 2: 1 v/v/v), which contained stable isotope label
as internal standard, was added. After vortexing for 30 s, steel balls were
added to the tube and the mixture was ground at 35 Hz for 4 min, fol­
lowed by ultrasonic extraction for 5 min in ice-cold water. The extrac­
tion procedures were repeated three times. The homogenate was
incubated for 60 min at 4 ◦ C, then centrifuged at 18,032×g (Centrifuge
5810R, Eppendorf, Hamburg, Germany) for 15 min at 4 ◦ C. The super­
natant (250 μL) was collected to a new tube and dried completely at 37
◦
C in a vacuum drying oven, and then redissolved in 500 μL of 50%
acetonitrile. After vortexing for 30 s, followed by sonication on ice for
10 min, the supernatant was centrifuged at 18,032×g for 15 min at 4 ◦ C.
Subsequently, 75 μL of the supernatant was transferred into a glass vial
for LC-MS analysis. Each tea sample was performed in triplicates.
The metabolite measurement was carried out on an Agilent 1290
ultra-high-pressure liquid chromatography (UHPLC) system (Agilent
Technologies, Palo Alto, CA, USA), coupled to a triple time-of-flight
(TOF) 6600 mass spectrometer (Sciex, Framingham, MA, USA) equip­
ped with a Waters ACQUITY UPLC BEH Amide column (2.1 mm × 100
mm, 1.7 μm). The mobile phase comprised phase A (25 mmol/L
ammonium acetate and 25 mmol/L ammonium hydroxide in water) and
phase B (100% acetonitrile). The linear gradient elution program was set
J. Xue et al.
as follows: 0–0.5 min, 95% B; 0.5–7 min, 95%− 65% B; 7− 8 min, 65%−
40% B; 8− 9 min, 40% B; 9–9.1 min, 40%− 95% B; 9.1–12 min, 95% B.
The sample cell temperature was kept at 4 ◦ C, the column oven was set at
25 ◦ C, the sample injection volume was 2 μL, and the flow rate was set at
0.5 mL/min. The mass spectrometry data acquisition used the
information-dependent acquisition (IDA) model, which consists of a full
MS1 scan and information-dependent trigger MS/MS fragmentation
events. The IDA criteria were set as follows: the 12 most intense ions
with an intensity threshold above 100 cps; the collision energy was set to
30 eV, and the cycle time was 0.56 s. The electrospray ionization (ESI)
source conditions were as follows: source temperature was 600 ◦ C;
nebulizer gas (Gas 1) and heater gas (Gas 2) were set at 60 and 30 psi,
respectively; curtain gas was set at 35 psi; the declustering potential was
60 V; the ion-spray voltage floating was set at 5 kV in positive ionization
mode and − 4 kV in negative ionization mode. Data acquisition was
performed on Analyst TF 1.7 software (Sciex, Concord, Ontario, Can­
ada). The raw data files (.wiff) were converted into mzXML format using
ProteoWizard software, then XCMS was used for peak alignment,
retention time correction, and peak area extraction. The minfrac and cut
off were set as 0.5 and 0.3, respectively. The identification of metabo­
lites was conducted by comparing their mass spectrometric information
and their retention times using an in-house database (Shanghai Biotree
Biotech Co., Ltd) and public databases (Massbank and Metlin).
2.4. Targeted absolute quantification of free amino acids (FAAs)
Ground tea leaves (0.1 g) were weighted into 15 mL tube and mixed
with 10 mL boiling water in 100 ◦ C water-bath for 30 min, subsequently,
centrifuged at 1307×g for 10 min at 4 ◦ C. Then transfer the supernatant
to a 10 mL volumetric flask and adjust the volume to 10 mL with water.
2 mL solution was added to 3 mL 4% sulfosalicylic acid, incubated at
room temperature for 30 min, and centrifuged at 15,364×g for 5 min.
The supernatant was filtered through a 0.22 μm membrane for analysis.
Each tea sample was performed in triplicates.
The determination of FAAs was performed on an automatic amino
acid analyzer (S-433D, Sykam Technologies, Munich, Bavaria, Ger­
many), equipped with a Na+ model sulfonic acid-based cation-exchange
resin (LCA K07/Li column, 4.6 mm × 150 mm). The column tempera­
ture was 40 ◦ C and the wavelengths were set at 570 nm and 440 nm. The
flow rate was 0.45 mL/min and the injection volume was 50 μL. The
mobile phases and gradient elution were accorded with the literature
(Hua et al., 2021).
The concentrations of total amino acids (TAAs) were calculated by
the following equation:
∑
TAAs = FAAi
The concentration of proteinaceous amino acids (PAAs) is the sum of
Asp, Thr, Ser, Glu, Ala, Tyr, His, Lys, Arg, Pro, Gln, Asn, Gly, Leu, Ile,
Val, Phe, and Cys.
2.5. Targeted absolute quantification of catechins and theaflavins by
UPLC-PDA-QDA
Ground tea leaves (0.2 g) were accurately weighted and extracted
with 5 mL 70% (v/v) methanol-water solution, vortexed for 30 s and
extracted for 10 min in 70 ◦ C water-bath, and then centrifuged for 10
min (1307×g, 4 ◦ C). Transfer the supernatant to a 10 mL volumetric
flask, and the extraction procedures were repeated two times. The two
extraction supernatants were merged, and adjusted to 10 mL with 70%
(v/v) methanol-water. Finally, the total supernatants were filtered
through a 0.22 μm membrane for UPLC-PDA-QDA analysis. Each tea
sample was performed in triplicates.
The analysis was performed on a UPLC system (Waters, Milford, MA,
USA), equipped with a PDA detector and a QDA single quadrupole mass
spectrometry detector. Chromatographic separation was achieved on a
3
LWT 156 (2022) 113010
Waters AQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) by
employing a gradient system of 0.1% formic acid in water (mobile phase
A) and 100% acetonitrile (mobile phase B), and the monitoring UV
wavelength was of 280 nm. The linear gradient elution was set as fol­
lows: 0–3 min (90% B), 3–6 min (90%–83% B), 6–12 min (83%–73% B),
12–15 min (73%–50% B), 15–15.5 min (50% B), 15.5–16 min (50%–
90% B), 16–20 min (90% B). The flow rate was 0.35 mL/min, and the
injection volume was 2 μL. The temperature of sample cell was main­
tained at 10 ◦ C and column temperature was set at 30 ◦ C. The scan range
for PDA was 200–500 nm. The capillary voltage was set at 0.8 kV and
cone voltage was 15 V, temperature of source and desolvation were
120 ◦ C and 600 ◦ C, respectively. Mass spectrometry for QDA detection
was conducted in negative mode using full-scan mass spectra (50–950
m/z).
The content of total catechins is the sum of C, CG, GC, GCG, EC, ECG,
EGC, and EGCG. Total theaflavins is the sum of TF, TF-3-G, TF-3ʹ-G and
TFDG.
2.6. Statistical analysis
One-way analysis of variance (ANOVA) followed by Tukey s-b(k)
(for ≥ three groups) test and student’s t-test (T-test, for two groups) were
conducted to analysis the significant difference levels using SPSS soft­
ware (version 26.0, SPSS Inc., Chicago, USA). Partial least squares
discriminant analysis (PLS-DA) analysis and orthogonal partial least
squares-discrimination analysis (OPLS-DA) were carried out on pareto-
scaling data after log transformation (log10) treatment by using Simca
14.1 software (Umetrics, Umea, Sweden). HCA (Hierarchical clustering
analysis) was generated using an integrative toolkit-TBtools (Chen,
Chen, et al., 2020). The line charts were drawn using the Origin 2018
(OriginLab, Northampton, MA). Pathway analysis was performed using
the online tool MetaboAnalyst (Pang, Zhou, Chong, & Xia, 2021).
3. Results and discussion
3.1. Dynamic changes in FAAs, catechins, and theaflavins during SBT
processing
The content of PAAs was first increased and then decreased during
SBT processing. Compared with fresh leaves, the content of PAAs in the
dried black tea increased 1.61-fold. The concentration of PAAs reached a
maximum (24.60 mg/g) in the fermented leaves. Chen’s experimental
results confirmed that the degradation of protein into PAAs occurred
during the fermentation process (Chen, Zeng, et al., 2020). It can be seen
protein degradation is the main reason for that the increase of PAAs in
fermentation process. Tea contains not only common PAAs but also
non-proteinaceous amino acids, for example, Thea and gamma-GABA.
Thea, a unique amino acid that is found almost exclusively in tea, is
considered a crucial contributor to the umami taste of tea (Zhang et al.,
2020) and accounts for approximately 37.55% of the TAAs in the dried
black tea (Table 1). The content of Thea gradually decreased during SBT
processing, especially during fermentation, which was consistent with
those of previous studies (Chen, Zeng, et al., 2020; Yu & Yang, 2020).
This phenomenon may be due to the formation of volatile compounds
via Strecker degradation from Thea and sugars during fermentation
(Guo, Ho, Schwab, Song, & Wan, 2019). Gamma-GABA is one of the
primary inhibitory neurotransmitters in the vertebrate central nervous
system and has antihypertensive and antianxiety effects (Abdou et al.,
2006; Shimada et al., 2009). Gamma-GABA is mainly biosynthesized via
the irreversible α-decarboxylation of Glu to gamma-GABA, which is
catalyzed by pyridoxal 5′ -phosphate (PLP)-dependent glutamate decar­
boxylase (GAD) in plants (Huang et al., 2018). The gamma-GABA con­
tent increased approximately 4-fold (from 0.10 mg/g in FL to 0.44 mg/g
in S3), indicating that mechanical damage caused by the shaking process
can lead to the accumulation of gamma-GABA. Moreover, the concen­
tration of gamma-GABA reached a maximum (1.06 mg/g) in the
J. Xue et al. LWT 156 (2022) 113010

Table 1
Changes in the contents of free amino acids, catechins, and theaflavins during shaken black tea processing.
Name Contents (mg/g)

FL S1 S2 S3 F D

Asp 1.57 ± 0.05 c 1.67 ± 0.00 b 1.68 ± 0.00 b 2.03 ± 0.01 a 1.3 ± 0.00 d 1.23 ± 0.03 e
Thr 0.23 ± 0.01 f 0.30 ± 0.00 e 0.33 ± 0.00 d 0.35 ± 0.00 c 0.50 ± 0.00 a 0.47 ± 0.00 b
Ser 0.76 ± 0.03 f 0.82 ± 0.00 e 0.91 ± 0.00 d 0.98 ± 0.00 c 1.87 ± 0.00 a 1.78 ± 0.04 b
Asn 0.18 ± 0.00 f 0.49 ± 0.00 e 0.73 ± 0.00 d 1.08 ± 0.00 c 5.05 ± 0.04 a 4.69 ± 0.10 b
Glu 1.96 ± 0.07 a 1.47 ± 0.00 b 1.44 ± 0.00 b 1.29 ± 0.00 c 0.93 ± 0.00 d 0.91 ± 0.01 d
Gln 6.40 ± 0.22 a 4.58 ± 0.07 b 3.83 ± 0.04 d 3.64 ± 0.07 d 4.16 ± 0.08 c 3.74 ± 0.12 d
Thea 18.91 ± 0.51 a 17.27 ± 0.07 b 16.25 ± 0.13 c 15.93 ± 0.17 c 15.08 ± 0.27 d 14.44 ± 0.38 d
Gly 0.04 ± 0.00 c 0.04 ± 0.00 c 0.04 ± 0.00 c 0.04 ± 0.00 c 0.07 ± 0.00 a 0.06 ± 0.00 b
Ala 0.31 ± 0.00 c 0.26 ± 0.00 d 0.25 ± 0.00 d 0.27 ± 0.00 d 0.63 ± 0.00 b 0.68 ± 0.01 a
Val 0.23 ± 0.00 c 0.25 ± 0.11 c 0.44 ± 0.00 b 0.54 ± 0.00 b 1.11 ± 0.01 a 1.09 ± 0.02 a
Cys 0.01 ± 0.00 e 0.02 ± 0.00 de 0.02 ± 0.00 d 0.03 ± 0.00 c 0.08 ± 0.00 a 0.07 ± 0.00 b
Ile 0.04 ± 0.00 e 0.14 ± 0.00 d 0.19 ± 0.00 c 0.25 ± 0.00 b 0.72 ± 0.00 a 0.72 ± 0.01 a
Leu 0.08 ± 0.00 e 0.22 ± 0.00 d 0.29 ± 0.00 c 0.37 ± 0.00 b 0.85 ± 0.00 a 0.86 ± 0.02 a
Tyr 0.36 ± 0.01 e 0.54 ± 0.02 d 0.60 ± 0.01 c 0.70 ± 0.01 b 0.98 ± 0.00 a 0.96 ± 0.02 a
Phe 0.21 ± 0.06 e 0.73 ± 0.00 d 0.84 ± 0.01 c 1.02 ± 0.02 b 1.56 ± 0.07 a 1.50 ± 0.020 a
gamma-GABA 0.10 ± 0.00 d 0.26 ± 0.00 c 0.32 ± 0.00 c 0.44 ± 0.00 b 1.06 ± 0.01 a 1.08 ± 0.07 a
His 0.06 ± 0.00 f 0.07 ± 0.00 e 0.09 ± 0.00 d 0.12 ± 0.00 c 0.23 ± 0.00 a 0.17 ± 0.00 b
Lys 0.15 ± 0.01 f 0.27 ± 0.03 e 0.34 ± 0.00 d 0.45 ± 0.00 c 1.02 ± 0.01 a 0.91 ± 0.01 b
Arg 1.58 ± 0.10 de 1.71 ± 0.02 d 1.49 ± 0.00 e 1.90 ± 0.00 c 2.65 ± 0.00 a 2.19 ± 0.16 b
Pro - 0.16 ± 0.00 d 0.24 ± 0.00 c 0.29 ± 0.01 b 0.82 ± 0.00 a 0.82 ± 0.02 a
PAAs 14.24 ± 0.10 d 13.81 ± 0.11 d 13.84 ± 0.08 d 15.42 ± 0.12 c 24.6 ± 0.23 a 22.92 ± 0.38 b
TAAs 33.26 ± 0.61 c 31.34 ± 0.14 de 30.41 ± 0.20 e 31.80 ± 0.29 d 40.75 ± 0.52 a 38.46 ± 0.50 b
Total catechins 126.20 ± 1.01 ab 130.52 ± 3.66 a 129.12 ± 0.25 ab 125.09 ± 2.00 b 17.59 ± 1.43 c 9.15 ± 0.10 d
Total theaflavins 1.93 ± 0.11 d 2.65 ± 0.46 d 3.17 ± 0.02 cd 4.26 ± 0.23 c 10.57 ± 0.79 a 7.26 ± 0.81 b

The data were represented as mean value ± standard deviation (mean ± SD). Different small letters in a row indicate significant differences between samples (p < 0.05,
Tukey s-b(k) test). FL, fresh leaves; S1, first shaking; S2, second shaking; S3, third shaking; F, fermentation; D, drying; PAAs, proteinaceous amino acids; TAAs, total
amino acids.

fermented leaves. It was mainly due to the enhancement of GAD enzy­ dynamic changes in the metabolite profiles of SBT samples at different
matic activity, which is induced by the low intracellular pH during stages of the manufacturing process. The metabolic profiles of 368
fermentation (Mustroph, Barding, Kaiser, Larive, & Bailey-Serres, compounds were displayed using PLS-DA score plots (Fig. 1A). The first
2014). As the precursor of gamma-GABA, Glu exhibited a completely
opposite trend to that of gamma-GABA during the entire manufacturing
process. Therefore, the accumulation of gamma-GABA is mainly induced
by stresses such as mechanical damage or pH (Yu & Yang, 2020).
Catechins are considered to be the most characteristic metabolites in
tea and contributes to the astringency of tea (Parmar, Mukesh, & Vijay,
2012; Zhang et al., 2020), which usually account for 12%− 24% of the
dry weight of fresh tea leaves (Wan, 2003, p. 9). During SBT processing,
the total content of catechins decreased dramatically, especially in the
fermentation stage. Compared with fresh leaves, the content of total
catechins in dried black tea decreased to 7.25%. The content of total
theaflavins increased significantly followed by subsequent decreased
slightly, reaching its maximum of 10.57 mg/g during fermentation
process. This phenomenon is caused by the high activity of endogenous
polyphenol oxidase (PPO) enzyme during fermentation, which leads to
the oxidation of catechins to form theaflavins (Baruah & Mahanta,
2003).

3.2. Dynamic changes in non-volatile metabolites during the SBT

manufacturing process

To define the dynamic changes in metabolites during the SBT

manufacturing process and to screen the crucial metabolites that are
responsible for metabolomics variation caused by the manufacturing
process, non-targeted metabolomics analysis based on UHPLC-QTOF-MS
coupled with multivariate statistical analysis (PLS-DA, HCA) was
applied to comprehensively analyze the characteristic metabolites in the
tea samples. A total of 368 identified metabolites were used for multi­
variate statistical analysis, including 334 compounds (198 in ESI+, 136
in ESI-) were detected by UHPLC-QTOF-MS, and 34 compounds that
were absolutely quantified using external standards (Table S1- S2). Fig. 1. Partial least squares discriminant analysis (PLS–DA) of tea samples. (A)
The PLS-DA score plot. (B) Permutation plot for PLS-DA model. FL, fresh leaves;
3.2.1. Multivariate statistical analysis S1, first shaking; S2, second shaking; S3, third shaking; F, fermentation;
The supervised PLS-DA was a useful method to investigate the D, drying.

4
J. Xue et al.
two principal components explained 80.55% of the total variations (PC1
= 74.10%, PC2 = 6.45%). Samples could be easily discriminated from
one another based upon different processing procedures, indicating that
the procedures result in samples with distinct chemical profiles and
significant differences. The PLS-DA model showed a good explained
variance (R2Y = 0.987) and high predictive capability (Q2 = 0.813).
Fig. 2. Heatmap of the 93 important differential compounds in tea samples during shaken black tea processing. (FL, fresh leaves; S1, first shaking; S2, second
shaking; S3, third shaking; F, fermentation; D, drying).
5
LWT 156 (2022) 113010
Moreover, permutation testing (Fig. 1B) with 200 iterations was used to
assess the established model, and the intercepts (R2 = 0.821, Q2 =
− 0.363) suggested reliability and avoidance of over-fitting of the PLS-
DA model. Furthermore, the variable importance in projection (VIP)
value was able to show the importance of the independent variable for
the model, which was widely used to measure the contributions of the
J. Xue et al.
variables to the data. According to the criteria of VIP >1 and p < 0.05
(Tukey s-b(k) test), 93 compounds were screened as potential markers,
which presented the greatest changes during the SBT processes and were
considered to play an important role in the discrimination processes of
PLS-DA (Table S3). These compounds were classified into nine cate­
gories according to their chemical structure, including 11 amino acids
and derivatives, 12 carbohydrates and carbohydrate conjugates, 12
flavonoids, 18 lipids and lipid-like molecules, 10 nucleosides and
nucleotide analogs, five organic acids and derivatives, 14 peptides, four
pyridines and derivatives and seven others.
To show the dynamic changes in metabolites in tea samples during
the SBT processes more clearly, a heat map of 93 differential compounds
was constructed (Fig. 2), in which red represented high contents of
compounds and blue represented low contents. The differential sub­
stances investigated were clearly divided into three groups: 11 com­
pounds in Class I, 57 compounds in Class II, and 25 compounds in Class
III.
The compounds in Class I are mainly flavonoids, including catechins
(EC, EGC, GCG, EGCG) and procyanidin C1. The content of these com­
pounds exhibited higher content in fresh leaves but decreased rapidly
during the fermentation process. Most substances were clustered in Class
II, the content of these substances increased significantly during the
fermentation process, especially theaflavins, amino acids, dipeptides,
and nucleosides. It is worth noting that the significantly increased
contents of amino acids and dipeptides after fermentation indicate that,
under the action of internal proteinases and peptidases, a fraction of the
protein had undergone extensive enzymatic degradation. The main
reason for this is that the cellular compartments of the tea leaves were
disrupted during the rolling process, and the proteases in the plastids
were activated, enabling protein capture (Chen, Zeng, et al., 2020). The
compounds in Class III mainly including dipeptides, carbohydrates, and
lipids, which have a direct effect on the formation of the final quality of
black tea. These compounds increased markedly during the drying
process, indicating that thermal reaction is the main influence on sig­
nificant changes in these compounds.
In conclusion, the contents of amino acids, dipeptides, theaflavins,
and lipids significantly increased during the whole process of SBT, while
the levels of catechins and procyanidin C1 dramatically decreased.
Fermentation is a critical process in which the contents of non-volatile
components undergo significant changes, followed by the drying and
shaking processes.
3.2.2. Alterations in metabolites during the shaking process
In order to clarify the influence of the shaking process on the changes
in tea metabolites, the supervised pairwise comparative OPLS-DA was
used to compare the contents of 368 metabolites in FL and S3. In OPLS-
DA score plots (Fig. S1), FL and S3 were separated significantly. Using
VIP >1, p value of <0.05 (T-test), and absolute Log2FC (fold change) > 1
as the screening criteria, 38 substances were identified that shifted
sharply during shaking process (34 upregulated and 4 downregulated)
in Table S4. These upregulated metabolites were mainly amino acids
(Pro, Ile, Asn, Phe, Leu, gamma-GABA, Lys, Val, Cys), dipeptides (Val-
Trp, Phe-Ala, Val-Phe, Ile-Trp), and theaflavins (TF, TFDG, and TF-3ʹ-G).
Pro, Ile, Phe had been reported as the key features metabolites of
shaking process (Zhang et al., 2019). In another report, the content of
free amino acids, especially Leu, Ile and Val increased during shaking
process (Wu et al., 2020).
Tea protein, especially chloroplast-localized proteins, can be
degraded to form amino acids and peptides under the action of various
proteolytic enzymes, this degradation occurs at various stages of the
enzymatic-catalyzed process of tea production (Lai et al., 2020; Yu &
Yang, 2020). Through the protein-metabolite association analysis, it was
found that the dynamic changes of amino acids content were related
with the abundance of their biosynthetic enzymes in different extents
during the shaking process in oolong tea (Wu et al., 2020), indicating
that the amino acids biosynthesis pathway was activated during shaking
6
LWT 156 (2022) 113010
process. Therefore, the accumulation of amino acids during shaking
process is the combined effect of protein degradation and biosynthesis.
The cell wall matrix of the leaf margin was disrupted during the shaking
process, and the outer membrane of chloroplast was broken (Chen, Liu,
et al., 2020). Catechins located in the chloroplasts and vacuoles contact
with the oxidative enzymes in the cytoplasm, and undergo oxidative
polymerization to synthesize theaflavins under the catalyzation of
endogenous enzymes (Chen, Liu, et al., 2020). The shaking process is
beneficial for the enrichment of amino acids, dipeptides and theaflavins.
It can be seen shaking process provided the material basis for the later
production of black tea and contributed to the formation of black tea
quality.
3.3. Dynamic changes in main taste compounds of tea samples during
SBT processing
The sensory evaluation of black tea infusion is its comprehensive
perception of bitterness, astringency, umami, and sweetness taste. In
this study, the SBT showed umami, sweet and kokumi taste character­
istics (Table S5), the comprehensive taste of which is closely related to
its different taste compounds and the chemical interactions between
them (Zhang et al., 2020). These main taste compounds in this study
were shown in Fig. 3, including amino acids, dipeptides, nucleosides,
carbohydrates, catechins and theaflavins.
3.3.1. Amino acids, dipeptides, nucleosides, and carbohydrates
Except for Gln, the content of 8 amino acids (Asn, Pro, Arg, Phe, Val,
Leu, Ile, and gamma-GABA) exhibited increased during shaking,
significantly increased in fermentation, and decreased during drying
process. Meanwhile, most of dipeptides showed increased contents
during the whole manufacturing processes, especially fermentation and
drying. Amino acids combine with carbonyl compounds by Maillard
reaction and Strecker degradation to form distinctive aroma aldehydes,
which contribute to the formation of unique aroma quality of black tea
(Ho, Zheng, & Li, 2015). This is the main reason for the reduction of
amino acids during drying process.
In this study, the amino acids were mainly presented umami (Asn,
Gln), sweet (Pro), bitter (Arg, Phe, Val, Leu and Ile), and astringency
taste (gamma-GABA) (Liu et al., 2018; Yu & Yang, 2020), while di­
peptides display umami (Val-Phe), bitter (Ile-Ile, Pro-Ala), salt enhancer
(Arg-Ser, Arg-Ala), and kokumi taste (gamma-Glu-Glu) (Asao, Iwamura,
Akamatsu, & Fujita, 1987; Kong et al., 2019; Schindler et al., 2011;
Yang, Bai, Zeng, & Cui, 2019). Studies have reported that kokumi-active
gamma-Glu-Glu can enhance the umami taste of monosodium glutamate
(MSG), and show synergistic effects in activating T1R3 (Yang et al.,
2021). Nucleotides like cytidine 5ʹ-monophosphate (5ʹ-CMP) are
considered significant contributors to the umami of tea infusions
(Manninen, Rotola-Pukkila, Aisala, Hopia, & Laaksonen, 2018). In
addition, carbohydrates like D-fructose also contribute to the sweetness
of black tea (Yu, Liu, Zhang, Luo, & Zeng, 2021; Zhang et al., 2020).
These substances were considered to be potential key factor for the
umami, and sweet mellow tastes in tea infusions, and further studies are
required to verify this inference.
3.3.2. Catechins and theaflavins
The important and well-known catechins in tea, EGCG, EGC, EC,
GCG, and CG, which taste strongly bitter and astringent (Zhang et al.,
2020). During SBT processing, the oxidation reaction of catechins oc­
curs, and its content decreased significantly. After the oxidation of these
compounds, the tea infusion tastes less bitter and more mellow (Zhang
et al., 2020). As reddish-orange pigments in black tea, theaflavins (TF,
TF-3-G, TF-3ʹ-G, and TFDG) contribute to the orange-yellow color of
infusion and briskness, astringent taste, which are essential tasting ele­
ments for black tea (Zhang et al., 2020). To a certain extent, the overall
acceptance of the taste of black tea has a certain positive correlation
with the bitter and astringent substances (Yu et al., 2021). When the
J. Xue et al. LWT 156 (2022) 113010

Fig. 3. The changes in main taste compounds of tea samples during shaken black tea processing. FL, fresh leaves; S1, first shaking; S2, second shaking; S3, third
shaking; F, fermentation; D, drying.

7
J. Xue et al.
content of these substances is low, the taste of black tea is thin.
3.4. Dynamic changes in metabolic pathways of key metabolites during
SBT processing
To more comprehensively evaluate the dominant pathways that
emerged during SBT processing, pathway enrichment analysis was
performed. The dynamic changes of the main differential metabolites
during SBT processing were mapped in metabolic pathways using the
Kyoto Encyclopedia of Genes and Genomes database (https://www.
kegg.jp/kegg/pathway.html). The results indicated that several path­
ways were enriched, including linoleic acid metabolism; vitamin B6
metabolism; phenylalanine metabolism; alanine, aspartate, and gluta­
mate metabolism; pyrimidine metabolism; riboflavin metabolism;
aminoacyl-tRNA biosynthesis; and biosynthesis of unsaturated fatty
acids (Fig. S2). These metabolic pathways are mainly related to the
biosynthesis of amino acids, flavonoids, and lipids. These metabolites
closely related to the formation of the taste and fragrance quality of tea
(Tan et al., 2016). In order to better understand the dynamic changes of
metabolites during the processing of SBT, the metabolism pathways of
main metabolites were plotted (Fig. 4).
In many studies of plant defense responses, it has been found that
amino acids, flavonoids, and linoleic acid biosynthesis metabolism are
enriched (Zhou et al., 2020). Amino acids metabolites are crucial reg­
ulators in plant immunity (Shan & He, 2018). Flavonoids are defensive
compounds, which promote the defense mechanism of plants (Samy­
nathan et al., 2021). Linoleic acid metabolism is important for the
generation of jasmonic acid, which plays an important role in the de­
fense response (Ahmad et al., 2016). In this study, amino acid, flavo­
noid, and linoleic acid biosynthesis metabolism are enriched, mainly
Fig. 4. Metabolism pathways of the main metabolites during shaken black tea processing. The bar plots show the metabolite contents in FL, S1, S2, S3, F and D from
left to right. (FL, fresh leaves; S1, first shaking; S2, second shaking; S3, third shaking; F, fermentation; D, drying; FMN, flavin mononucleotide; 2ʹ,3ʹ-Cyclic CMP,
cytidine 2ʹ,3ʹ-cyclic phosphate; CDP, cytidine 5ʹ-diphosphate).
8
LWT 156 (2022) 113010
because the shaken leaves should be regarded as a living organism under
stress (dehydration, mechanical damage), and the dynamic changes of
their endogenous metabolites were due to stress response. Pyruvic acid
formation from phosphoenolpyruvate (PEP) is involved in the biosyn­
thesis of branched-chain amino acids (Val, Leu, Ile). The contents of the
downstream metabolite products of Glu, such as Pro, gamma-GABA, and
Arg, were also increased, but Gln decreased, indicating that the trans­
formation of Glu to Gln was inhibited during shaking processing.
Phenylalanine ammonialyase, cinnamate 4-hydroxylase, flavanone
3-hydroxylase and flavonoid 3ʹ -monooxygenase are upstream enzymes
of flavonoid biosynthesis pathway, which are involved in plant response
to dehydration stress (Baba & Ashraf, 2019; Sanchez-Rodriguez, Mor­
eno, Ferreres, Rubio-Wilhelmi, Mdel, & Ruiz, 2011). Under the catalysis
of these enzymes, Phe was guided into the biosynthesis of flavonoids to
form catechins (Wu et al., 2020). Compared with fresh leaves, the
content of theaflavins in shaken leaves increased, but the content of
catechins did not change significantly, indicating that the biosynthesis
of catechins occurred in this process. In other words, the fresh leaves
produced flavonoids in response to stress during the shaking process.
In enzymatic-catalyzed process, catechins undergo oxidative poly­
merization to form theaflavins and procyanidins. In this study, the main
substances of catechins (EGC, EGCG and EC) were significantly
decreased during the fermentation process, indicating that epi-catechins
consumption largely occurs during the fermentation process. Similar
changes were found for ECG, C, and GC, but they were not marked,
indicating that these compounds were partially retained in tea. The re­
sults are consistent with those reported in the literature (Chen et al.,
2021). PPO is an oxidoreductase enzyme that uses oxygen to oxidize
catechins. EGC was the most sensitive to oxygen, followed by EGCG and
EC, while ECG was least sensitive to oxygen (Chen et al., 2021).
J. Xue et al.
Theaflavins (TF, TF-3-G, TF-3′ -G and TFDG), which are the dimeric
products of catechins, showed an opposite trend to catechins. Mean­
while, there was a slight increase in the levels of trans-catechins (GCG
and CG), indicating that catechins undergo different transformation
pathways during the fermentation process, including oxidation and
epimerization, with oxidation being the main pathway.
The contents of gamma-linolenic acid and linoleic acid were shown
to decrease during the fermentation process and to increase during
drying. During the fermentation process, the cell membrane releases a
large quantity of unsaturated fatty acids (gamma-linolenic acid and
linoleic acid). These substances produce cyclic aromatic substances,
such as methyl jasmonate, cis-jasmone, and jasmine lactones via
lipoxygenase-mediated fat oxidation pathways. These volatiles are
principal floral aromatic compounds and give important contributions
to the aroma of black tea (Ho et al., 2015; Zhang et al., 2021).
4. Conclusions
The non-volatile metabolites during SBT production were investi­
gated based on UHPLC-QTOF-MS in this study. Ninety-three important
differential compounds were screened from the tea samples during the
processing of SBT by PLS-DA analysis. During the whole process of SBT,
the levels of amino acids, dipeptides, theaflavins, and lipids remarkably
increased, while the levels of catechins and procyanidin C1 reduced, all
of which have implications for the quality of black tea. HCA indicated
that fermentation was the crucial process that caused dynamic changes
in metabolites, followed by drying and shaking. The dynamic changes
were mainly caused by enzymatic reactions in the processes of shaking
and fermentation, and by thermal reactions during the drying process.
Amino acids, flavonoids, and linoleic acid biosynthesis metabolism are
enriched, due to the response of fresh tea leaves to stress (dehydration,
mechanical damage) during shaking processing. The degradation of
protein occurs during the whole processes of SBT, especially in
fermentation, which is the main reason for the increase of PAAs and
dipeptides during this stage. In conclusion, the taste of black tea can be
ultimately attributed to the final metabolites in the tea product, which
result from the enzymatic and non-enzymatic chemical transformation
of the metabolites in tea leaves during the manufacturing process. This
study provides a reference for the development of black tea products
with special characteristics and provides a novel theoretical basis for the
development of SBT flavors, which provides a practical guide for quality
control during SBT processing.
CRediT authorship contribution statement
Jinjin Xue: Conceptualization, Methodology, Software, Formal
analysis, Investigation. Panpan Liu: Resources, Visualization. Guiyi
Guo: Resources, Investigation, Visualization. Weiwei Wang: Investi­
gation, Software. Jianyong Zhang: Investigation. Wei Wang: Software.
Ting Le: Investigation. Junfeng Yin: Writing – review & editing.
Dejiang Ni: Writing – review & editing. Heyuan Jiang: Conceptuali­
zation, Funding acquisition, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the Central Public-interest Scientific
Institution Basal Research Fund (Y2021GH06), the National Natural
Science Foundation of China (Projects No. 31670692) and the Open
Fund of Henan Key Laboratory of Tea Comprehensive utilization in
South Henan (Projects No. HNKLTOF2018004). The authors thank
Shanghai Biotree Biotech Co., Ltd. (Shanghia, China) for technical
support.
9
LWT 156 (2022) 113010
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2021.113010.
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Asp,: L-aspartic acid
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Thr: L-threonine; Ser, L-serine;
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Glu: L-glutamic acid
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Thea: L-theanine;
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Ala: L-alanine;
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Tyr: L-tyrosine;
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Gamma-GABA: gamma-aminobutyric acid
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His: L-histidine;
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Lys: L-lysine;
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Arg: L-arginine;
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Gln: L-glutamine;
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Asn: L-asparagine;
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Gly: L-glycine;
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Leu: L-leucine;
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Ile: L-isoleucine;
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Val: L-valine;
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Cys: L-cystine;
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FL,: fresh leaves
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S3: third shaking
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F: fermentation
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D: drying
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IDA: information-dependent acquisition
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ESI: electrospray ionization
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FAAs: free amino acids
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TAAs: total amino acids
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ANOVA: analysis of variance
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T-test: student’s t-test
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PLS-DA: partial least squares discriminant analysis
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HCA: hierarchical clustering analysis
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PLP: pyridoxal 5ʹ-phosphate
proteinaceous and non-proteinaceous amino acid formation in tea (Camellia sinensis)
GAD: glutamate decarboxylase
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PPO: polyphenol oxidase
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VIP: variable importance in projection
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FC: fold change
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MSG: monosodium glutamate
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5ʹ-CMP: cytidine 5ʹ-monophosphate
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PEP: phosphoenolpyruvate

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