LWT - Food Science and Technology 116 (2019) 108567

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LWT - Food Science and Technology

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The influence of processing conditions on catechin, caffeine and chlorophyll T

contents of green tea (Camelia sinensis) leaves and infusions
Natthawuddhi Donlaoa,b, Yukiharu Ogawac,∗
a
Unit of Innovative Food Packaging and Biomaterials, School of Agro-Industry, Mae Fah Luang University, Chiang Rai, 57100, Thailand
b
Tea and Coffee Institute, Mae Fah Luang University, Chiang Rai, 57100, Thailand
c
Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, 271-8510, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Green tea samples were produced from different roasting temperatures (200 and 300 °C) and drying temperature
Drying (80, 120 and 160 °C). Individual catechins, total catechins, caffeine, and chlorophylls content were all evaluated
Roasting in dried green tea leaves and green tea infusions. The color of dried tea leaves and tea infusions were also
Caffeine evaluated. Results showed variation of individual catechins, total catechins, caffeine, and chlorophylls content
Catechins
among both the dried tea leaves and of the tea infusions. Epimerization and thermal degradation were reported
Chlorophylls
to be the main cause of change in tea catechins during processing. A correlation analysis indicated that total
catechins content in dried tea leaves had significant effect with the content in tea infusions. Chlorophyll was
considered an important compound for determining the greenness of dried tea leaves, but it had no impact on the
color of the tea infusions. Results from this study suggest that strict temperature control is important for
maintaining chemical constituents in green tea.

1. Introduction natural substances: (+)-catechin (C), (−)-catechin gallate (CG),

Tea is a popular non-alcoholic beverage worldwide (Cabrera,
Giménez, & López, 2003; Guo et al., 1999; Koo & Noh, 2007; Okello,
McDougall, Kumar, & Seal, 2011). Tea is prepared by pouring hot water
over the dried leaves of the tea plant (Camellia sinensis) (Cabrera,
Artacho, & Giménez, 2006; Liang, Lu, Zhang, Wu, & Wu, 2005; Yang &
Landau, 2000). It has been consumed as an aromatic beverage and as a
traditional medicine for over a thousand years, especially in East Asia
(Cabrera et al., 2003; Lorenzo & Munekata, 2016; Sato et al., 1989).
More recently, tea drinking has widely associated with having health
benefits such as helping to prevent of some diseases, which brought a
great deal of new attention to this common drink (Higdon & Frei,
2003).
Several studies have worked to explain the health-related properties
of tea and tea products. Experimental data has shown evidence that tea
exhibits significant health protecting activity due to its bioactive com-
pounds (Manzocco, Anese, & Nicoli, 1998; Okello et al., 2011). Flavo-
noids are a class of plant called secondary metabolites. The main fla-
vonoids found in tea are flavan-3-ols, and more precisely tea catechins
(Ananingsih, Sharma, & Zhou, 2013; Kim et al., 2007; Labbé, Tremblay,
& Bazinet, 2006; Pérez-Burillo, Giménez, Rufián-Henares, & Pastoriza,
2018). The tea catechins are composed of a family of eight abundant
(−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epicatechin gallate
(ECG), (−)-epigallocatechin (EGC), (−)-gallocatechin gallate (GCG),
and (−)-epigallocatechin gallate (EGCG) (Ananingsih et al., 2013;
Dalluge & Nelson, 2000). Other flavonoids such as flavonols (i.e.,
quercetin, kaempferol, and myricetin) and flavones (i.e., apigenin and
luteolin) are also found in tea, whereas the amount of these compounds
are lesser than the flavan-3-ol (Peterson et al., 2005). Moreover, among
the tea flavonoids, only catechins show a significant bioavailability
following ingestion (Bronner & Beecher, 1998). The antioxidant activity
and free radical-scavenging capacity of tea catechins have been re-
ported as one of the most important benefits of tea (Almajano, Carbó,
Jiménez, & Gordon, 2008; Sheila A. Wiseman, 2009). These compounds
provide some potential physiological functions such as those affecting
duodenum, colon, skin, lung, breast, esophageal, pancreatic and pros-
tate cancer (Suganuma et al., 1999). In addition, the tea catechins also
have a wide range of beneficial medicinal properties, including in-
hibition of carcinogenesis, tumorigenesis, and mutagenesis, as well as
the inhibition of tumor growth and metastasis (Dalluge & Nelson,
2000).
Green tea is produced from a process without fermentation
(Ananingsih et al., 2013; Astill, Birch, Dacombe, Humphrey, & Martin,
2001; Liang et al., 2005; Wang, Kim, & Lee, 2000). Consequently,

∗
Corresponding author.
E-mail addresses: natthawuddhi.don@mfu.ac.th (N. Donlao), ogwy@faculty.chiba-u.jp (Y. Ogawa).

https://doi.org/10.1016/j.lwt.2019.108567
Received 31 March 2019; Received in revised form 3 July 2019; Accepted 27 August 2019
Available online 30 August 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
N. Donlao and Y. Ogawa LWT - Food Science and Technology 116 (2019) 108567

among the three main types of commercial teas, green tea generally Table 1
shows significantly higher catechins content than all others (Kaur, Processing conditions applied to tea leaves and drying times required to obtain
Jayasekera, & Moughan, 2014; Wang et al., 2000; Zhu, Zhang, Tsang, the final moisture content.a
Huang, & Chen, 1997). Besides, on a mass equivalent basis, green tea Processing conditions Drying Moisture content
also represents stronger antioxidant activity than oolong tea and black time (g/100 g)b
tea (Gadow, Joubert, & Hansmann, 1997; Kaur et al., 2014; Manzocco Abbreviations Roasting Drying (min)
temperature temperature
et al., 1998). It is well known that the fresh tea shoots are very rich in
(°C) (°C)
catechins and its derivatives. However, in green tea processing, there
remains some oxidations during the withering phase. The amount of R200D80 200 80 65 4.90 ± 0.05a
flavonoids (~20–30%) in green tea may be changed to oxidized ca- R200D120 120 45 3.06 ± 0.17c
R200D160 160 20 3.36 ± 0.17c
techin polymers (Higdon & Frei, 2003). Although the fresh shoots are
R300D80 300 80 60 4.78 ± 0.15a
typically steamed using non-pressurized steam, or they are roasted R300D120 120 35 3.89 ± 0.31b
using a roasting kettle to deactivate polyphenol oxidases (Gulati, R300D160 160 18 3.88 ± 0.11b
Rawat, Singh, & Ravindranath, 2003), these operations also result in
a
the reduction of total catechins in the final product. From Donlao and Ogawa (2018) © Elsevier (2018); reproduced with per-
Apart from catechins, tea leaves also contain a certain amount of mission from Elsevier.
b
Mean ± standard deviation (n = 3). Different letters in the same column
caffeine, and people are much more interested in its stimulatory effects
indicate significant differences (P < 0.05).
(Park et al., 2009). However, tea is known to have various quantities of
caffeine, which may or may not be affected by processing conditions. In
production of green tea samples. Firstly, the two-steps withering were
addition, tea infusion color is one of the most important attributes for
preceded in the presence of air. The tea leaves were withered on
tea grading and chlorophyll have been found to contribute to the
bamboo tray at room temperature for 12 h. The leaves were subse-
greenness of tea infusion to some extent (Wang, Park, Chung, Baik, &
quently withered under sunlight for 2 h. After the completion of with-
Park, 2004). However, there is little information on the variations of
ering, the withered leaves were passed through a drum roaster (Yuan
chlorophyll content in dried green tea leaves and green tea infusions
Chang Machinery, Taoyuan, Taiwan). The leaves were roasted at two
after subjected to processing conditions. In our previous study, we
different temperatures (200 °C or 300 °C) for 6 min. Purpose of roasting
observed that the antioxidant characteristic of green tea infusions, and
was to stop the enzyme activities. Further, the roasted leaves were
the ability of the antioxidants in the tea infusions to withstand in vitro
passed through a rolling machine (Yuan Chang Machinery) for
digestion were also altered by the roasting and drying temperatures
crushing. The leaves were rolled in the same duration i.e., 10 min
used during processing. Those variations may be related to the observed
rolling time. After that, each tea leaf was exposed to hot air in an
differences in catechin profiles among the tea infusions made under
electric dryer (Kluay Nam Thai, Bangkok, Thailand) to reduce moisture
different processing conditions (Donlao & Ogawa, 2018). Therefore we
to lower than 8% wet basis (w.b.). The drying temperature regimes
were interested in investigating whether roasting temperature and
were 80 °C, 120 °C and 160 °C. Detailed operating conditions used
drying temperature during green tea processing can alter nutritional
during this study are illustrated Table 1. After drying, the coarse dried
and qualitative characteristics of green tea such as color, chlorophylls,
leaves were directly ground in a mill (NM-200; Nakasa, Osaka, Japan).
caffeine, and individual catechins contents of the dried green tea leaves
The ground tea was then passed through a 2.5-mm screen, packed in
and green tea infusions. Moreover, evaluation of the variation in in-
aluminum foil bags. The storage temperature was maintained at 4 °C.
dividual catechins can support the results of our previous study.

2. Materials and methods

2.4. Determination of moisture content
2.1. Chemicals
The moisture content of tea sample was analyzed according to the
procedure described by AOAC (1997). Briefly, the tea sample (~1 g)
The standards for (+)-catechin (C), (−)-epicatechin (EC),
was exposed to hot air in a hot-air dryer (WFD-400; Eyela, Tokyo,
(−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-epicatechin
Japan). The drying temperature and drying time were set at 105 °C and
gallate (ECG), (−)-catechin gallate (CG), (−)-epigallocatechin gallate
24 h, respectively. The moisture content was calculated based on the
(EGCG), (−)-gallocat-echin gallate (GCG), and caffeine (CF) were
different in the sample weight before and after drying.
purchased from Sigma-Aldrich (St-Louis, MO, USA). Methanol and
acetone were obtained from Wako Pure Chemical (Osaka, Japan).
Trifluoroacetic acid was purchased from Sigma-Aldrich (St-Louis, MO,
2.5. Preparation of extracts
USA). Acetonitrile and methanol, HPLC-grade were purchased from
Merck (Darmstad, Germany). Deionized water was obtained from an in-
2.5.1. Methanol extracts preparation
house Milli-Q water purification system (Millipore, Bedford, MA, USA).
The extraction method described below was based on a previous
study (Donlao & Ogawa, 2018). Briefly, ground dried green tea leaves
2.2. Plant material
(1 g each) were extracted two times with 20 ml of 70% (v/v) methanol
(Wako Pure Chemical, Osaka, Japan) at 70 °C for 10 min. The methanol
Leaves of the tea plant (C. sinensis var. sinensis) were harvested at
extract liquids were combined into the 50 ml volumetric flask and ad-
Boon Rawd Farm, Chiang Rai province located in the north of Thailand.
justed to scale with cold 70% (v/v) methanol.
The leaves were packed in a plastic mesh bag and transported to the tea
processing plant.

2.5.2. Aqueous extracts preparation (tea infusions)

2.3. Green tea processing
The aqueous extract was prepared by infusing green tea sample in
hot water for a fixed time (Donlao & Ogawa, 2018). The tea samples
The following experiments were conducted on tea processing plant
(5 g each) were infused with 500 ml of water at 95 °C for 5 min. The
at Mae Fah Luang University, Chiang Rai, Thailand during October
samples were then filtered through Whatman no. 1 filter paper (GE
2015. Traditional commercial green tea processing includes withering,
Healthcare UK, Buckinghamshire, UK) and the residue were filtered off.
roasting, rolling and drying. This technique was utilized for the

2
N. Donlao and Y. Ogawa
2.6. HPLC analysis of green tea catechins and caffeine
HPLC analysis of green tea catechins and caffeine were carried out
by the method described by the International Organization for
Standardization (ISO14502-2, 2005). A 0.45 μm nylon syringe filter
(Whatman International Ltd.) was used to filter methanol and aqueous
green tea extracts. The tea liquors were then transferred to glass au-
tosampler vials (Aijiren Technology Co., Ltd., Zhejiang, China) before
being injected into a HPLC (Waters Alliance e2695 Separations Module,
Waters Corporation, MA, USA). The injection volume for all of the
samples was 10 μl. The gradient mobile phase consisted on a solution of
A (0.05% (v/v) trifluoroacetic acid in water) and B (99.9% acetoni-
trile). Solution A was filtered through a 0.45 μm cellulose acetate filter
(Whatman International Ltd.) and solution B was filtered through a
0.45 μm nylon membrane filter (Whatman International Ltd.). The
gradient was as follows: 87% solution A and 13% solution B. Separation
and quantification of tea catechins and caffeine were achieved by
commercially available column, a platinum C18-EPS 3μ
(53 mm × 7 mm) supplied by GRACE (W. R. Grace & Co.-Conn, Mary-
land, USA). The temperature of the column has been maintained at
30 °C. Elution was performed at a solvent flow rate of 2.0 ml/min for
10 min. A Photodiode Array (PDA) Detector (Waters® 2996, Waters
Corporation) was used to detect the eluent at 210 nm. Standard curves
were calculated at various concentrations from a mixture of catechins
and caffeine standards. Catechins and caffeine were prepared at nine
different concentrations (0.20–100.00 μg/ml) and analyzed in dupli-
cate. Identification of catechins and caffeine was carried out by com-
paring retention times and spectral data with those of the authentic
standards and the quantification were made by the external standard
method. The amounts of catechins and caffeine in tea extracts were
expressed as g/100 g dried sample.
2.7. Determination of chlorophyll
Determination of chlorophyll was performed based on the colori-
metric method as previously described by Huang, Sheng, Yang, and Hu
(2007) with minor modification. For dried green tea leaves, the mate-
rial was further grounded in an electric mill (NM-200, Nakasa) and
passed through 500-μm sieves. The tea powder (~0.1 g) was mixed
with 25 ml of 80% (v/v) acetone and the mixture was then mixed
thoroughly on a tube mixer (Trio TM-2F, As One, Osaka, Japan) for
5 min. The sample was then filtered through Whatman no. 1 filter paper
(GE Healthcare UK). For green tea infusion, 1 ml of tea infusion was
mixed with 4 ml of 100% acetone and the mixture was then mixed
thoroughly on a tube mixer (As One) for 5 min. The sample was then
filtered through Whatman no. 1 filter paper (GE Healthcare UK). The
amounts of chlorophyll were calculated from the readings of absorption
at 663 nm (chlorophyll a) and 645 nm (chlorophyll b) using UV-VIS
Spectrophotometer (V-630, Jasco, Tokyo, Japan). The contents were
calculated using the following formulas:
Content of chlorophyll a (mg/l) = 12.7A663 – 2.95A645,
Content of chlorophyll b (mg/l) = 22.9A645 – 4.67A663,
Total chlorophyll content (mg/l) = Content of chlorophyll a + Content
of chlorophyll b
where A663 and A645 are the absorbance of the sample at 663 nm and
645 nm, respectively.
Finally, the total chlorophyll contents were calculated as mg/g dried
sample.
2.8. Measurement of colors
20 g of dried leaves were ground (prepared using the same methods
as described in the previous section) and fresh powder samples were
3
LWT - Food Science and Technology 116 (2019) 108567
analyzed. A spectrophotometer (ColorQuest XE, HunterLab, VA, USA)
was used to measure the colors of tea powder and tea infusion.
According to CIE (L*, a*, b*) system, the value of L* represents light-
ness, the value of a* indicates redness and greenness, and the value of
b* indicates yellowness and blueness, respectively.
2.9. Statistical analysis
All data are reported as means ± standard error over three re-
plications. Statistical analyses were conducted using Minitab 16
Statistical Software (Minitab Pty Ltd., Sydney, Australia). The differ-
ence between samples was determined using one-way ANOVA and
Tukey's test at a significance level of P < 0.05. A two-way ANOVA
procedure (with roasting temperature and drying temperature identi-
fied as factors) was analyzed to determine how green tea quality
changed depending on the processing conditions. Significant differ-
ences were reported when P < 0.05. The correlation matrix for total
chlorophyll content and color values of different types of samples was
investigated using a Pearson correlation analysis, as well as the corre-
lation of total catechins, total chlorophyll, and color values between
different types of samples.
3. Results and discussion
3.1. Moisture content and drying times
The results of final moisture content (MC) of green tea leaves after
subjected to processing conditions are shown in Table 1. The results
show that the expected final MC for dried green tea leaves (lower than
8 g/100 g w.b.) was achieved in all of tested processing conditions. The
final MC for all samples was in the range from 3.06 to 4.90 g/100 g w.b.
MC in tea is well known to be an important quality and shelf - life
indicator. Normally, the commercial green tea has a target moisture
content range of 5.00–6.00 g/100 g w.b., according to the Thai in-
dustrial standard for tea (Ministry of Public Health, 1994). Anything
higher leads to degradation in quality. Therefore, the MC of all tea
samples produced in this study were regarded as suitable MC compared
with the standard value.
3.2. Impacts of processing conditions on individual catechins, total
catechins and caffeine contents in dried green tea leaves
Table 2 shows the mean values for the contents of various catechins
(GC, EGC, C, EC, EGCG, GCG, ECG and CG), caffeine (CF), and total
catechins (TC) as sum of individual determined catechins in dried green
tea leaves made under different processing conditions. For catechin
fractions, EGC accounted for a large proportion (varying from 4.64 to
8.36 g/100 g dried sample), followed by EGCG (varying from 3.82 to
8.35 g/100 g dried sample). By contrast, CG was the smallest fraction,
which indicates the values varied from 0.08 to 0.13 g/100 dried sample.
The TC content varied from 11.40 to 19.58 g/100 g dried sample. The
individual catechins, TC, and CF contents of dried green tea leaves
varied widely among samples. It was evident that the contents of seven
catechins (GC, EGC, EC, EGCG, GCG, ECG and CG) and the TC of the
leaves that subjected to roasting at 200 °C exceeded those samples
produced under roasting at 300 °C. This is likely to reflect higher
thermal degradation at higher roasting temperatures of phenolic com-
pounds. These results are consistent with the values of total phenolic
content (TPC) in the dried green tea leaves that were reported pre-
viously (Donlao & Ogawa, 2018). ANOVA test showed significant dif-
ferences in the contents of seven catechins (GC, EGC, C, EC, EGCG, GCG
and ECG), TC, and CF. However, the content of CG was not significantly
affected by the processing conditions.
At the same roasting temperature, significant changes in the mea-
sured compounds were also observed. Interestingly, these changes
showed two different patterns: a decrease with increasing drying
N. Donlao and Y. Ogawa LWT - Food Science and Technology 116 (2019) 108567

catechin gallate; GCG, (−)-gallocatechin gallate; ECG, (−)-epicatechin gallate; CG, (−)-catechin gallate; TC, total catechins; CF, caffeine. RT, roasting temperature; DT, drying temperature; RT × DT, interaction between
The different letters after data within a column represent significant difference at 95% probability level. GC, (−)-gallocatechin; EGC, (−)-epigallocatechin; C, (+)-catechin; EC (+)-epicatechin; EGCG, (−)-epigallo-
temperature (i.e., EGC, EC, EGCG, and TC) and an increase with in-

0.03 ab

0.01 ab
0.01 ab
0.05 ab
creasing drying temperature (i.e., GC, and GCG). However, the contents

0.02b
0.01a
of C and CG in the dried tea leaves processed under different drying
temperatures were not significantly different (P > 0.05). These find-
±
±
±
±
±
±
0.14
0.12
0.20
0.14
0.15
0.15
ings could reflect the epimerization of catechins during green tea pro-

NS
CF

*
*
cessing. Generally, epimerization is the transformation of the catechins
to their isomers, and this mechanism usually occurs during processing
(Wang & Helliwell, 2000). In this study, the epimerization of individual
0.74d
0.10b
0.04b
0.38a
0.05a
0.08c

catechins was more pronounced at higher drying temperature. This

±
±
±
±
±
±
behavior is indicated by the amount of catechins decreasing while their
19.23
19.58
15.07
16.31
16.58
11.40

isomers increased as the temperature rose. It can be seen that the

***
***
NS
TC

change seems to be epimerization from the epistructure (i.e., EGC and

EGCG) to the non-epistructure (i.e., GC and GCG). It is well known that
CF is a major plant alkaloid in green tea (Park et al., 2009). The data in
0.04a
0.02a
0.03a
0.01a
0.01a
0.01a

this study shows that the drying temperature significantly affected the
CF content of the dried green tea leaves. A relative increase in the CF
±
±
±
±
±
±
0.12
0.13
0.11
0.08
0.08
0.10

content was found in the drying at the higher temperature, and the
CG

NS
NS
*

reason for this behavior remains unclear. It might be related to both

temperature and time during the drying process. In the production of
black tea, the increase in CF was mostly observed during the withering
0.00d
0.01b

0.00b
0.01a
0.02a

0.00c

stage; however, the decrease in CF was normally found during the

±
±
±
±
±
±

fermentation and drying stage. It has also been reported that the rise in
0.78
0.79
0.62
0.58
0.61
0.50
ECG

CF content takes place during the withering stage; however, slightly

***
***
***

subsequent decrease in CF is usually observed during the shaping and

firing stages (Astill et al., 2001). However, the results from this study
impart convincing evidence that drying also alters the CF content in
0.01d

0.00d
0.01b
0.00a
0.00e
0.01c

green tea.
±
±
±
±
±
±

Apart from epimerization, there was a downward trend in the TC

roasting temperature and drying temperature. Significance: NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
GCG

0.12
0.17
0.48
0.09
0.12
0.42

with rising processing temperature due to the degradation of catechins.

***
***
***
Effects of processing conditions on individual catechins, total catechins and caffeine contents in dried green tea leaves.

This suggests that most of the catechins were altered by thermal pro-
cessing, and that they might be responsible for the changes in TC.
Generally, the degradation and epimerization of tea catechins might
0.095a

0.03b
0.01b
0.01b
0.07a

0.79c

take place at the same time during thermal processing (Ananingsih

et al., 2013). Among various processing conditions, R200D120 condi-
±
±
±
±
±
±
EGCG

8.17
8.35
6.60
6.53
6.79
3.82

tion had the highest TC content (as a consequence of its higher EGC,
***
***
*

EGCG, and ECG levels). R300D160 condition had the lowest TC content
(as a result of its lower EGC, EC, EGCG and ECG levels). These results
demonstrate that the severity of the tea processing is associated with
0.00 ab
0.01cd
0.01bc
0.00bc
0.01d
0.09a

almost all the amount of chemical constituents in the dried tea leaves.
In this work, two-way ANOVA was also carried out with the ob-
±
±
±
±
±
±

jective to examine the interactions of two main process variables

0.71
0.68
0.51
0.59
0.59
0.44

***
***
NS
EC

(roasting and drying temperatures) on the contents of eight individual

catechins, TC, and CF. The results demonstrate that the contents of six
catechins (GC, EGC, EC, EGCG, GCG, and ECG) and TC were sig-
0.00 ab
0.06 ab
0.06 ab

0.05 ab

nificantly affected by both roasting temperature (RT) and drying tem-

0.02b
0.07a

perature (DT). However, the value of CG was significantly affected by

±
±
±
±
±
±

only roasting temperature (RT). For the contents of six catechins (GC,
0.44
0.37
0.33
0.33
0.30
0.37

EGC, C, ECGC, GCG, and ECG) and CF, significant interactions

NS
NS
C

(P < 0.05) between roasting temperature and drying temperature

Compounds content (g/100 g dried sample)

(RT × DT) were observed, indicating that there was an effect of drying
0.01d
0.01b
0.02b
0.14a
0.02a

temperature, which varied with respect to roasting temperature.

0.02c

Likewise, the changes in the constituents among roasting temperatures

±
±
±
±
±
±

were not consistent and varied with drying temperatures.

8.27
8.36
5.16
7.51
7.42
4.64
EGC

***
***
***

3.3. Influences of processing conditions on the contents of individual

catechins, total catechins and caffeine in green tea infusions
0.05de

0.00cd
0.02b
0.01a
0.01e
0.00c

Findings related to the hot-water extraction of green tea catechins

±
±
±
±
±
±

are particularly interesting because the chemical constituents in the

0.63
0.73
1.25
0.60
0.67
1.12

aqueous extracts is projected to reflect that of the global green tea

***
***
GC

**

consumption (Nkhili et al., 2009). Moreover, measuring the quantity of

catechins in green tea infusions is expected to indicate potential health
Significance

benefits. In this investigation, the same batch of freshly plucked tea

Conditions

R200D120
R200D160

R300D120
R300D160
R200D80

R300D80

RT × DT

leaves was made into green tea under different processing conditions.
Table 2

Individual catechins, TC, and CF determinations using HPLC were

DT
RT

conducted on samples taken after hot-water extraction. Table 3 shows a

4
N. Donlao and Y. Ogawa LWT - Food Science and Technology 116 (2019) 108567

catechin gallate; GCG, (−)-gallocatechin gallate; ECG, (−)-epicatechin gallate; CG, (−)-catechin gallate; TC, total catechins; CF, caffeine. RT, roasting temperature; DT, drying temperature; RT × DT, interaction between
The different letters after data within a column represent significant difference at 95% probability level. GC, (−)-gallocatechin; EGC, (−)-epigallocatechin; C, (+)-catechin; EC (+)-epicatechin; EGCG, (−)-epigallo-
breakdown of the impacts of these processing conditions on the content

0.37 ab
0.02bc
of compounds measured in the green tea infusion. This data was again

0.02a
0.00c

0.00c
0.00c
calculated for comparative purposes based on a dry weight basis. For

±
±
±
±
±
±
the catechin fractions, unlike the dried green tea leaves, it was obvious
0.00
0.02
0.10
0.00
0.01
0.07
that only EGC showed the largest fraction (ranging from 3.64 to 6.96 g/

***
***
CF

**
100 g dried sample) in the tea infusion. However, the smallest fractions
were GCG and CG, indicating that the dried sample values varied from
11.07 ± 0.13b

11.22 ± 0.53b
10.99 ± 0.02b
12.51 ± 0.12a

0.03 to 0.11 and 0.02–0.12 g/100 g, respectively. The individual ca-

8.33 ± 0.12c

8.00 ± 0.19c techins, TC, and CF contents of the tea infusion also changed widely
among the tested samples. The data from one-way ANOVA showed that
green tea processing conditions significantly affected all of the inter-
NS
TC

**

*
ested constituents in the tea infusion, except for the content of C. On
average, the contents of all measured compounds in the tea infusion
made from the tea leaves roasted at 200 °C was relatively higher when
0.04 ab
0.04 ab

0.00 ab
0.02b

0.03b
0.00a

compared with those made from the tea leaves roasted at 300 °C. At the
same roasting temperature, significant changes in the measured com-
±
±
±
±
±
±

pounds were also observed. It appears from these results that, as ex-
0.12
0.07
0.06
0.02
0.07
0.04
CG

NS
**

pected, the changes of chemical constituents in the tea infusion had

*

similar patterns as those observed in the dried tea leaves. That is the
content of one group (i.e., EGC, EC, EGCG, ECG, and TC) decreased
0.02 ab
0.01b
0.01a
0.11a

0.02a
0.01a

with increasing drying temperature. The other group (i.e., GC, GCG,
and CF) increased with increasing drying temperature.
±
±
±
±
±
±

The TC content in the tea infusions varied from 8.00 to 12.51 g/

0.24
0.16
0.02
0.19
0.20
0.13
ECG

100 g dried sample. These findings further demonstrate that both

NS
**
*

roasting and drying temperatures were affected the TC content of the

tea infusions. The decrease in values of TC was more obvious in higher
0.02 ab

0.02 ab

temperature treatments. Among the six different processing treatments,

0.03b

0.01b
0.02b
0.01a

the R200D80 condition had the highest TC content (owing to its higher
roasting temperature and drying temperature. Significance: NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
±
±
±
±
±
±

EGC, EC, EGCG, ECG and CG contents), whereas the R300D160 con-
GCG

0.07
0.05
0.11
0.03
0.05
0.06

dition showed the lowest TC content (as a consequence of its lower

***
NS
*
Effects of processing conditions on individual catechins, total catechins and caffeine contents in green tea infusions.

EGC, EC and EGCG contents). In general, the differences in composition

of the tea infusion are due in part to the differences in chemical com-
0.00cd

position in the dried tea leaves used as raw material. Therefore, the
0.06d
0.02b

0.04a
0.05c

0.06c

variations of catechins in the dried green tea leaves, altered by the

processing conditions, are considered to be a dominant factor influen-
±
±
±
±
±
±
EGCG

cing the compositional differences in the final tea infusion. As de-

2.15
1.93
2.52
1.90
1.87
1.76

***
***
***

monstrated by the data shown in Tables 2 and 3, the TC contents of the

tea infusions were relatively low when compared with its content in the
dried tea leaves. Nevertheless, it can be observed that a large amount of
0.02d
0.00b
0.01a

0.01e
0.10c
0.01c

catechins (~55%–70% of the TC contents in the dried tea leaves) can be

released into the water by the first-brew. This is probably due to the fact
±
±
±
±
±
±
2.17
1.99
0.32
1.73
1.76
1.17

that the samples were prepared with very fine ground leaf particles
***
***
EC

**

(< 5 mm), which would increase the surface area, making it easier for
more material to get into contact with hot water during the brewing
process. This could lead to the release of more catechins in the first-
0.01a
0.02a
0.06a
0.01a
0.00a
0.03a

brew extraction of hot-water. However, the surprising fact is that the

±
±
±
±
±
±

tested green tea infusions contained a very low CF (the maximum

0.28
0.27
0.24
0.29
0.30
0.30

NS
NS

content was only 0.10 g/100 g dried sample); moreover, the tea infu-
C

sions made under R200D80 and R300D80 conditions did not show to
Compounds content (g/100 g dried sample)

contain any caffeine. Generally, significant key factors of final compo-

0.35 ab
0.02bc

sition of green tea infusion are the brewing conditions, such as the
0.01d

0.07d
0.06a
0.16c

amount of tea used, the brewing time, the brewing temperature (Astill
±
±
±
±
±
±

et al., 2001; Bancirova, 2010; Sharpe, Hua, Schuckers, Andreescu, &

6.96
6.09
4.06
6.61
6.20
3.64
EGC

Bradley, 2016). Labbé et al. (2006) and Pérez-Burillo et al. (2018)

***
*

studied the influence of water temperature and infusion time on the

extraction of caffeine. These authors found out that the caffeine content
0.00cd

in tea infusion slightly increased with increasing brewing temperature

0.02d

0.04b
0.01a
0.02c

0.02c

and infusion time. Moreover, the caffeine content increased greatly

when infusion time was above 10 min. In this study, however, the tea
±
±
±
±
±
±
0.52
0.52
0.99
0.46
0.54
0.90

samples were infused with water at 95 °C for 5 min. In the case of such
***
GC

**

**

infusion condition, it can be observed that the tea samples release very
low levels of caffeine beyond their first brew. This is likely the factor
Significance

that causes tea infusion to have low caffeine content.

Conditions

R200D120
R200D160

R300D120
R300D160
R200D80

R300D80

RT × DT

The results of the two-way ANOVA further suggest that the contents
Table 3

of six catechins (i.e., GC, EGC, EC, EGCG, GCG, and ECG) and CF were
DT
RT

all significantly affected by both roasting temperature (RT) as well as

5
N. Donlao and Y. Ogawa LWT - Food Science and Technology 116 (2019) 108567

Table 4
Effects of processing conditions on total chlorophylls content and color values of dried green tea leaves and green tea infusions.
Conditions Dried green tea leaves Green tea infusions

Total chlorophyll Color values Total chlorophyll Color values

content (mg/g dried content (mg/g dried
sample) L* a* b* sample) L* a* b*

R200D80 3.80 ± 0.18b 53.45 ± 0.09bc −1.35 ± 0.03d 14.56 ± 0.04c 0.21 ± 0.05a 70.22 ± 0.36a −5.50 ± 0.02d 24.85 ± 0.39bc
R200D120 2.48 ± 0.09c 56.02 ± 0.64a −0.82 ± 0.07c 16.01 ± 0.21a 0.18 ± 0.06a 69.30 ± 0.40a −4.66 ± 0.06c 25.90 ± 0.48b
R200D160 2.12 ± 0.14c 54.15 ± 0.64b 0.74 ± 0.04a 14.53 ± 0.36c 0.24 ± 0.04a 64.73 ± 0.85b −0.27 ± 0.03a 33.95 ± 0.32a
R300D80 4.44 ± 0.11a 53.76 ± 0.45bc −2.69 ± 0.09f 15.98 ± 0.05a 0.24 ± 0.03a 70.67 ± 0.37a −6.23 ± 0.04f 24.36 ± 0.30cd
R300D120 3.57 ± 0.13b 54.40 ± 0.14b −2.32 ± 0.06e 15.40 ± 0.07b 0.20 ± 0.06a 70.69 ± 0.46a −5.73 ± 0.11e 23.17 ± 0.94d
R300D160 2.49 ± 0.22c 52.49 ± 0.56c 0.05 ± 0.04b 13.28 ± 0.14d 0.21 ± 0.04a 64.59 ± 0.61b −1.03 ± 0.15b 33.96 ± 0.45a

Significance
RT *** ** *** NS NS * *** **
DT *** *** *** *** NS *** *** ***
RT × DT ** ** *** *** NS NS ** **

The different letters after data within a column represent significant difference at 95% probability level. RT, roasting temperature; DT, drying temperature; RT × DT,
interaction between roasting temperature and drying temperature. Significance: NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

drying temperature (DR). However, only roasting temperature (RT) had
a significant impact on C value. In addition, significant interaction ef-
fects between roasting temperature and drying temperature (RT × DT)
on the contents of GC, EGC, EC, EGCG, CG, TC, and CF were all ob-
tained. However, no significant interaction effects were found with the
contents of C, GCG, and ECG (P < 0.05), indicating that there was an
independent effect of both roasting temperature and drying tempera-
ture on these contents for green tea infusion. Findings of this study
show that the roasting and drying temperatures during the tea pro-
cessing possess several effects on the contents of the tea infusions of
individual catechins, TC, and CF, as they do for the dried tea leaves.
Therefore, the results of this research confirm the assumptions of our
previous study (Donlao & Ogawa, 2018), as the variation in catechin
profiles between tea infusions reflect the initial antioxidant activity of
tea infusions, as well as the ability of antioxidants in tea infusions to
tolerate simulated gastric and intestinal digestions.
3.4. Impacts of processing conditions on total chlorophylls content and color
values of dried green tea leaves and green tea infusions
Table 4 shows total chlorophyll content and variations in color of
dried green tea leaves and green tea infusions made under different
processing conditions. The data from one-way ANOVA showed that the
tea processing conditions significantly impacted all of the values in both
dried leaves and infusions, except for the total chlorophyll content of
the tea infusions. The total values of chlorophyll content for dried tea
leaves ranged from 2.12 to 4.44 mg/g dried sample and with both
roasting and drying temperatures, the total chlorophyll content of the
dried tea leaves showed wide variation. The amount of chlorophyll in
the samples decreased with increasing drying temperature. However, it
is important to point out that the chlorophyll content of the leaves
roasted at 300 °C exceeded those samples roasted at 200 °C, indicating
lower chlorophyll loss at higher roasting temperature. According to the
data presented in Table 1, it was obvious that the drying time was also
related to the roasting temperature. That is the roasting at 300 °C re-
quired shorter drying time compared to the roasting temperature at
200 °C. Generally, thermal degradation during drying process is de-
pendent on both temperature and time. Therefore, a shorter drying time
may cause lower chlorophyll degradation. When comparing the chlor-
ophyll contents of the dried tea leaves and the tea infusions from all
treatments, the result shows that during the infusion process, a very
small amount of chlorophyll was released from the dried leaves into the
water. In spite of the fact that chlorophyll is a water-insoluble pigment,
it is released during hot-water extraction from tea leaves and may exist
as an emulsion form in tea infusion (or as a small green particle floating
in tea infusion). This emulsion could also contribute to some extent to
the greenness of the infusion; however, it may also increase the tur-
bidity of the tea beverage (Wang et al., 2004), and this increase might
represent an adverse effect on the sensory characteristics of the tea. The
results in this experiment demonstrates that the chlorophyll content
and L* value, as well as the a* value of the dried leaves were sig-
nificantly influenced by both roasting (RT) and drying (DT) tempera-
tures. Moreover, the interaction of these two factors (RT × DT) was
also significant. However, the b* value was significantly affected by
drying temperature (DT) and the interaction of the two factors
(RT × DT). For the tea infusion, it was found that all the color values
were significantly affected by both roasting (RT) and drying (DT)
temperatures; however, only a* and b* values were significantly
changed by the interactions of these two factors (RT × DT).
In this study, we further evaluated the Pearson correlation coeffi-
cients among the chlorophyll content and the color values of both the
dried tea leaves and the tea infusions (data are given in Table 5). For
the dried green tea leaves, the chlorophyll content was inversely cor-
related with L* and a* values, and positively associated with b* value.
However, only the correlation between chlorophyll content and a*
value was statistically significant (P < 0.05). Moreover, a significant
direct correlation between L* and b* values, and an inverse correlation
between b* and a* values, were also found. For the tea infusion, no
significant correlation was found between the chlorophyll content and
color values. Concerning the very low levels of chlorophyll in all tea
infusion samples, chlorophyll may not be the predominant factor which
contributes to the color of green tea infusion. However, there were
strong negative correlations between L* and a* values, and between L*
and b* values. According to the high correlation between the chlor-
ophyll content and a* value of the leaves, the implication is that
chlorophyll is the major compound determining the greenness of the tea
leaves. On the contrary, such lack of correlation between the chlor-
ophyll content and a* value in the tea infusion is surprising. It indicates
that the method of maximizing chlorophyll levels cannot be an im-
portant way for enhancing the color of green tea infusion, therefore,
chlorophyll is not crucial for the greenness of the tea infusion.
3.5. Pearson correlations of total catechins content, total chlorophylls
content, and color values between dried green leaves and green tea infusions
The Pearson correlation coefficients of total catechin content, total
chlorophylls content, and color values among the dried leaves and in-
fusions are all given in Table 6. Positive correlations were found be-
tween total content of catechin, total content of chlorophyll, L* value,
and a* value of dried tea leaves and tea infusions. In addition, the
negative correlations were also observed between the b* value of dried
tea leaves and tea infusions. However, only the correlations between

6
N. Donlao and Y. Ogawa LWT - Food Science and Technology 116 (2019) 108567

Table 5
Correlation matrix for chlorophyll content and color values of dried green tea leaves and green tea infusions made from various processing conditions.
Sample Pearson correlation coefficient

Dried tea leave Chlorophyll content L* value a* value b* value

Total chlorophylls content 1

L* value −0.146 (NS) 1
a* value −0.904 (***) −0.116 (NS) 1
b* value 0.434 (NS) 0.759 (***) −0.638 (**) 1
Tea infusion Chlorophyll content L* value a* value b* value

Total chlorophylls content 1

L* value −0.158 (NS) 1

a* value 0.099 (NS) −0.980 (***) 1
b* value 0.125 (NS) −0.981 (***) 0.980 (***) 1

NS, correlation is not significant; *, correlation is significant at P < 0.05 levels; **, correlation is significant at P < 0.01 levels; ***, correlation is significant at
P < 0.001 levels.

Table 6 temperature of 80 °C, both the lowest temperatures. Under these con-
Correlation of total catechins content, total chlorophylls content and color va- ditions, the tea infusions can extract a large amount of total catechins
lues between dried green tea leaves and green tea infusions. while obtaining a clear greenish-yellow color.
Relationship Pearson correlation coefficient Significance
Conflicts of interest
Total catechins content 0.841 ***
Total chlorophylls content 0.119 NS
Natthawuddhi Donlao declares that he has no conflict of interest.
L* value 0.393 NS
a* value 0.937 *** Yukiharu Ogawa declares that he has no conflict of interest.
b* value −0.737 ***
Ethical approval
NS, correlation is not significant; *, correlation is significant at P < 0.05 levels;
**, correlation is significant at P < 0.01 levels; ***, correlation is significant at This article does not contain any studies with human participants or
P < 0.001 levels.
animals performed by any of the authors.

total catechins, a* value, and b* value were shown to be statistically

significant (P < 0.05). It is noteworthy that a strong correlation be-
tween the a* value of the dried leaves and infusions. This data indicates
that the greener color of dried green tea leaves could also result in the
greener color of tea infusions. Although the greenness of tea infusion
cannot be contributed by chlorophyll, the color of tea infusions could be
associated with other flavanols in green tea. This could be particularly
true for quercetin, the principal flavanol found in the tea infusion. The
color of quercetin is bright green with a little yellow, and it is the
particular important flavonol closely associated with the greenness of
the tea infusions (Wang et al., 2004). Aditionally, quercetin has been
notified to be degraded intensely during thermal processing (Buchner,
Krumbein, Rohn, & Kroh, 2006). As a result, the degradation of quer-
cetin at different degrees may produce variations in the greenness of the
tea infusions.
4. Conclusion
Current study aimed to examine the effects of roasting and drying
temperatures in dried green tea leaves and green tea infusions on ca-
techin, caffeine, and chlorophyll concentrations. The results demon-
strate considerable variation between both the leaves of dried tea and
the tea infusions. It is reported that epimerization and thermal de-
gradation are the key reason of change in green tea catechins during the
processing. There is also evidence that the variations of chemical con-
stituents in dried green tea leaves are considered to be a dominant
factor influencing all compositional differences in tea infusions, with
the exception of chlorophyll content. Although chlorophyll is the major
compound determining the greenness of the dried tea leaves, it is not
important for the greenness of tea infusion. These results indicate the
importance of strict temperature control measured to maintain the
chemical constituents in the tea leaf undergoing roasting and hot-air
drying. In this experiment, the best condition for the processing of
green tea infusion was a roasting temperature of 200 °C and a drying
Acknowledgments
The authors would be glad to cordially thank Mae Fah Luang
University for their contribution to this research study. The authors'
sincere thanks also go to Mr. Kittipong Wongaudomwicha for his
kindness and support. In particular, the authors also would like to ex-
press their gratitude to Boonrawd Farm Co., Ltd. for providing the fresh
tea shoots. This manuscript was edited for grammatical accuracy by
Matthew Robert Ferguson of Mahidol University International College,
Bangkok, Thailand.
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