foods

Article
Enzymatic Reaction-Related Protein Degradation and
Proteinaceous Amino Acid Metabolism during the
Black Tea (Camellia sinensis) Manufacturing Process
Yiyong Chen 1,2 , Lanting Zeng 2,3 , Yinyin Liao 2 , Jianlong Li 1 , Bo Zhou 1 , Ziyin Yang 2,3 and
Jinchi Tang 1, *
1 Tea Research Institute, Guangdong Academy of Agricultural Sciences & Guangdong Provincial Key
Laboratory of Tea Plant Resources Innovation and Utilization, Dafeng Road 6, Tianhe District,
Guangzhou 510640, China; chenyiyong@gdaas.cn (Y.C.); skylong.41@163.com (J.L.); zhoubo@gdaas.cn (B.Z.)
2 Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement &
Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy
of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China; zenglanting@scbg.ac.cn (L.Z.);
honey_yyliao@scbg.ac.cn (Y.L.); zyyang@scbg.ac.cn (Z.Y.)
3 Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, Xingke Road 723, Tianhe
District, Guangzhou 510650, China
* Correspondence: tangjinchi@126.com; Tel.: +86-20-8516-1049




Received: 9 October 2019; Accepted: 20 December 2019; Published: 8 January 2020


Abstract: Amino acids contribute to the nutritional value and quality of black tea. Fermentation is the
most important stage of the black tea manufacturing process. In this study, we investigated protein
degradation and proteinaceous amino acid metabolism associated with enzymatic reactions during
fermentation in the black tea manufacturing process. The results showed that the concentrations of
both protein and free amino acids decreased during fermentation. We also confirmed that proteins
were broken down into free amino acids by artificially synthesized dipeptide benzyloxycarbonyl
glutamyl-tyrosine (Z-Glu-Tyr). Metabolites of the amino acid metabolic pathway increased
significantly during fermentation. Furthermore, we confirmed that free amino acids were degraded
to volatile compounds in a tracer experiment with the isotope precursor. These results provide
information that will help black tea manufacturers improve the quality of black tea.

Keywords: amino acids; black tea; Camellia sinensis; fermentation; volatile compounds

1. Introduction
Amino acids (AAs) have many important physiological and nutritional functions in humans,
which are not only closely related to body growth and development, but also health and disease.
Traditionally, 20 proteinaceous amino acids involved in constructing protein skeletons are classified
as nutritionally essential or nonessential amino acids [1]. In plants, amino acids perform various
critical functions. In addition to their roles in protein synthesis, various physiological processes in
plants have been reported to be related to amino acids [2–7]. First, amino acids are important nutrients
and regulatory substances for plant growth and development, and act as intracellular pH regulators.
Second, metabolic energy or redox power generation in plants is related to amino acids metabolism.
Finally, amino acids are plant resistant factors against both abiotic and biotic stress. For example,
amino acids have been reported to have significant effects on pathogen infection. On one hand,
amino acid metabolites can serve as important defense compounds, on the other hand, they also act
as indispensable nitrogen sources for many biotrophic pathogens [8–10]. Furthermore, the role of
amino acids during signaling in plants has been discussed [4–11]. Essential amino acids significantly

Foods 2020, 9, 66; doi:10.3390/foods9010066 www.mdpi.com/journal/foods

Foods 2020, 9, 66 2 of 15

contribute to the nutritional quality of plant-originated foods. Fruit taste might be influenced by
some free amino acids. A well-known example is the contribution of glutamate (L-Glu), which has
delicious savory taste, to the umami flavor of fruits and vegetables [12]. Furthermore, alanine (Ala)
and lysine (Lys) contribute to the sweetness of food, while phenylalanine (L-Phe) and tyrosine (Tyr)
taste bitter [13].
A close relationship exists between amino acid metabolism and carbohydrate metabolism.
The substrates used for amino acid synthesis are derived from carbohydrate metabolites, and some
products of amino acid degradation can act as energy substances in the citric acid cycle. Furthermore,
amino acid metabolism is closely related to nitrogen absorption and utilization. Inorganic nitrogen
absorbed from plant roots can be synthesized into organic molecules through the glutamate synthesis
pathway, and all other nitrogen-containing organic compounds are synthesized from glutamate and
glutamine [14]. Additionally, amino acid metabolism products are important substances for protein
synthesis and secondary metabolism.
Tea manufactured from Camellia sinensis leaves as the raw material is the most consumed
plant-derived beverage globally. Leaves, flowers, fruits, roots, stems, and even some whole wild
plants can be processed into teas, known as tea substitutes in China [15]. Though tea-substitute plant
resources and related products are common in China, kudingcha and Lycium barbarum tea are the
most common tea substitutes made from leaves [16,17]. However, only Camellia sinensis tea has a
long history of consumption worldwide. The production of Camellia sinensis has important value
in agriculture and commerce, owing to its peculiar flavor and nutritional functions. Amino acids,
caffeine, and other substances in tea leaves endow tea with many physiological and pharmacological
properties [18]. The relationship between amino acid content and tea quality has been studied by
Yang et al. (2012) [19]. Free amino acids have been reported to make important contributions to the
quality and function of tea leaves. Some free amino acids are key precursors of tea aroma compounds,
for example, the aromatic amino acids can be converted into the aroma components of tea [19–21].
Theanine is the most abundant and most characteristic free amino acid in tea leaves. Theanine not only
affects the taste and aroma of tea, but also has many physiological functions in the body. The functions
of theanine in relaxation and against cancer have been reported [20,22].
Manufacturing practices play a decisive role in the formation of optimum tea flavor and quality.
Tea leaves used for manufacturing are usually freshly picked, and can comprise the bud or the first two
leaves of the tea shoot. After harvesting, the tea leaves are immediately transported to a manufacturing
factory for subsequent processing. According to the different degrees of fermentation in tea processing,
manufactured tea leaves are generally classified into three types; tea leaves without fermentation are
processed into green tea, those that are partially fermented are processed into oolong tea, and those that
are fully fermented are processed into black tea. Enzymatic oxidation is the key biochemical reaction
in all types of tea processing, and is positively correlated with the degree of fermentation. In black
tea processing, the enzymatic oxidation process during fermentation determines the quality of black
tea [23]. Free amino acids contribute significantly to the flavor features of black tea, and their contents
change significantly during fermentation. Previous research has shown that the concentrations of
some free amino acids—such as glutamic acid, glutamine, leucine, serine, isoleucine, phenylalanine,
threonine, and theanine—decrease appreciably during fermentation, while other amino acids show
little change [24]. In 1965, Wickremasinghe and Swain reported that free amino acid contents were
overall decreased during black tea fermentation [25]. These results suggested that free amino acids may
be converted into other metabolic components during fermentation. Several studies have suggested
that some important volatile aroma components in black tea are derived from the conversion of amino
acids [26,27]. However, little is known about the metabolism of free amino acids during black tea
fermentation. In the present study, we aimed to analyze the changes in free amino acid profiles
and contents at different enzyme reaction stages of the black tea manufacturing process. We also
investigated whether protein degradation contributed to the changes in free amino acids and verified
the conversion of free amino acids into the volatile aroma components of black tea during fermentation.
Foods 2020, 9, 66 3 of 15

2. Materials and Methods

2.1. Plant Materials and Black Tea Manufacturing

Tea leaf samples used in this study comprised one bud and two leaves from C. sinensis cv.
Yinghong No.9. Tea plants were planted in Yingde Tea Experimental Station of Tea Research Institute,
Guangdong Academy of Agricultural Sciences (23◦ N, 113◦ E, Yingde, China). Samples were collected
in July 2018, and the tea leaves were immediately transported to the manufacture factory, the method for
black tea manufacture was according to the general processing (Figure 1A). Fresh tea leaves (plucking)
were withered indoor at temperature about 25 ◦ C for 10 h [28]. The withered tea leaves (withering)
were then rolled 40 min using a roller (6CR-10, Fuyang Machinery Co. Ltd., Hangzhou, China) at
room temperature. After rolling (rolling), the tea leaves were placed in an environment control room at
a temperature of 25 ◦ C, and relative humidity of 90% for fermentation. After 4 h, the fermented tea
leaves (fermentation) were transferred into a tea-firing roller machine (JY-6CHZ-7B, Fujian Jiayou Tea
Machine Intelligent Technology Co. Ltd., Anxi, China), parched at 250 ◦ C for 30 min to fix sample
(firing). Finally, the tea leaves were dried using a hot air drier at 120 ◦ C (JY-6CHZ-7B, Fujian Jiayou Tea
Machine Intelligent Technology Co. Ltd., Anxi, China). Tea samples were collected at each stage and
immediately frozen with liquid nitrogen. Three independent biological replicates were processed for
each step 9,ofx black
Foods 2020, tea REVIEW
FOR PEER manufacturing. 6 of 14

Figure 1. Changes
Figure 1. Changes ofof protein
protein content
content and
and amino
amino acids
acids content
content during
during the
the enzymatic
enzymatic reaction
reaction of
of black
black
tea manufacturing process. P, Plucking; W, Withering; R, Rolling; F, Fermentation. (A) Five process
tea manufacturing process. P, Plucking; W, Withering; R, Rolling; F, Fermentation. (A) Five process
stage of black tea (Camellia sinensis) manufacturing; (B,C) the changes of protein content and total
stage of black tea (Camellia sinensis) manufacturing; (B,C) the changes of protein content and total
amino acids content during the enzymatic reaction of black tea manufacturing process. Bars indicate
amino acids content during the enzymatic reaction of black tea manufacturing process.
the means ± S.D. (n = 3) of three biological replicates, and bars with different letters are significantly
different at p ≤ 0.05 according to Duncan’s multiple range test.
In theory, due to protein degradation, the free amino acid content should increase during the
black tea manufacturing process. However, the total amino acid content decreased during the
manufacturing process (Figure 1C). Thirty free amino acids were detected in tea leaves during the
black tea manufacturing process. Notably, 15 free amino acids (including threonine, serine, valine,
cysteine, methionine, isoleucine, leucine, tyrosine, and phenylalanine) showed significantly
increased contents during withering (p < 0.05), but decreased contents in subsequent steps of the
Foods 2020, 9, 66 4 of 15

2.2. Determination of Total Amino Acids and Total Soluble Protein Content at Each Step of Black
Tea Manufacture
Determination of total free amino acids content in black tea leaves was used the method of
ninhydrin colorimetry [29]. Tea leaf samples (0.5 g) were finely crushed with a mortar and pestle,
and extracted with 10 mL distilled water in boiling water bath for 1 h. After cooling to room temperature,
the mixture was centrifuged using an Allegra 64R centrifuge (Beckman Coulter Inc., Fullerton, CA,
USA), at 10,000× g for 10 min. Ninhydrin reagent (2%, w/v) and phosphate buffer (pH 8.0), respectively
0.5 mL, were mixed with 1 mL extracted supernatant, and reacted in a boiling-water bath for 15 min.
the reaction mixture was transferred to a 10 mL volumetric flask and made up to volume with distilled
water, before standing at room temperature for 10 min. The absorbance of each sample was measured
using an ultraviolet spectrophotometer (MetashUV-5200, Shanghai Metash Instruments Co., Ltd.,
Shanghai, China) at 570 nm. Glutamate was used as the standard substance to construct the standard
curve. The protein content was measured according to the Bradford assay method, and the bovine
serum albumin (BSA) calibration line was established to quantify the protein content [30]. The moisture
contents of samples at each step of the black tea manufacturing were determined using standard
method GB 5009.3-2010 [31]. Total amino acids and protein content were expressed as dry weight (DW).

2.3. Free Amino Acid Content Analysis for Each Step of Black Tea Manufacture
To analyze the content of each free amino acid in tea samples, the method previously described
by Mei et al. (2016) was used [32]. Tea leaves from each process step during black tea manufacture
were finely powdered with a mortar and pestle. One hundred milligrams tea leaves powder was
placed in a 2 mL centrifuge tube and 0.5 mL of pre-cooled methanol used as the extraction solution
added. the mixture was vortex 2 min using a vortex mixer (Scientific Industries, Inc., Suffolk, MA,
USA), and subjected to ultrasonic extracted in an ice bath for 15 min. Chloroform (0.5 mL) and distilled
water (0.2 mL) were added to the tube and mixed. Centrifugation followed at 5000× g, 4 ◦ C for 10 min,
with the extraction solution in the tube undergoing phase separation. The upper layer was obtained,
and dried by a vacuum centrifugal enrichment at 45 ◦ C. Next, 5% sulfosalicylic acid solution was added
to the tube to redissolve the dried crude extraction, followed by filteration through a 0.45 µm nylon filter
membrane. An automatic amino acid analyzer (Sykam S-430D, Eresing, Germany) was used to analyze
the concentrations of each free amino acid in the samples. The mobile phase was a physiological
Li C4 system containing lithium citrate buffer at pH 2.9, 4.2, and 8.0. According to the operating
instructions of the instrument, the flow velocity of the mobile phase was set as 0.45 mL/min, and the
flow velocity of the derived reagent ninhydrin was set as 0.25 mL/min. The injection volume was 50
µL. The temperatures of the sodium cation-exchange column and post-column reaction equipment
were respectively set at 38 ◦ C and 130 ◦ C. UV-Vis detection was used to measure the peaks of each free
amino acids at wavelengths of 570 nm and 440 nm. The auto-sampler temperature was set at 5 ◦ C.

2.4. Simulation of Enzyme Reaction Step during Black Tea Manufacturing Processes and Analysis of Free
Amino Acid Content
Fleshly plucked tea leaves were completely crushed with liquid nitrogen to simulate the rolling
process of black tea manufacture. Non-crushed tea leaves were used as control. Tea samples were
placed in an artificial incubator for 6 h of fermentation, and the temperature was set at 25 ◦ C with
a relative humidity of 95%. Tea samples were collected at 0 h and 6 h and immediately frozen
with liquid nitrogen. The total amino acid and protein contents were measured using the methods
mentioned above.

2.5. Investigation of Protein Degradation during Black Tea Fermentation with Artificially Synthesized
Dipeptide Benzyloxycarbonyl Glutamyl-Tyrosine (Z-Glu-Tyr)
Tea leaves were completely crushed with liquid nitrogen, finely powdered tea leaves (100 mg) and
25 µL 50 g/L dipeptide Z-Glu-Tyr were mixed together in a centrifugal tube, then before fermenting in
Foods 2020, 9, 66 5 of 15

an incubator for 6 h at 25 ◦ C and a relative humidity of 95%. The control comprised crushed tea leaf
power and 25 µL 50 g/L dipeptide Z-Glu-Tyr fermented separately for 6 h and then mixed. Each free
amino acid was extracted and detected using the methods described above.

2.6. Analysis of Black Tea Aroma Compounds during the Manufacturing Processes
Aroma compounds of black tea during the manufacturing processes were analyzed, and the
method referred to the research reported by Zeng et al. (2019) [33]. Tea leaf sample powder (0.5 g) and
extracting buffer (dichloromethane (2 mL) contain 0.5 nmol ethyl n-decanoate as an internal standard)
were added into a 5 mL glass vial, and the samples were extracted overnight on an orbital shaker.
The extraction solution was then passed through a sodium sulphate anhydrous column to remove
residual water and collected in a 2 mL glass tube. The tube was transported to a termovap sample
concentrator (NDK200-1, MIU Instruments Co., Ltd., Shanghai, China). All samples were concentrated
to 200 µL, transferred to GC-MS sample bottles, and the aroma compounds in these samples were
analyzed used the instrument GC-MS QP2010 SE (Shimadzu Corporation, Kyoto, Japan) according to
manufacturer instructions. A SUPELCOWAX 10 column (30 m × 0.25 mm × 0.25 µm, Supelco Inc.,
Bellefonte, PA, USA) was equipped to separate aroma compounds. The GC injection port temperature
was set at 230 ◦ C and held for 1 min. Splitless control mode was selected. High purity helium gas was
used as the carrier, and the flow velocity was set at 16.667 µL/s. As for the temperature program settings,
the initial temperature of GC oven was set at 60 ◦ C, helding for 3 min, and then ramped up to a final
temperature of 240 ◦ C at a rate of 0.067 ◦ C/s, helding for 30 min. For MS program, full scan acquisition
mode was selected for mass spectra collection, starting at m/z 40, and ending at m/z 200. Identification
and quantitative analyses of benzaldehyde, benzeneacetaldehyde, benzyl alcohol, and phenylethyl
alcohol were conducted by direct comparison with authentic standards and calibration curves.

2.7. Investigation of the Amino Acid Catabolism during Black Tea Fermentation with Supplementation of [2 H8 ]
L-phenylalanine
Fresh tea leaves (0.2 g) were crushed with liquid nitrogen in a mortar. Then 100 µL of 10 g/L
labeled mixed [2 H8 ] L-phenylalanine solution was added, followed by fermentation in an incubator at
25 ◦ C for 6 h. The control was tea leaf powder without fermentation. After fermentation, the samples
were collected and extracted with dichloromethane. The volatiles among labeled phenylpyruvic acid
products were analyzed by GC-MS. The method for volatiles analysis products of tea leaf samples is
described above. Three independent experiments were performed.

2.8. Statistical Analysis

All experiments in the study were repeated three times, and Excel 2010 and SPSS Statistics 23.0
software were used for calculation and statistical analysis. The experimental results were expressed
in the form of mean ± standard deviation (S.D.). Student’s t-test was used to calculate significant
differences between two treatments (* p ≤ 0.05, ** p ≤ 0.01). For three or more treatments, the differences
were calculated by one-way ANOVA followed by Duncan’s multiple comparison tests. Significant
differences between groups were defined at the probability level of 5% (p ≤ 0.05).

3. Results and Discussion

3.1. Changes in Proteins and Free Amino Acids during the Black Tea Manufacturing Process
Generally, teas are divided into different categories according to different processing technologies,
and the difference in fermentation degree is the most important characteristic of all types of teas.
The three major categories of tea, in order of increasing degrees of fermentation, are green tea,
oolong tea, and black tea [20]. In an orthodox manufacturing process, fresh tea leaves are picked
from the shoots of tea plants, after four independent processing steps—namely, withering, rolling,
fermentation, and drying—black tea is produced (Figure 1A). The withering process during black tea
Foods 2020, 9, 66 6 of 15

manufacturing results in partial moisture removal (Table S1). In this stage, the degree and duration
of withering must be well controlled to ensure that the tea leaves are in a good physiological state,
which is suitable for the subsequent manufacturing stages. Many biochemical reactions that seem
to affect product quality, such as the breakdown of proteins to amino acids, have been reported to
occur during the withering stage [24]. The cellular compartments of tea leaves are disrupted during
the subsequent processing stage of rolling, with the cytoplasmic polyphenol oxidase coming into
contact with catechin substrates as monomers in the vacuole. This is followed by the fermentation
step, during which the most important reaction is the polyphenol oxidase and peroxidase catalyzed
oxidation of tea polyphenols. In the biochemical reaction of black tea fermentation, a large amount of
heat is released, which might cause unwanted secondary metabolism reactions and, therefore, affect
the quality of tea components. Therefore, the temperature during the fermentation process must be
controlled. These biochemical reactions during fermentation are terminated during subsequent drying,
with some flavor characteristics formed at this stage.
Tea protein has recently been reported to have biological functions, such as antioxidant,
antimutation, and radiation protection functions [34]. Previous studies have mainly focused on
the metabolic transformation process of tea polyphenols and catechins, with tea proteins having seldom
been investigated until recently. In the present study, the soluble protein content of tea leaves decreased
during the black tea manufacturing process (Figure 1B). The main reason for this decrease in protein
content was tea leaf protein degradation. Black tea manufacture processes from fresh tea leaf plucking
to rolling also involved partial fermentation. During these processes, tea leaf withering and crushing
induced oxidation reaction. The cellular compartments of tea leaves were disrupted, and proteases in
plastids were activated to more easily capture proteins.
In theory, due to protein degradation, the free amino acid content should increase during the
black tea manufacturing process. However, the total amino acid content decreased during the
manufacturing process (Figure 1C). Thirty free amino acids were detected in tea leaves during the black
tea manufacturing process. Notably, 15 free amino acids (including threonine, serine, valine, cysteine,
methionine, isoleucine, leucine, tyrosine, and phenylalanine) showed significantly increased contents
during withering (p < 0.05), but decreased contents in subsequent steps of the manufacturing process
(Table 1). The free amino acids formed were likely important for determining tea quality [27]. Total
free amino acid content usually accounts for 1–4% of the dry weight of tea leaves, and the types of free
amino acids and their proportion in tea are closely related to tea aroma and taste [35]. Free amino acids
not only contribute to a different oral taste of tea, such as bitterness, sweetness, and umami-like of tea,
but can also be converted into other quality components [36]. The plucked shoot tips of the tea plant can
be considered senescent plant tissues. The enzymatic breakdown of proteins is a well-known feature of
senescence [37], and the increase in free amino acids in tea leaves during withering demonstrates this
general phenomenon. The overall decrease in free amino acid content that occurs during fermentation
suggests that they are converted into other substances. In previous research, results have suggested
that free amino acids are partially converted into volatile compounds in tea. Some of these volatile
compounds are considered to be important constituents of tea aroma. During the drying stage, amino
acids can lead to the formation of aromatic substances under heating conditions, such as indoles,
alcohols, and aldehydes after a series of complex reactions [38].
Foods 2020, 9, 66 7 of 15

Table 1. Contents of free amino acids in black tea leaves during the manufacturing process.

Amino Acid µg/g (Dry Weight) Plucking Withering Rolling Fermentation Drying
a a a a
P-Ser 146.89 ± 9.21 136.29 ± 14.76 154.64 ± 2.69 156.27 ± 11.46 96.39 ± 34.04 b
PEA 79.13 ± 16.46 b 127.90 ± 7.40 a 94.57 ± 6.85 b 50.69 ± 5.75 c 39.06 ± 15.45 c
Asp 13.97 ± 8.92 b 20.61 ± 4.59 b 21.85 ± 4.12 ab 30.57 ± 5.52 b 40.04 ± 14.91 a
Thr 140.04 ± 25.22 c 277.19 ± 6.76 a 264.51 ± 39.46 ab 229.25 ± 15.56 b 130.85 ± 3.96 c
Ser 564.34 ± 53.16 c 880.84 ± 74.88 a 736.48 ± 56.08 b 663.63 ± 46.75 b 383.09 ± 21.37 c
Asn 0.00 ± b 382.22 ± 662.02 ab 678.97 ± 145.24 a 556.45 ± 78.34 ab 391.01 ± 84.43 ab
Glu 2331.12 ± 310.66 a 1609.88 ± 101.01 b 1423.61 ± 22.43 bc 1224.51 ± 96.73 c 773.41 ± 12.54 d
Thea 6874.15 ± 1226.16 a 5284.69 ± 572.10 b 5100.84 ± 110.42 b 4618.89 ± 420.34 b 2559.11 ± 18.16 c
α-AAA 21.32 ± 5.85 d 126.19 ± 20.76 a 114.37 ± 11.77 ab 97.48 ± 10.95 b 52.69 ± 9.79 c
Gly 67.38 ± 11.69 a 39.63 ± 2.61 b 35.06 ± 4.82 bc 17.42 ± 14.47 cd 8.21 ± 7.17 d
Ala 248.51 ± 39.53 c 402.25 ± 12.70 b 480.03 ± 15.91 a 470.10 ± 23.93 a 289.01 ± 29.66 c
α-ABA 15.24 ± 2.67 a 14.70 ± 2.44 a 12.89 ± 1.51 ab 9.07 ± 3.52 b 7.77 ± 3.27 b
Cit 2.88 ± 0.28 b 17.34 ± 7.74 a 13.81 ± 3.77 ab 16.62 ± 7.14 a 10.53 ± 3.15 ab
Val 102.20 ± 21.55 d 581.33 ± 68.70 a 479.27 ± 33.04 b 465.69 ± 22.36 b 282.32 ± 25.48 c
Cys 16.11 ± 10.17 c 107.07 ± 19.84 a 64.65 ± 8.54 b 56.20 ± 16.62 b 21.31 ± 2.85 c
Met 1.92 ± 2.61 b 46.54 ± 39.84 a 10.31 ± 2.71 b 6.55 ± 0.21 b 1.82 ± 0.32 b
Ile 14.38 ± 2.06 d 385.67 ± 41.44 a 327.93 ± 36.95 b 307.94 ± 4.83 b 202.77 ± 14.63 c
Leu 11.58 ± 7.69 d 391.21 ± 24.24 a 338.60 ± 27.36 b 307.84 ± 13.90 b 198.18 ± 15.12 c
Tyr 0.00 ± c 555.68 ± 95.14 a 542.77 ± 21.18 a 498.81 ± 35.46 a 334.49 ± 22.82 b
Phe 45.78 ± 6.33 e 1806.72 ± 162.61 a 1396.06 ± 63.67 b 1220.48 ± 60.33 c 734.05 ± 60.09 d
β-Ala 6.61 ± 0.94 b 39.11 ± 27.67 ab 76.65 ± 38.10 a 56.08 ± 10.26 a 31.48 ± 3.90 ab
β-ABA 6.48 ± 0.84 b 4.37 ± 3.80 b 5.86 ± 5.27 b 23.65 ± 12.82 a 8.90 ± 4.76 b
GABA 2.01 ± 0.53 d 59.97 ± 23.77 bc 91.26 ± 6.45 a 78.41 ± 12.86 ab 42.44 ± 4.54 c
His 9.40 ± 1.27 c 80.51 ± 8.41 a 34.53 ± 4.80 b 15.17 ± 1.19 c 19.49 ± 15.88 bc
3Mehis - - - 10.48 ± 11.16 a 1.43 ± 1.37 ab
1Mehis 2.63 ± 0.29 a 1.97 ± 0.51 a 0.90 ± 0.08 a 3.83 ± 3.88 a 1.17 ± 1.35 a
Trp 37.24 ± 20.68 c 296.47 ± 44.63 a 287.80 ± 102.25 a 174.23 ± 65.19 b 72.30 ± 9.56 c
ORN 91.31 ± 99.49 c 360.88 ± 66.09 a 318.92 ± 42.89 ab 173.48 ± 151.58 bc 13.30 ± 6.04 c
Lys 38.72 ± 17.26 d 368.64 ± 59.13 a 267.90 ± 23.81 b 154.20 ± 53.25 c 56.45 ± 7.42 d
Arg 17.76 ± 5.03 a 42.87 ± 23.45 a 26.61 ± 26.08 a 18.45 ± 8.78 a 15.89 ± 8014 a
Data are expressed as mean ± S.D. (n = 3). P-Ser, Phosphoserine; PEA, Phosphorylethanolamine; Asp, Aspartic acid; Thr, Threonine; Ser, Serine; Asn, Asparagine; Glu, Glutamate; Thea,
Theanine; α-AAA, α-amino acetic acid; Gly, Glycine; Ala, Alanine; α-ABA, α-Aminobutyric acid; Cit, Citrulline; Val, Valine; Cys, Cystine; Met, Methionine; Ile, Isoleucine; Leu, Leucine;
Tyr, Tyrosine; Phe, Phenylalanine; β-Ala, β-Alanine; β-ABA, β-Aminobutyric acid; GABA, γ-Aminobutyric acid; His, Histidine; 3Mehis, 1-Methyl histidine; 1Mehis, 1-Methyl histidine;
Trp, Tryptophan; ORN, Ornithine; Lys, lysine; Arg, Arginine. Different means with different letters in the same row are significantly different from each other (p ≤ 0.05).
Foods 2020, 9, 66 8 of 15

3.2. Protein Degradation during Black Tea Fermentation with Artificially Synthesized Dipeptide
Benzyloxycarbonyl Glutamyl-Tyrosin (Z-Glu-Tyr)
Proteins are reportedly broken down into amino acids by enzyme peptidase during the black tea
manufacturing process [39,40]. The enzyme reaction is a long process beginning with tea leaf plucking
and ending with firing. Particularly during the withering and fermentation processes, black tea
leaves make use of sufficient enzyme action [41]. In 1964, Sanderson and Roberts reported that the
progressive increase in free amino acids in tea leaves during withering was due to the hydrolytic
activity of endogenous peptidase [42]. During the fermentation process, most research has focused on
endogenous oxidative enzymes, such as the conversion of selected flavanol combinations to theaflavins
and thearubigins [43]. However, less attention has been paid to proteolytic enzymes. Furthermore,
previous studies have only detected changes in protein and amino acid contents during the black
tea manufacturing process, while direct evidence is lacking. In this study, we selected artificial
synthetic dipeptide Z-Glu-Tyr as a representative substrate to directly identify protein degradation
into amino acids during black tea fermentation (Figure 2A). Z-Glu-Tyr is a complex with two aromatic
moieties, namely, the phenyl group in Z and phenol group in tyrosine, of which the former is more
hydrophobic [44]. Therefore, tyrosine is more readily broken down by Z-Glu-Tyr. In this study,
Z-Glu-Tyr was added to the crushed tea leaves, and the black tea fermentation step was simulated
for 6 h. A larger amount of tyrosine was detected in the tea leaves after fermentation, while tyrosine
was not detected in the control. These results suggested that protein degradation occurred during
black tea fermentation, and that tyrosine was obtained from the breakdown of Z-Glu-Tyr. Interestingly,
the glutamate content showed no significant change in this study (Figure 2B), which might be due to
the phenyl group of Z not being able to breakdown glutamate, and the glutamate in group Z cannot be
detected. In the simulation of black tea fermentation, compared with the plucked leaves, the protein
content was significantly decreased in crushed tea leaves after fermentation for 6 h, without obvious
changes in leaf integrity (Figure 3). A crushing process has been designed to simulate the rolling of
black tea, with complete disruption of tea leaf cells in this process leading to interactions between
substrates and enzymes [45]. The results suggested that tea leaf protein was degraded to free amino
acids during the withering, rolling, and fermentation processes. However, the amino acid content did
not increase, but decreased (Figure 3). Most free amino acids also showed decreased contents after
fermentation for 6 h compared with fresh tea leaves (Table S2). This evidence suggested that the free
amino acids were converted into other metabolites during black tea fermentation.

3.3. Proteinaceous Amino Acid Metabolism during Black Tea Fermentation

Free amino acids undergo appreciable changes during the black tea manufacturing process
(Table 1). The free amino acid contents decreased overall during fermentation, suggesting that
amino acids were converted into other substances. Some amino acids have been reported to increase
tea aroma [46]. There are two possible pathways for converting free amino acids into tea aroma
compounds or volatile compounds. The first involves combination with orthoquinone, an oxidized
form of catechin, which plays an important role in determining the black tea aroma [47,48]. The second
pathway, undergone by some amino acids, including phenylalanine, leucine, isoleucine, and valine,
involves partial conversion to the aldehydes expected from a Strecker degradation [47]. In this
study, we selected phenylalanine as the representative amino acid to investigate free amino acid
degradation during black tea fermentation, because the phenylalanine degradation metabolites
(including benzaldehyde, benzyl alcohol, and methyl benzoate) contribute to the main aroma properties
of black tea flavor [49]. The phenylalanine content decreased significantly during the fermentation
stage of black tea manufacture (Table 1). To investigate changes in the volatile compounds during the
black tea manufacturing process, samples from five manufacturing stages were analyzed by GC-MS.
Four volatile compounds (benzaldehyde, phenylacetaldehyde, benzyl alcohol, and phenylethyl alcohol)
involved in the phenylalanine degradation pathway were detected and identified by direct comparison
with authentic standards. The concentrations of each compound were calculated based on calibration
 Foods 2020, 9, x FOR PEER REVIEW 8 of 14

during black tea fermentation, and that tyrosine was obtained from the breakdown of Z-Glu-Tyr.
Interestingly, the glutamate content showed no significant change in this study (Figure 2B), which
might
Foods be9,due
2020, 66 to the phenyl group of Z not being able to breakdown glutamate, and the glutamate in
9 of 15
group Z cannot be detected. In the simulation of black tea fermentation, compared with the plucked
leaves, the protein content was significantly decreased in crushed tea leaves after fermentation for 6
curves. The results showed that all four volatile compounds had significantly increased contents at
h, without obvious changes in leaf integrity (Figure 3). A crushing process has been designed to
the fermentation stage compared with the plucking stage (Figure 4A, Figure S1). Previous reports
simulate the rolling of black tea, with complete disruption of tea leaf cells in this process leading to
showed that these four compounds were derived from L-phenylalanine via trans-cinnamic acid
interactions between substrates and enzymes [45]. The results suggested that tea leaf protein was
or directly from L-phenylalanine in tea leaves [49,50]. Kraujalytė et al. (2016) reported that
degraded to free amino acids during the withering, rolling, and fermentation processes. However,
aldehydes were the most abundant group comprising 55% of total identified volatiles in black
the amino acid content did not increase, but decreased (Figure 3). Most free amino acids also showed
tea, phenylacetaldehyde,
decreased and benzaldehyde
contents after fermentation respectively
for 6 h compared contribute
with honey-like
fresh tea leaves (Tableodor and evidence
S2). This almond
odor in black tea [51]. Benzyl alcohol was also the prominent scent compounds
suggested that the free amino acids were converted into other metabolites during black in black, and largely
tea
contributes
fermentation. to the floral aroma [49].

Figure2.2. Identification
Figure Identification of
of protein
protein degradation
degradation during
during black
black tea
tea fermentation
fermentation with with ananartificially
artificially
synthesized
synthesizeddipeptide
dipeptidebenzyloxycarbonyl
benzyloxycarbonylglutamyl-tyrosin
glutamyl-tyrosin(Z-Glu-Tyr).
(Z-Glu-Tyr). (A) (A)Schematic
Schematicdiagram
diagramofof
experimental
experimentaldesign.
design. Control: Z-Glu-Tyr and
Control: Z-Glu-Tyr andtea
tealeaves
leavespowder
powderwerewerefermentation
fermentationrespectively
respectively atat
25
25 ◦ C for 6 h, and then mixed to detect the contents of free Glu and Tyr. Treatment: Z-Glu-Tyr and tea
°C for 6 h, and then mixed to detect the contents of free Glu and Tyr. Treatment: Z-Glu-Tyr and tea
leaves ◦ C for 6 h step 1, Z-Glu-Tyr
leavespowder
powderwere
weremixed
mixedfirst,
first,and
andthen
thencompanied
companiedfermentation
fermentationatat25 25 °C for 6 h step 1, Z-Glu-Tyr
and ◦
andteatealeaves
leavespowder
powderwere
werefermentation
fermentationatat25 C for
25 °C for66hhrespectively
respectively(control)
(control)or ormixed
mixed(treatment);
(treatment);
step
step 2, Z-Glu-Tyr mixed with tea leaves powder. (B) Changes of Glu and Tyr content afterenzymatic
2, Z-Glu-Tyr mixed with tea leaves powder. (B) Changes of Glu and Tyr content after enzymatic
reaction
reactionduring
duringthe
thefermentation.
fermentation.Data
Datashown
shownasasthe mean±±SD
themean (n =
SD (n ** pp <<0.01
3). **
= 3). 0.01vs.
vs.Control.
Control.
 Foods 2020, 9, 66 10 of 15
Foods 2020, 9, x FOR PEER REVIEW 9 of 14

Figure 3. Changes of protein content (A) and amino acids content (B) in enzymatic reaction of black tea.
Figure 3. Changes
Bars indicate of protein
the means content
± S.D. (n = 3)(A) and amino
of three acidsreplicates,
biological content (B) inbars
and enzymatic reactionletters
with different of black
are
tea.
significantly different at p ≤ 0.05 according to Duncan’s multiple range test.

3.3. Proteinaceous Aminothe

To further confirm Acid Metabolismofduring
degradation Black TeatoFermentation
phenylalanine volatile compounds during the fermentation
stage, [2 H8amino
Free ] L-phenylalanine
acids undergo was appreciable
added to the changes
crushed tea leavesthe
during and the fermentation
black step of black
tea manufacturing tea
process
manufacturing was simulated. After fermentation for 6 h, [ 2 H ] phenylacetaldehyde ([2 H ] PAld) and
7
(Table 1). The free amino acid contents decreased overall during fermentation, suggesting that amino 7
[2 H7 ] were
acids 2-phenylethanol ([2 H7other
converted into ] 2PE)substances.
were detected
Some(Figure
amino 4C). PAld
acids andbeen
have 2PE are considered
reported important
to increase tea
constituents for black tea quality owing to their rose-like aroma. A previous
aroma [46]. There are two possible pathways for converting free amino acids into tea aroma study showed that 2PE
could be derived
compounds from L-Phe via the The
PAldfirst
route and PLA route in Rose [52]. After fermenting for 6 h,
Foods 2020,
2 H 9, x FORor volatile
PEER compounds.
REVIEW involves combination with orthoquinone, an oxidized
2H ] 10 of 14
[form7 of catechin, which plays an important role in determining the black tea aroma [47,48].[ The
] PAld was detected in large quantities, but was not detected in the control. Furthermore, 7
2PE was
second detectedundergone
pathway, both in theby treatment and control
some amino groups, with
acids, including no significant
phenylalanine, differences
leucine, in content
isoleucine, and
via the(Figure
PLA route4C). (Figure
This 4B), that
suggested which 2PE can occur
might be rapidly.
derived from These
L-Pheresults
via theconfirmed
PLA route that free
(Figure amino acids
4B),
valine, involves partial conversion to the aldehydes expected from a Strecker degradation [47]. In this
can bestudy,
degraded
which can to volatile
occur
we selected rapidly. compounds
These
phenylalanine results during
confirmed
as the black teaamino
that free
representative amino fermentation.
acids
acid can be degraded
to investigate to volatile
free amino acid
compounds during black tea fermentation.
degradation during black tea fermentation, because the phenylalanine degradation metabolites
(including benzaldehyde, benzyl alcohol, and methyl benzoate) contribute to the main aroma
properties of black tea flavor [49]. The phenylalanine content decreased significantly during the
fermentation stage of black tea manufacture (Table 1). To investigate changes in the volatile
compounds during the black tea manufacturing process, samples from five manufacturing stages
were analyzed by GC-MS. Four volatile compounds (benzaldehyde, phenylacetaldehyde, benzyl
alcohol, and phenylethyl alcohol) involved in the phenylalanine degradation pathway were detected
and identified by direct comparison with authentic standards. The concentrations of each compound
were calculated based on calibration curves. The results showed that all four volatile compounds had
significantly increased contents at the fermentation stage compared with the plucking stage (Figure
4A, Figure S1). Previous reports showed that these four compounds were derived from L-
phenylalanine via trans-cinnamic acid or directly from L-phenylalanine in tea leaves [49,50].
Kraujalytė et al. (2016) reported that aldehydes were the most abundant group comprising 55% of
total identified volatiles in black tea, phenylacetaldehyde, and benzaldehyde respectively contribute
honey-like odor and almond odor in black tea [51]. Benzyl alcohol was also the prominent scent
compounds in black, and largely contributes to the floral aroma [49].
To further confirm the degradation of phenylalanine to volatile compounds during the
fermentation stage, [2H8] L-phenylalanine was added to the crushed tea leaves and the fermentation
step of black tea manufacturing was simulated. After fermentation for 6 h, [2H7] phenylacetaldehyde
([2H7] PAld) and [2H7] 2-phenylethanol ([2H7] 2PE) were detected (Figure 4C). PAld and 2PE are
considered important constituents for black tea quality owing to their rose-like aroma. A previous
study showed that 2PE could be derived from L-Phe via the PAld route and PLA route in Rose [52].
After fermenting for 6 h, [2H7] PAld was detected in large quantities, but was not detected in the
control. Furthermore, [2H7] 2PE was detected Figure 4. Cont.
both in the treatment and control groups, with no
significant differences in content (Figure 4C). This suggested that 2PE might be derived from L-Phe
 Foods 2020, 9, 66 11 of 15

Figure 4. Aroma compound concentrations in phenylalanine degradation pathway Phe, phenylalanine;

Figure 4. Aroma compound concentrations in phenylalanine degradation pathway Phe,
PAld, phenylacetaldehyde; 2PE, 2-phenylethanol; PAR, phenylacetaldehyde reductase. (A) Aroma
phenylalanine; PAld, phenylacetaldehyde; 2PE, 2-phenylethanol; PAR, phenylacetaldehyde
compound concentrations in phenylalanine degradation pathway during the enzymatic reaction of
reductase. (A)(B)
black tea; Aroma compound
The known pathwayconcentrations
of L-phenylalaninein phenylalanine
degradation; (C) degradation pathway
Identification the during the
transformation
enzymatic
of L-phenylalanine to aroma compound with [ H8 ] L-phenylalanine. Data shown as the mean ± SD (C)
reaction of black tea; (B) The known
2 pathway of L-phenylalanine degradation;
Identification
(n = 3). ** pthe transformation
< 0.01 of L-phenylalanine
vs. CK. Bars indicate to aroma
the means ± S.D. (n = 3)compound with [replicates,
of three biological 2H8] L-phenylalanine.
and bars
Datawith
shown as theletters
different meanare± SD (n = 3). **different
significantly p < 0.01atvs.
p ≤CK.
0.05 according to Duncan’s multiple range test.

4. Conclusions
4. Conclusions
Our results showed that the decrease in protein concentration during the fermentation stage of
Our results
black showed was
tea manufacture that due
the to
decrease in protein concentration
protein degradation during
(Figure 5). We also the fermentation
confirmed stage of
that the proteins
blackwere
tea manufacture was due to protein degradation (Figure 5). We also confirmed that
broken down to free amino acids using artificially synthesized dipeptide Z-Glu-Tyr. However, the proteins
wereresults
broken down
from to free amino
the automatic amino acids
acidusing artificially
analyzer synthesized
showed that, during thedipeptide Z-Glu-Tyr. However,
black tea fermentation stage,
results
thefrom the automatic
free amino amino aciddid
acid concentrations analyzer showed
not increase, but that, during
decreased the black
overall. tea fermentation
Metabolites of the aminostage,
the free
acidamino acidpathway
metabolic concentrations did not
were detected increase,
to have but decreased
significantly increasedoverall. Metabolites
contents of the amino
during fermentation.
We also verified that free amino acids were degraded into volatile compounds using a tracer experiment
with an isotope precursor. Our results confirmed protein degradation and proteinaceous amino acid
metabolism during the fermentation stage of black tea manufacture and provided information that
will help black tea manufacturers improve black tea quality.
acid metabolic pathway were detected to have significantly increased contents during fermentation.
We also verified that free amino acids were degraded into volatile compounds using a tracer
experiment with an isotope precursor. Our results confirmed protein degradation and proteinaceous
amino acid metabolism during the fermentation stage of black tea manufacture and provided
Foods 2020, 9, 66 12 of 15
information that will help black tea manufacturers improve black tea quality.

Figure 5. Summary
Summaryon
onenzymatic
enzymaticreaction
reaction related
related protein
protein degradation
degradation and
and proteinaceous
proteinaceous amino acids
metabolism during the black tea (Camellia sinensis) manufacturing process.
process.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Table S1. The changes
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/1/66/s1.
of water content in tea leaves during black tea manufacturing process. Table S2: Contents of free amino acids in
Table S1. The changes of water content in tea leaves during black tea manufacturing process. Table S2: Contents
tea leaves
of free of simulated
amino theleaves
acids in tea black tea fermentation.
of simulated Figuretea
the black S1.fermentation.
GC-MS chromatograms of aromachromatograms
Figure S1. GC-MS compound in tea
of
leaves
aroma during
compoundblackintea
teamanufacturing process.
leaves during black tea manufacturing process.
Z.Y. and J.T. conceived and designed
Author Contributions: Z.Y. designed the
the experiments;
experiments; Y.C.,
Y.C., L.Z.,
L.Z., Y.L.,
Y.L., J.L.,
J.L., and
and B.Z.
B.Z.
conducted the
conducted the experiments;
experiments; Z.Y.,
Z.Y., Y.C.,
Y.C., and
and L.Z.
L.Z. analyzed
analyzed the
the results
results and
and wrote
wrote the
the manuscript. All authors
manuscript. All authors have
have
read and agreed to the published version of the manuscript.
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Funding: This research received no external funding.
Acknowledgments: This study was supported by the financial support from the National Key Research
Acknowledgments: This study
and Development Program was supported
of China by the financial
(2018YFD1000601), support from
the Guangdong the National
Special Support Key
PlanResearch and
for Training
High-Level Talents
Development (2016TQ03N617),
Program the Guangdong Natural
of China (2018YFD1000601), Science Foundation
the Guangdong for Distinguished
Special Support Young Scholar
Plan for Training High-
(2016A030306039),
Level and the Guangdong
Talents (2016TQ03N617), Provincial
the Guangdong Special
Natural Fund for
Science Modern Agriculture
Foundation Industry
for Distinguished Technology
Young Scholar
Innovation Teams (2019LM1143).
(2016A030306039), and the Guangdong Provincial Special Fund for Modern Agriculture Industry Technology
Conflicts ofTeams
Innovation Interest: The authors declare no competing financial interests.
(2019LM1143).

Conflicts of Interest: The authors declare no competing financial interests.

Abbreviations
AA Amino acids
Abbreviations
ANOVA Analysis of variance
AA
BSA Aminoserum
Bovine acids albumin
ANOVA
DW Analysis
Dry weightof variance
BSA
GC-MS Bovine
Gas serum albumin spectrometry
chromatography-mass
DW
1PE Dry weight
1-Phenylethanol
GC-MS
2PE Gas chromatography-mass spectrometry
2-Phenylethanol
1PE
Z-Glu-Tyr 1-Phenylethanol glutamyl-tyrosin
Benzyloxycarbonyl
2PE 2-Phenylethanol
Z-Glu-Tyr Benzyloxycarbonyl glutamyl-tyrosin
Foods 2020, 9, 66 13 of 15

References
1. Wu, G.; Bazer, F.W.; Dai, Z.; Li, D.; Wang, J.; Wu, Z. Amino acid nutrition in animals: Protein synthesis and
beyond. Annu. Rev. Anim. Biosci. 2014, 2, 387–417. [CrossRef] [PubMed]
2. Fagard, M.; Launay, A.; Clement, G.; Courtial, J.; Dellagi, A.; Farjad, M.; Krapp, A.; Soulié, M.C.;
Masclaux-Daubresse, C. Nitrogen metabolism meets phytopathology. J. Exp. Bot. 2014, 65, 5643–5656.
[CrossRef]
3. Galili, G.; Avin-Wittenberg, T.; Angelovici, R.; Fernie, A.R. The role of photosynthesis and amino acid
metabolism in the energy status during seed development. Front. Plant Sci. 2014, 5, 447. [CrossRef] [PubMed]
4. Häusler, R.E.; Ludewig, F.; Krueger, S. Amino acids—A life between metabolism and signaling. Plant Sci.
2014, 229, 225–237. [CrossRef] [PubMed]
5. Pratelli, R.; Pilot, G. Regulation of amino acid metabolic enzymes and transporters in plants. J. Exp. Bot.
2014, 65, 5535–5556. [CrossRef]
6. Watanabe, M.; Balazadeh, S.; Tohge, T.; Erban, A.; Giavalisco, P.; Kopka, J.; Mueller-Roeber, B.; Fernie, A.R.;
Hoefgen, R. Comprehensive dissection of spatio-temporal metabolic shifts in primary, secondary and lipid
metabolism during developmental senescence in Arabidopsis thaliana. Plant Physiol. 2013, 162, 1290–1310.
[CrossRef]
7. Zeier, J. New insights into the regulation of plant immunity by amino acid metabolic pathways. Plant Cell
Environ. 2013, 36, 2085–2103. [CrossRef]
8. Douglas, A.E. The nutritional quality of phloem sap utilized by natural aphid populations. Ecol. Entomol.
1993, 18, 31–38. [CrossRef]
9. Solomon, P.S.; Tan, K.C.; Oliver, R.P. The nutrient supply of pathogenic fungi; a fertile field for study. Mol.
Plant Pathol. 2003, 4, 203–210. [CrossRef]
10. Rico, A.; Preston, G.M. Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced
nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast. Mol. Plant
Microbe 2008, 21, 269–282. [CrossRef]
11. Szabados, L.; Savouré, A. Proline: A multifunctional amino acid. Trends Plant Sci. 2010, 15, 89–97. [CrossRef]
[PubMed]
12. Lindemann, B. Receptors and transduction in taste. Nature 2001, 413, 219–225. [CrossRef] [PubMed]
13. Belitz, H.D.; Grosch, W.; Schieberle, P. Lehrbuch der Lebensmittelchemie: 1.2 Aminosäuren, 5th ed.;
Springer-Verlag: Berlin, Germany, 2001.
14. Bernard, S.M.; Habash, D.Z. The importance of cytosolic glutamine synthetase in nitrogen assimilation and
recycling. New Phytol. 2009, 182, 608–620. [CrossRef] [PubMed]
15. Li, X.; Chen, G.M.; Wang, M.Q. The development and utilization of tea-substituting plant resources in China.
Beverage Ind. 2008, 11, 4–6. (In Chinese)
16. Liu, B.; Zhang, H.K.; Gong, H.Y. Determination of rutin and quercetin in tea of Lycium barbarum leaves by
RP-HPLC. Chin. J. Exp. Tradit. Med. Formul. 2011, 14, 31. (In Chinese)
17. Jia, Z.Y.; Luo, S.X.; Yan, H.; Wang, Y.J.; He, Y.Y.; Wang, J. Research on the bioavailability of Cd in Ilex
kudingcha CJ Tseng Soil. Adv. Mater. Res. 2012, 356, 165–171.
18. Thippeswamy, R.; Gouda, K.M.; Rao, D.H.; Martin, A.; Gowda, L.R. Determination of theanine in commercial
tea by liquid chromatography with fluorescence and diode array ultraviolet detection. J. Agric. Food Chem.
2006, 54, 7014–7019. [CrossRef]
19. Yang, Z.Y.; Kobayashi, E.; Katsuno, T.; Asanuma, T.; Fujimori, T.; Ishikawa, T.; Tomomura, M.; Mochizuki, K.;
Watase, T.; Nakamura, Y.; et al. Characterisation of volatile and non-volatile metabolites in etiolated leaves
of tea (Camellia sinensis) plants in the dark. Food Chem. 2012, 135, 2268–2276. [CrossRef]
20. Wan, X.C. Tea Biochemistry, 3rd ed.; China Agriculture Press: Beijing, China, 2003. (In Chinese)
21. Yang, Z.Y.; Baldermann, S.; Watanabe, N. Recent studies of the volatile compounds in tea. Food Res. Int. 2013,
53, 585–599. [CrossRef]
22. Juneja, L.R.; Chu, D.C.; Okubo, T.; Nagato, Y.; Yokogoshi, H. L-theanine—A unique amino acid of green tea
and its relaxation effect in humans. Trends Food Sci. Technol. 1999, 10, 199–204. [CrossRef]
23. Fowler, M.S.; Leheup, P.; Cordier, J.L. Cocoa, coffee and tea. In Microbiology of Fermented Foods; Springer:
Boston, MA, USA, 1998; pp. 128–147.
Foods 2020, 9, 66 14 of 15

24. Roberts, G.R.; Sanderson, G.W. Changes undergone by free amino-acids during the manufacture of black tea.
J. Sci. Food Agric. 1966, 17, 182–188. [CrossRef] [PubMed]
25. Wickremasinghe, R.; Swain, T. Studies of the quality and flavour of Ceylon tea. J. Sci. Food Agric. 1965, 16,
57–64. [CrossRef]
26. Wickremasinghe, R.L.; Swain, T. The flavour of black tea. Chem. Ind. 1964, 37, 1574–1575.
27. Bokuchava, M.A.; Popov, V.R.; Bakh, A.N. The significance of amino acids in the formation of tea aroma in
its interaction with tannin substances under conditions of elevated temperature. Dokl. Akad. Nauk. SSSR
1954, 99, 145–148.
28. Chen, X.H.; Chen, D.J.; Jiang, H.; Sun, H.Y.; Zhang, C.; Zhao, H.; Li, X.S.; Yan, F.; Chen, C.; Xu, Z.M. Aroma
characterization of Hanzhong black tea (Camellia sinensis) using solid phase extraction coupled with gas
chromatography–mass spectrometry and olfactometry and sensory analysis. Food Chem. 2019, 274, 130–136.
[CrossRef]
29. Doi, E.; Shibata, D.; Matoba, T. Modified colorimetric ninhydrin methods for peptidase assay. Anal. Biochem.
1981, 118, 173–184. [CrossRef]
30. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef]
31. GB 5009.3-2010. National Food Safety Standard Determination of Moisture in Foods; Ministry of Health: Beijing,
China, 2010.
32. Mei, X.; Chen, Y.Y.; Zhang, L.Y.; Fu, X.M.; Wei, Q.; Grierson, D.; Zhou, Y.; Huang, Y.H.; Dong, F.; Yang, Z.Y.
Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in
tea (Camellia sinensis) leaves exposed to multiple stresses. Sci. Rep. 2016, 6, 23685. [CrossRef]
33. Zeng, L.T.; Wang, X.Q.; Dong, F.; Watanabe, N.; Yang, Z.Y. Increasing postharvest high-temperatures lead to
increased volatile phenylpropanoids/benzenoids accumulation in cut rose (Rosa hybrida) flowers. Postharvest
Biol. Technol. 2019, 148, 68–75. [CrossRef]
34. Zhang, Y.; Chen, H.X.; Zhang, N.; Ma, L.S. Antioxidant and functional properties of tea protein as affected by
the different tea processing methods. J. Food Sci. Technol. 2015, 52, 742–775. [CrossRef]
35. Harbowy, M.E.; Balentine, D.A.; Davies, A.P.; Cai, Y. Tea chemistry. Crit. Rev. Plant Sci. 1997, 16, 415–480.
[CrossRef]
36. Hufnagel, J.C.; Hofmann, T. Orosensory-directed identification of astringent mouthfeel and bitter-tasting
compounds in red wine. J. Agric. Food Chem. 2008, 56, 1376–1386. [CrossRef] [PubMed]
37. Varner, J.E. Biochemistry of senescence. Annu. Rev. Plant Physiol. 1961, 12, 245–264. [CrossRef]
38. Qu, F.; Zhu, X.; Ai, Z.; Ai, Y.; Qiu, F.; Ni, D. Effect of different drying methods on the sensory quality and
chemical components of black tea. LWT 2019, 99, 112–118. [CrossRef]
39. Dev, C.; Bajaj, K.L. Role of chlorophylls, amino acids and sugars in tea. Two Bud 1980, 27, 16–20.
40. Kumar, R.S.S.; Murugesan, S.; Kottur, G.; Gyamfl, D. Black tea: The plants, processing/manufacturing and
production. In Tea in Health and Disease Prevention; Preedy, V., Ed.; Academic Press: San Diego, CA, USA,
2012; pp. 41–57.
41. Kumazawa, K.; Wada, Y.; Masuda, H. Characterization of epoxydecenal isomers as potent odorants in black
tea (Dimbula) infusion. J. Agric. Food Chem. 2006, 54, 4795–4801. [CrossRef]
42. Sanderson, G.W.; Roberts, G.R. Peptidase activity in shoot tips of the tea plant (Camellia sinensis L.). Biochem. J.
1964, 93, 419–423. [CrossRef]
43. Stodt, U.W.; Blauth, N.; Niemann, S.; Stark, J.; Pawar, V.; Jayaraman, S.; Koek, J.; Engelhardt, U.H.
Investigation of processes in black tea manufacture through model fermentation (oxidation) experiments.
J. Agric. Food Chem. 2014, 62, 7854–7861. [CrossRef]
44. Yamamura, H.; Rekharsky, M.V.; Ishihara, Y.; Kawai, M.; Inoue, Y. Factors controlling the complex architecture
of native and modified cyclodextrins with dipeptide (Z-Glu-Tyr) studied by microcalorimetry and NMR
spectroscopy: Critical effects of peripheral bis-trimethylamination and cavity size. J. Am. Chem. Soc. 2004,
126, 14224–14233. [CrossRef]
45. Gui, J.D.; Fu, X.M.; Zhou, Y.; Katsuno, T.; Mei, X.; Deng, R.F.; Xu, X.L.; Zhang, L.Y.; Dong, F.; Watanabe, N.;
et al. Does enzymatic hydrolysis of glycosidically bound volatile compounds really contribute to the
formation of volatile compounds during the oolong tea manufacturing process? J. Agric. Food Chem. 2015,
63, 6905–6914. [CrossRef]
Foods 2020, 9, 66 15 of 15

46. Owuor, P.O.; Cheruiyot, D.K. Effects of nitrogen fertilizers on the aluminium contents of mature tea leaf and
extractable aluminium in the soil. Plant Soil. 1989, 119, 342–345. [CrossRef]
47. Co, H.; Sanderson, G.W. Biochemistry of tea fermentation: Conversion of amino acids to black tea aroma
constituents. J. Food Sci. 1970, 35, 160–164. [CrossRef]
48. Bhattacharyya, N.; Seth, S.; Tudu, B.; Tamuly, P.; Jana, A.; Ghosh, D.; Bandyopadhyay, R.; Bhuyan, M.
Monitoring of black tea fermentation process using electronic nose. J. Food Eng. 2007, 80, 1146–1156.
[CrossRef]
49. Wang, X.Q.; Zeng, L.T.; Liao, Y.Y.; Zhou, Y.; Xu, X.L.; Dong, F.; Yang, Z.Y. An alternative pathway for the
formation of aromatic aroma compounds derived from L-phenylalanine via phenylpyruvic acid in tea
(Camellia sinensis (L.) O. Kuntze) leaves. Food Chem. 2019, 270, 17–24. [CrossRef] [PubMed]
50. Dong, F.; Yang, Z.; Baldermann, S.; Kajitani, Y.; Ota, S.; Kasuga, H.; Imazeki, Y.; Ohnishi, T.; Watanabe, N.
Characterization of L-phenylalanine metabolism to acetophenone and 1-phenylethanol in the flowers of
Camellia sinensis using stable isotope labeling. J. Plant Physiol. 2012, 169, 217–225. [CrossRef] [PubMed]
51. Kraujalytė, V.; Pelvan, E.; Alasalvar, C. Volatile compounds and sensory characteristics of various instant
teas produced from black tea. Food Chem. 2016, 194, 864–872. [CrossRef] [PubMed]
52. Watanabe, S.; Hayashi, K.; Yagi, K.; Asai, T.; Mactavish, H.; Picone, J.; Turnbull, C.; Watanabe, N.
Biogenesis of 2-phenylethanol in rose flowers: Incorporation of [2 H8 ] L-phenylalanine into 2-phenylethanol
and its b-D-glucopyranoside during the flower opening of Rosa ‘Hoh-Jun’ and Rosa damascena Mill.
Biosci. Biotechem. Biochem. 2002, 66, 943–947. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).