Food Chemistry 220 (2017) 177183

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Characterisation of odorants in roasted stem tea using gas

chromatographymass spectrometry and gas chromatography-
olfactometry analysis
Tetsuya Sasaki a, Erina Koshi b, Harumi Take a, Toshihide Michihata a, Masachika Maruya c,
Toshiki Enomoto b,
a
Chemistry/Food Department, Industrial Research Institute of Ishikawa, Kanazawa, Ishikawa 920-8203, Japan
b
Department of Food Science, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan
c
Maruhachi Seichajo Co., Ltd., Kaga, Ishikawa, 922-0331, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Roasted stem tea has a characteristic flavour, which is obtained by roasting tea stems, by-product of
Received 19 May 2016 green tea production. This research aims to understand the characteristic odorants in roasted stem tea
Received in revised form 14 September by comparing it to roasted leaf tea. We revealed potent odorants in commercial roasted stem tea using
2016
gas chromatographymass spectrometry (GCMS) and gas chromatography-olfactometry with aroma
Accepted 30 September 2016
Available online 1 October 2016
extract dilution analysis (AEDA). The difference between roasted stem and leaf tea derived from the same
tea plants were investigated using GCMS. Pyrazine compounds exhibited a roasted odour and high
flavour dilution (FD) factors, as determined via AEDA. Roasted stem tea was richer in these pyrazines than
Keywords:
Roasted tea
roasted leaf tea. Geraniol and linalool exhibited high FD factors and a floral odour, and roasted stem tea
Stem tea was richer in these compounds than roasted leaf tea. These results may have a positive impact on the
Odorant development of tea products.
Gas chromatography-olfactometry 2016 Elsevier Ltd. All rights reserved.

1. Introduction Although tea stems are considered as worthless in commercial

Roasted stem tea has a characteristic flavour (savoury, roast or
sweet), which is obtained by roasting tea stems, by-product of the
manufacturing process of green tea, Sencha. A well-known roasted
tea, Houjicha, is generally manufactured from the leaves of green
tea and is mainly consumed in Japan (Mizukami, Sawai, &
Yamaguchi, 2008). Houjichas popularity and the perception of its
quality are lower than those of green teas such as Sencha, Maccha
and Gyokuro because roasted tea is typically made using lower
grades of green tea harvested as the third- or fourth-flush teas
between summer and autumn (Yamanishi, Shimoji, Ukita,
Kawashima, & Nakatani, 1973); however, roasted stem tea is com-
monly produced from the stems of the first-flush tea, which is the
best-grade Japanese green tea. Roasted stem tea is the preferred
tea in Kanazawa, the capital city of the Ishikawa Prefecture, Japan.
This city is considered to be the origin of roasted stem tea, and indi-
vidual products are manufactured and consumed in many tea
houses.
Corresponding author.
E-mail address: enomoto@ishikawa-pu.ac.jp (T. Enomoto).
tea production, roasted stem tea has a characteristic flavour that
attracts many people. Tea stems are unsuitable for the production
of general green tea, e.g. Sencha, because of the lack of astringency
(Hara, Fukatsu, & Ina, 1993); however, tea stems are suitable for
roasting tea, and we believe that characterisation of odorants in
roasted stem tea may reveal the reasons behind this. Furthermore,
odorant characterisation may help enhance the quality of roasted
tea. Additionally, odorant characterisation is expected to clarify
the effects of roasted stem tea because a relaxing effect of its
flavour components has been reported (Aoshima et al., 2006;
Fukumoto et al., 2008). Black and oolong tea are consumed world-
wide and have characteristic flavours. Similar to green tea, the
stems of black and oolong tea leaves are also removed during their
respective manufacturing processes. Determining odorants in
roasted stem tea could lead to the stems being utilised to add
flavour to black and oolong teas.
Many researchers have analysed odorants in green tea (Kato &
Shibamoto, 2001; Katsuno et al., 2014; Kumazawa & Masuda,
1999, 2002; Yamanishi, Nose, & Nakatani, 1970; Yamaguchi &
Shibamoto, 1981), black tea (Joshi & Gulati, 2015; Kumazawa &
Masuda, 2001; Schuh & Schieberle, 2006) and oolong tea

http://dx.doi.org/10.1016/j.foodchem.2016.09.208
0308-8146/ 2016 Elsevier Ltd. All rights reserved.
178 T. Sasaki et al. / Food Chemistry 220 (2017) 177183
(Kawakami, Ganguly, Banerjee, & Kobayashi, 1995; Pripdeevech &
Machan, 2011; Schuh & Schieberle, 2006) using gas chromatography
(GC). The volatile compounds from roasted tea have been studied
and are reported to contain pyrazine, pyrrole and furan compounds
and organic acids (Kawakami & Yamanishi, 1999; Mizukami et al.,
2008; Yamanishi et al., 1973). Recently, GC-olfactometry (GC-O)
with aroma extract dilution analysis (AEDA) (Grosh, 1993) was uti-
lised to clarify the odorants in tea and many potent odorants were
identified (Kumazawa & Masuda, 1999, 2001, 2002). Potent odor-
ants in roasted tea have also been identified by AEDA (Mizukami
et al., 2008); however, the flavour characteristics of roasted stem
tea have not yet been reported.
The purpose of this study was to understand the characteristic
odorants in roasted stem tea. We reveal potent odorants in roasted
stem tea using GCmass spectrometry (GCMS) and GC-O with
AEDA. Moreover, we evaluated the characteristics of roasted stem
tea by comparing the major odorants in roasted stem tea to leaf
tea. Pyrazine compounds that contribute to the roasted odour are
produced from amino acids in the heating process (Amrani-
Hemaimi, Cerny, & Fay, 1995). Free amino acids and catechins are
major taste components in tea. Because these compounds are
important for the production of odorants and the quality of tea, free
amino acids and catechins were also evaluated when comparing
roasted stem and leaf teas. Additionally, we revealed the changes
in the amounts of these components caused by the roasting process.
2. Materials and methods
2.1. Materials
Tea samples: A commercial roasted stem tea product (Kenjyou
Kaga Boucya; Maruhachi Seichajo Co. Ltd., Ishikawa, Japan) was
obtained in the tea shop of this product. The stems and leaves of
green tea (Camellia sinensis var. sinensis cv. Yabukita) were derived
from the same tea plants in Kagoshima Prefecture and were sepa-
rated from the first-flush tea. Stems and leaves tea samples were
harvested in the same tea farm where the ingredients of commer-
cial roasted stem tea were harvested. The roasting tea was
obtained by roasting the stems and leaves of green tea at 200 C
for 30 s using roasting machinery (Sindohiireki; Yamamasu, Ltd.,
Shizuoka, Japan). The roasting conditions are the same used for
commercial roasted stem tea products. One or more kilograms of
the green and roasted teas of both the stems and leaves were
obtained and parts used in this study.
Chemicals: Compounds 1, 3, 57, 10, 12, 13, 17, 2123, 2527
and 3335 (Table 1) were purchased from Tokyo Chemical Indus-
try Co., Ltd. (Tokyo, Japan) for the identification of the odorants.
N-alkanes (n = 625) were also purchased from Tokyo Chemical
Industry Co., Ltd. for determining the retention indices (RIs) of
the compounds in GC analyses. Cyclohexanol was purchased from
Wako Pure Chemical Industries Ltd. (Tokyo, Japan) and was used as
the internal standard for GC analyses. Amino acids and tea cate-
chins were purchased from Wako Pure Chemical Industries Ltd.
and Nagara Science Co., Ltd. (Gifu, Japan), respectively.
2.2. Sample preparation
Boiling distilled water (260 mL) was added to the tea material
(6 g) in a teapot. After standing for 45 s, the mixture was filtered
to obtain the tea brew.
2.3. Stir bar sorptive extraction
Odorants in the tea brews were analysed with GCMS and GC-O
by a stir bar sorptive extraction (SBSE) method as described by
Baltussen, Sandra, Frank, and Carel (1999). Ten mL of tea brew
was added to cyclohexanol as an internal standard to obtain a final
cyclohexanol concentration of 1 mg/L. NaCl (3 g) was added to the
tea brew. A 15-mm stir bar with a 500 lm polydimethylsiloxane
layer was added to the tea brew and equilibrated for 1 h at room
temperature with constant stirring. Thereafter, the stir bar was
removed from the tea brew and dried with filter paper.
2.4. Thermal desorption
For the GCMS and GC-O analyses, the stir bar was inserted into
a thermal desorption unit (TDU; Gerstel Inc., Mlheim an der Ruhr,
Germany) that was connected to a cold trap injector (CIS; Gerstel
Inc.) and a GC system. The extracted odorants were desorbed from
the stir bar at 230 C using the thermal desorption system, cryofo-
cused at 120 C using liquid N2, volatilised at 230 C in the CIS
and injected into a capillary column.
2.5. Gas chromatographymass spectrometry and gas
chromatography-olfactometry
Odorants were analysed using a GC system (Agilent 7890A;
Agilent Technologies Inc., Palo Alto, CA) equipped with a mass
spectrometer (Agilent 5975C; Agilent Technologies Inc.) and a
sniffing port (ODP2; Gerstel Inc.) according to the method of Gao,
Fan, and Xu (2014) with some modifications. The column was a
60 m  0.25 mm (internal diameter) DB-Wax fused silica capillary
column (J&W Scientific Inc., Folsom, CA) with a film thickness of
0.25 lm. The oven temperature was programmed from 40 C
(10 min hold) to 230 C (12 min hold) at a rate of 4 C/min. The
split ratio was 1:10 for the main experiment or was optimised
for AEDA. Helium was used as the carrier gas at a linear flow rate
of 1.2 mL/min. The split ratio of the flow to the mass spectrometry
system and sniffing port was 1:1. The mass spectra were obtained
by electron-impact ionisation under the following conditions: Ion-
isation voltage, 70 eV; ion source temperature, 150 C. The analysis
mode of the mass spectra was the SCAN mode. Nitrogen gas was
pumped into the sniffing port at 50 mL/min and the RIs were cal-
culated using the method of Kovts (1958). The relative concentra-
tions of odorants were calculated by comparing the integrated
peak area of each compound with that of the internal standard
(1 mg/L of cyclohexanol) by GCMS.
2.6. Identification of odorants
Odorants were identified by comparison to reference mass
spectra in the Wiley and NIST databases, the RI of authentic com-
pounds or in published papers and the odour quality was assessed
by reference to previous research on tea odorants (Kumazawa &
Masuda, 2002; Mizukami et al., 2008).
2.7. Aroma extract dilution analysis
The extracted odorants on the stir bars were diluted to 1:16,
1:64, 1:256, 1:1024 and 1:4096 by the GC injector and TDU, with
a split function according to the method of Feng et al. (2015) with
some modifications. The accuracy of the split ratio was confirmed
by comparing it to the split setting value and peak area of cyclo-
hexanol. The flavour dilution (FD) factor and odour quality were
confirmed by three panellists who were trained using the T&T
olfactometer (Daiichi Yakuhin Sangyo Ltd., Tokyo, Japan) and Open
Essence (Wako Pure Chemical Industries Ltd.), to check the sensi-
tivity and identification ability of olfactory sense for the diagnostic
of olfactory disturbance.
 T. Sasaki et al. / Food Chemistry 220 (2017) 177183 179

Table 1
Potent odorants in roasted stem tea.

No. RIa Odorant Odour quality FD factor IDb

1 722 Dimethylsulfide Sulfury 16 MS, RI(a,l1)
2 1175 1-ethylpyrrole Roast 16 MS,RI(l2)
3 1196 Trimethyloxazole Roast 16 MS, RI(a,l3)
4 1239 Unknown Roast 64
5 1300 Acetol Roast 16 MS, RI(a,l3)
6 1324 2,5-dimethylpyrazine Roast 16 MS, RI(a,l4)
7 1335 Ethylpyrazine Roast 64 MS, RI(a,l3)
8 1368 (Z)-1,5-octadien-3-one Metallic 256 RI(l5)
9 1388 2-ethyl-6-methylpyrazine Roast 256 MS, RI(l2)
10 1408 2,3,5-trimethylpyrazine Roast 256 MS, RI(a,l5)
11 1422 Propylpyrazine Roast 64 MS,RI(l6)
12 1438 2,6-diethylpyrazine roast 16 MS, RI(a,l2,6)
13 1446 (E)-linalool oxide (furanoid) Sweet 16 MS, RI(a,l7)
14 1450 2-ethyl-3,6-dimethylpyrazine Roast 1024 MS, RI(l5,7)
15 1466 2-ethyl-3,5-dimethylpyrazine Roast 4096 MS, RI(l5,7)
16 1496 2,3-diethyl-5-methylpyrazine Roast, sweet 256 MS, RI(l5,7)
17 1541 Linalool Floral 64 MS, RI(a,l5,7)
18 1587 Unknown Floral 16
19 1735 Unknown Sweet 16
20 1762 Linalool oxide (pyranoid)c Sweet 16 MS,RI(l4)
21 1789 Methyl salicylate Sweet 16 MS, RI(a,l7)
22 1831 1-(2-furanylmethyl)-1H-pyrrole Sweet 16 MS, RI(a,l6)
23 1844 Geraniol Floral 256 MS, RI(a,l5)
24 1871 Unknown Sweet, caramel-like 16
25 1954 b-ionone Sweet 16 MS, RI(a,l8)
26 1962 (Z)-jasmone Sweet 16 MS, RI(a,l5)
27 2036 Furaneol Sweet, caramel-like 64 MS, RI(a,l9)
28 2073 Unknown Sweet 16
29 2202 Unknown Sweet 16
30 2269 Unknown Floral 16
31 2284 c-jasmone lactone Floral 16 MS, RI(l5,8)
32 2335 Geranic acid Sweet, caramel-like 16 MS, RI(l10)
33 2350 (Z)-methyl jasmonate Sweet 16 MS, RI(a,l5,8)
34 2463 Indole Metallic 16 MS, RI(a,l5,8)
35 2485 Cumarin Sweet 16 MS, RI(a,l5,8)
a
RI: retention index on DB-Wax column (60 m  0.25 mm i.d.; coated with a film thickness of 0.25 lm) observed for GC/MS.
b
Method of identification: MS, identification by comparison with mass spectra stored in the NIST and Wiley libraries. RI(a), identification by comparison to the retention
indices of authentic reference compounds. RI(l), identification by comparison to the retention indices from the literature (1, Guen, Prost, & Demaimay, 2000; 2, Shimoda,
Shiratsuchi, Nakada, Wu, & Osajima, 1996; 3, Umano, Hagi, Nakahara, Shyoji, & Shibamoto, 1995; 4, Ito, Sugimoto, Kakuda, & Kubota, 2002; 5, Kumazawa & Masuda, 2002; 6,
Lpez-Galilea, Fournier, Cid, & Guichard, 2006; 7, Mizukami et al., 2008; 8, Kawakami & Yamanishi, 1999; 9, Kumazawa, Kubota, & Masuda, 2005; 10, Bureau, Razungles, &
Baumes, 2000).
c
Optical isomer is unclear.

2.8. Analyses of free amino acids and catechins 2.9. Statistical analysis

The free amino acid content in the tea brew was analysed using
an amino acid autoanalyser (L-8900; Hitachi High-Technologies
Corporation, Tokyo, Japan). Catechins were analysed using a
high-performance liquid chromatography (HPLC) with mass spec-
trometric (MS) system (LC-2010A; Shimadzu Corporation, Kyoto,
Japan) equipped with a C18 column (250  4.6 mm, 5 lm particle
size; Shinwa Chemical, Tokyo, Japan) according to the method of
Saijyo and Takeda (1999) with some modifications. The solvent
compositions used were (A) 0.1% formic acid in water-
tetrahydrofuran (95:5) and (B) acetonitrile. The mobile phase com-
position started at 97% solvent A and 3% solvent B with a linear
increase to 35% solvent B over 10 min. The external standard
method was used: the calibration curve was made from standard
solutions of catechins ranging from 0.1 to 1.0 mg/100 mL, injecting
1 lL into the HPLC. The tea samples were diluted appropriately
with solvent A. The electrospray ionisation probe was operated
in the negative mode: curved desolvation line (CDL) temperature,
200 C; block temperature, 200 C; CDL voltage, 25.0 kV; probe
voltage, 3.5 kV; nitrogen gas flow, 1.5 L/min. Selective ion moni-
toring mode (m/z 289, 305, 441 and 457) was used for quantitative
analysis.
Differences between each sample score were analysed using the
Students t-test. This statistical analysis was performed using a
spreadsheet programme (Microsoft Excel 2013; Microsoft Corpora-
tion, Redmond, WA).
3. Results and discussion
3.1. Potent odorants in roasted stem tea
To evaluate the characteristics of odorants in roasted stem tea,
we analysed tea brew using GCMS and GC-O with the SBSE
method. Potent odorants were screened by AEDA and the results
are summarised in Table 1. The application of AEDA revealed 35
potent odorants of which 28 were identified, showing FD factors
in the range of 164096. These odorants mainly consisted of roast
(no. 27, 912 and 1416), floral (no. 17, 18, 23, 30 and 31) and
sweet (no. 13, 1922, 2429, 32, 33 and 35) components. The
chemical structures of most of the roast odorants were pyrazines,
such as 2-ethyl-3,5-dimethylpyrazine (15) and 2-ethyl-3,
6-dimethylpyrazine (14), which showed the highest FD factors of
4096 and 1024, respectively. In a previous study of roasted tea
180 T. Sasaki et al. / Food Chemistry 220 (2017) 177183
(Mizukami et al., 2008), pyrazines showed the highest FD factors,
and these were found in large amounts in an expensive product.
Roasted stem tea also contained many pyrazines that are believed
to confer a strong roast aroma to the tea.
Various potent floral and sweet odorants were also found in
roasted stem tea. Of the floral and sweet odorants, the following
had high FD factors (P64): geraniol (floral, no. 23, FD factor:
256), linalool (floral, no. 17, FD factor: 64) and furaneol (sweet,
caramel-like, no. 27, FD factor: 64). In addition, 16 floral or sweet
odorants were detected with FD factors of 16. Various floral and
sweet odorants contribute to the rich flavour of roasted stem tea,
and odorants with high FD factors have particularly strong influ-
ences on the flavour (no. 17, 23 and 27).
A metallic odorant that was not detectable by GCMS was
detected by GC-O and had a high FD factor of 256. Comparison
with the RIs from the literature identified this odorant to be (Z)-
1,5-octadien-3-one (Kumazawa & Masuda, 2002). Dimethylsulfide
is reportedly found in expensive green tea such as Gyokuro and
contributes to the quality of the tea flavour (Yamaguchi &
Shibamoto, 1981). These odorants were therefore presumed to
strongly influence the aromas of both roasted stem and green teas.
3.2. Characteristic odorants in roasted stem tea as compared with leaf
tea
An important characteristic of roasted stem tea is that it is made
from the stem of the tea plant, in contrast to general roasted tea
that is mainly made from the leaf. To evaluate the characteristics
of roasted stem tea, we compared the GCMS peak intensity of
the major odorants in roasted stem and leaf teas. Major odorants
were selected if they had high FD factors in the three odour quality
groups of roast, floral and sweet, which were found to be com-
prised of five pyrazines, geraniol and linalool and furaneol, respec-
tively. Table 2 shows the peak areas and ratios of these odorants
in roasted stem and leaf teas. In the case of 2-ethyl-3,5-
dimethylpyrazine, which had the highest FD factor, the peak area
ratio of stem to leaf was 1.5. Other roast odorants also had high
ratios of 1.31.9 (mean ratio: 1.6). Roasted stem tea was approxi-
mately 1.6 times richer in these pyrazines than roasted leaf tea,
suggesting that roasted stem tea has a richer flavour than leaf
tea. In a previous study, high concentrations of pyrazines were
found in an expensive roasted tea product (Mizukami et al.,
2008). Therefore, the commodity value of roasted stem tea is high.
In terms of the floral odorants, geraniol and linalool showed
high FD factors of 256 and 64, respectively. Additionally, there
Table 2
The peak ratio of major odorants in roasted stem and leaf teas.
FD factor Odorant
Roast odorants
4096 2-ethyl-3,5-dimethylpyrazine
1024 2-ethyl-3,6-dimethylpyrazine
256 2-ethyl-6-methylpyrazine
256 2,3,5-trimethylpyrazine
256 2,3-diethyl-5-methylpyrazine
Floral odorants
256 Geraniol
64 Linalool
Sweet odorant
64 Furaneol
The major odorants were selected if they had high FD factors in the three odour quality groups of roast, floral and sweet. The experiments were performed using three
individual samples, and the values are shown as the mean standard error.
a
Relative concentration was calculated from the peak area of the internal standard (cyclohexanol).
b
The peak ratio (stem/leaf) was calculated by dividing the peak area of the roasted stem tea by that of the roasted leaf tea.
were 4.1 and 3.6 times more geraniol and linalool in roasted stem
tea than in roasted leaf tea, respectively. The levels of furaneol, a
sweet odorant, were 1.5 times higher in roasted stem tea than in
roasted leaf tea. The roasted stem tea contained high levels of floral
and sweet odorants. In particular, the levels of floral odorants in
roasted stem tea were approximately four times those of roasted
leaf tea. These odorants were presumed to contribute strongly to
the flavour of roasted tea due to their high FD factors.
3.3. Changes in characteristic odorants by roasting stem and leaf teas
To evaluate the creation of the major odorants, we investigated
the changes of odorants in stem and leaf teas caused by the
roasting process. Fig. 1 shows the GCMS peak intensities of the
major odorants (2-ethyl-3,5-dimethylpyrazine, 2-ethyl-3,6-
dimethylpyrazine, geraniol, linalool and furaneol) in tea brews
obtained from both roasted teas and the green stem and leaf raw
materials. Neither pyrazines (2-ethyl-3,5-dimethylpyrazine and
2-ethyl-3,6-dimethylpyrazine as shown in Fig. 1A and B, respec-
tively) were detected in the green stem and leaf teas, whereas they
were detected in the roasted teas. Pyrazines are generated in the
roasting process, and these display a roast odour. Furthermore,
the levels of pyrazines were higher in the roasted stem tea than
in the roasted leaf tea.
Pyrazines are produced by the heating process via the Maillard
reaction (Amrani-Hemaimi et al., 1995). The most direct route for
pyrazine formation results from the interaction of a-amino acids
and reducing sugars through Strecker degradation. The condensa-
tion of two a-aminocarbonyl compounds, which result from the
Strecker reaction, produce dehydropyrazines that are intermediate
products in pyrazine formation. Additionally, amides containing
free amino acids such as glutamine (Gln) can convert to high levels
of pyrazines compared to other free amino acids (Hwang, Hartman,
Rosen, & Ho, 1993). The amide nitrogen, possibly through deami-
dation, is available for amino/carbonyl interactions that lead to
pyrazine generation. Theanine, which is one of the amide contain-
ing amino acids, is known to be present in green tea as a major
taste compound. Green stem tea, the ingredient of roasted stem
tea, is expected to contain many free amino acids, especially amide
containing amino acids and sugars, because pyrazines were found
in higher amounts in the roasted stem tea than in the roasted leaf
tea.
Regarding the floral odorants, the concentrations of geraniol
(Fig. 1C) and linalool (Fig. 1D) were 35 times higher in the stem
tea than in the leaf tea, whether they were roasted or not. In
a
Relative concentration (mg/L) Peak ratiob (stem/leaf)
Roasted stem tea Roasted leaf tea
0.61 0.04 0.42 0.04 1.5
2.60 0.04 1.66 0.17 1.6
0.43 0.01 0.26 0.03 1.7
0.63 0.02 0.47 0.07 1.3
0.67 0.02 0.36 0.03 1.9
0.48 0.03 0.12 0.004 4.1
0.52 0.01 0.15 0.01 3.6
0.0033 0.0002 0.0021 0.0001 1.5
 T. Sasaki et al. / Food Chemistry 220 (2017) 177183 181

A 2-Ethyl-3,5-dimethylpyrazine B 2-Ethyl-3,6-dimethylpyrazine
0.8 Stem 3 Stem
a
Relative concentration (mg/L)
Leaf Leaf

Relative concentration (mg/L)

a
0.6
2
b b
0.4

1
0.2

N.D. N.D.
0.0 0
Green Tea Roasted Tea Green Tea Roasted Tea

C Geraniol D Linalool
0.6 Stem 0.6 Stem
Leaf c Leaf

Relative concentration (mg/L)

Relative concentration (mg/L)

0.4 0.4

a
a
0.2 0.2 d
d
b
b
0.0 0.0
Green Tea Roasted Tea Green Tea Roasted Tea

E Furaneol
0.004 Stem
Leaf
Relative concentration (mg/L)

c
0.003

d
0.002

0.001
b
a
0.000
Green Tea Roasted Tea

Fig. 1. The effects of roasting on odorants in stem and leaf teas. Data from (A) 2-ethyl-3,5-dimethylpyrazine, (B) 2-ethyl-3,6-dimethylpyrazine, (C) geraniol, (D) linalool and
(E) furaneol. Green tea is the raw ingredient of roasted tea before the roasting process. Relative concentration was calculated from the peak area of the internal standard
(cyclohexanol). The experiments were performed using three individual samples, and the values are shown as the mean standard error. N.D. stands for not detected.
Different letters indicate significant difference between samples at p 6 0.05.

addition, the amounts of these odorants increased in the roasting Furaneol, which exhibits a sweet odour, was generated by the
process, strongly suggesting that the roasting process damages roasting process (Fig. 1E). Similar to the pyrazines, furaneol is
the cellular tissue of the raw tea material, thus increasing the effi- known to be generated in the heating process via the Maillard
ciency of geraniol and linalool extraction. Hara et al. (1993) also reaction (Blank, Devaud, Matthey-Doret, & Robert, 2003; Wang &
reported that the amounts of geraniol and linalool in green stem Ho, 2008). Furaneol can be formed from hexose such as glucose
tea were higher than those in green leaf tea. Additionally, geraniol via methylglyoxal reduction which may proceed by the Strecker
and linalool exist as terpenoid glycosides in green tea that increase reaction with amino acids. On the other hand, furaneol can be
in the heating process through thermal decomposition (Kumazawa formed through cyclisation of an intact hexsose in the presence
& Masuda, 2001; Wang, Yoshimura, Kubota, & Kobayashi, 2000). of amino acids. Green tea contains substantial levels of furaneol
Therefore, we believe that the roasting process of tea stems results that is generated in the drying process during manufacturing of
in formation of large amounts of linalool and geraniol that are green tea. Moreover, the results show that the roasted stem tea
found in the roasted stem tea. contained 1.5 times more furaneol than the roasted leaf tea. Thus,
182 T. Sasaki et al. / Food Chemistry 220 (2017) 177183

Table 3
The concentrations of free amino acids in green and roasted teas of the stems and leaves.

Green tea Roasted tea

Stem Leaf Stem Leaf
Asp 2.5 0.1a 2.3 0.1b 1.5 0.04c 1.2 0.04c
Ser 1.3 0.1a 1.2 0.04b 0.6 0.02c 0.4 0.02d
Glu 2.3 0.1a 2.0 0.2b 0.5 0.04c 0.3 0.02c
Gln 10 0.2a 4.2 0.3b 0.3 0.1c 0.1 0.01d
Ala 0.7 0.02a 0.7 0.03a 0.3 0.01b 0.3 0.02b
Arg 3.1 0.1a 3.8 0.2a 1.6 0.1b 1.7 0.04b
Theanine 17 0.4a 12 0.4b 4.8 0.2c 2.3 0.1d

Green tea is the raw ingredient of roasted tea before the roasting process. Asp: aspartic acid, Ser: serine, Glu: glutamic acid, Gln: glutamine, Ala: alanine Ala, Arg: arginine. The
values are expressed in mg/100 mL. The experiments were performed using three individual samples, and the values are shown as the mean standard error. Different letters
indicate significant difference between samples at p 6 0.05.

Table 4
The concentrations of catechin compounds in green and roasted teas of the stems and leaves.

Green tea Roasted tea

Stem Leaf Stem Leaf
EC 9.3 0.3a 10 0.4a 4.8 0.2b 3.9 0.2c
EGC 29 0.5a 43 1.1b 13 0.4c 15 0.4d
ECg 3.3 0.2a 5.6 0.4b 2.5 0.2a 3.4 0.2ab
EGCg 20 0.9a 38 2b 13 0.6c 16 0.9a
C 0.8 0.06a 0.8 0.04a 2.4 0.08b 3.0 0.1c
GC 1.4 0.06a 2.1 0.05b 8.8 0.3c 14 0.3d
Cg 0.1 0.03a 0.1 0.04a 0.5 0.04b 1.5 0.07c
GCg 0.3 0.03a 0.5 0.03b 5.0 0.03c 10 0.4d
Total 64 100 50 67

Green tea is the raw ingredient of roasted tea before the roasting process. EC: epicatechin, EGC: epigallocatechin, ECg: epicatechin gallate, EGCg: epigallocatechin gallate, C:
catechin, GC: gallocatechin, Cg: catechin gallate, GCg: gallocatechin gallate. The values are expressed in mg/100 mL. The experiments were performed using three individual
samples, and the values are shown as the mean standard error. Different letters indicate significant difference between samples at p 6 0.05.

green stem tea is expected to contain many amino acids and
sugars.
3.4. Changes in free amino acids and catechins by roasting stem and
leaf tea
Free amino acids and catechins are major taste components in
tea, and free amino acids are precursors for the Maillard reaction
that produce pyrazines and furaneol. These compounds are impor-
tant for production of odorants and the quality of tea; therefore, we
investigated these components. Table 3 shows the concentrations
of free amino acids in the green and roasted teas of both the stems
and leaves. The major free amino acids described in Table 3 include
aspartic acid (Asp), serine (Ser), glutamic acid (Glu), Gln, alanine
(Ala), arginine (Arg) and theanine. In the green teas, we found
higher levels of theanine and Gln relative to other free amino acids,
and these levels were higher in stem tea than in leaf tea. Theanine
reportedly induces relaxation since the intake of which generates
a-waves on the occipital and parietal regions of the brain surface
(Juneja, Chu, Okubo, & Yokogoshi, 1999; Kimura, Ozeki, Juneja, &
Ohira, 2007); thus, consumption of stem tea may result in
increased relaxation. The free amino acid levels were lower in
the roasted teas than in the green teas as they were decomposed
by the roasting process or converted to odorants such as pyrazines
and furaneol by the Maillard reaction. As above, amide containing
amino acids could change to high levels of pyrazines compared to
other free amino acids (Hwang et al., 1993). Amide containing
amino acids in green tea were theanine, Gln and Asp and these
account for the large part of free amino acids in green tea. Addi-
tionally, green stem tea contains richer theanine and Gln than
green leaf tea (theanine: 1.4 times, Gln: 2.4 times). Thus, the rea-
son for the high levels of pyrazines in the roasted stem tea is that
green stem tea contains high levels of theanine and Gln, amide
containing free amino acids.
Table 4 shows the concentrations of catechin compounds in
green and roasted stem and leaf teas. From the table, the catechin
compounds found in the teas include epicatechin (EC), epigallocat-
echin (EGC), epicatechin gallate (ECg), epigallocatechin gallate
(EGCg), catechin (C), gallocatechin (GC), catechin gallate (Cg) and
gallocatechin gallate (GCg). The green teas contained high levels
of EGC, EGCg and EC and the concentrations of the total catechin
compounds in green stem and leaf teas were 64 and
100 mg/100 mL, respectively. The total amount of catechin com-
pounds in the green leaf tea was higher than in the green stem
tea because catechin compounds were produced from the theanine
in the tea leaf (Kito, Kokura, Izaki, & Sasaoka, 1968). From the
roasting process, the concentrations of the total catechin com-
pounds in the roasted stem and leaf teas decreased to 50 and
67 mg/100 mL, respectively. Conversely, C, GC, Cg and GCg
increased in both the stem and leaf teas after roasting. The roasting
process resulted in the decomposition or epimerisation of the cat-
echin compounds (Chen, Zhu, Tsang, & Huang, 2001; Seto,
Nakamura, Nanjo, & Hara, 1997). In both the green and roasted
teas, the total levels of catechins in the stem teas were lower than
those in the leaf teas. This result indicates that the astringency of
the stem teas was weaker than that of the leaf teas, which may
mean that roasted stem tea is easy to swallow.
4. Conclusion
We revealed potent odorants in roasted stem tea using GCMS
and GC-O with AEDA and investigated the differences of odorants
between roasted stem and leaf teas using GCMS. Some pyrazine
compounds, such as 2-ethyl-3,5-dimethylpyrazine, exhibited high
 T. Sasaki et al. / Food Chemistry 220 (2017) 177183
FD factors and were found in higher amounts in roasted stem tea
compared to roasted leaf tea because the raw ingredient, green
stem tea, contains many free amino acids that are the precursors
of pyrazines. Geraniol and linalool, which had high FD factors
and a floral odour, were also found in higher concentrations in
roasted stem tea compared to roasted leaf tea. Geraniol and lina-
lool increased in the roasting process, which might have damaged
the cellular tissue of the raw tea material and decomposed the ter-
penoid glycosides. Roasted stem tea was revealed to be rich in fla-
vour with a savoury, roast or sweet odour even though tea stems
are considered to be waste by-product of the commercial tea pro-
duction process. Therefore, tea stems may be useful for adding fla-
vour to other types of teas, such as black and oolong teas.
Acknowledgement
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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