Food Chemistry 296 (2019) 69–77

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

Profiling of volatile and non-phenolic metabolites—Amino acids, organic T

acids, and sugars of green tea extracts obtained by different extraction
techniques
⁎
Protiva Rani Dasa, Youngmok Kimb, Seong-Jin Honga, Jong-Bang Euna,
a
Department of Food Science and Technology, Graduate School of Chonnam National University, Gwangju 61186, South Korea
b
Synergy Research and Development Center, Synergy Flavors, Inc., Hamilton, OH, 45011 USA

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

Keywords: Volatile compounds and non-phenolic metabolites (amino acids, organic acids, and sugars) of aqueous green tea
Green tea volatiles extracts obtained by ultrasonic extraction (UE), agitation extraction (AE), hot water extraction (HWE), and
Non-phenolic metabolites conventional extraction (CE) were determined using SPME-GC–MS and HPLC, respectively. Significantly higher
Green tea amino acids (P < 0.05) yields of volatiles and non-phenolic metabolites were obtained via UE and AE than via HWE and CE.
Green tea organic acids
UE, AE, HWE, and CE released 212, 201, 103, and 65 volatiles, respectively. Sum total of amino acid and organic
Green tea sugars
acid in extracts was 54.57, 54.35, 27.11, and 12.67 (mg/100 g), and 5.96, 6.19, 3.81, and 1.68 (mg/100 g) for
UE, AE, HWE, and CE, respectively. Volatiles except nitrogen-containing compounds had higher positive cor-
relations with L-theanine, sucrose, malic acid, and catechins yields. Findings of the current study suggest that an
efficient extraction technique may significantly increase volatile and non-phenolic metabolite yields in aqueous
green tea extract.

1. Introduction
Green tea is the most widely consumed beverage globally. Its po-
pularity is credited to attractive aroma, taste, and the presence of an-
tioxidants. In addition to bioactive phenolic compounds, non-phenolic
metabolites such as amino acids, organic acids, and sugars comprise the
organoleptic characteristics of green tea infusions (Horie, Yamauchi, &
Kohata, 1998; Kausar, Akram, & Kwon, 2013; Saeed et al., 2017).
Amino acids are crucial to the taste and flavor attributes of tea and
impart numerous biological benefits, including anti-inflammatory, an-
timicrobial, and neurological effects (Saeed et al., 2017). Furthermore,
quality attributes of tea can be affected by organic acids that are present
as minor constituents in green tea infusion (Horie et al., 1998). Daily
intake of beneficial organic acids can lessen the risk of cardiovascular
diseases, improve cellular energy, metabolism, digestion, and nutritive
values, and maintain a healthy gut (Gundogdu et al., 2014). The pre-
sence of free sugar in tea also enhances the quality attributes, crucial for
the synthesis of catechin, contributes soluble solids, and enhances the
formation of flavor compounds during processing (Podadera & Sabato,
2007).
Overall consumer acceptance of green tea depends on the compo-
sition and extent of release of predominant aroma compounds during
green tea extraction (Jeon et al., 2017; Kraujalyte, Pelvan, & Alasalvar,
2016; Yang, Baldermann, & Watanabe, 2013; Yener et al., 2016). The
release of volatile compounds during tea infusion preparation has been
positively correlated with extracted yields of phenolic compounds,
amino acids, organic acids, sugar content, and other components
(Senanayake, 2013). Traditionally, different distillation methods, in-
cluding steam distillation and hydro-distillation, have been used to
recover volatiles from plant materials. These methods are time con-
suming and degrade thermo-sensitive compounds due to lengthy
thermal treatments (Da Porto, Decorti, & Natolino, 2014). Therefore,
the application of innovative ecofriendly extraction techniques such as
ultrasound extraction, supercritical fluid extraction, micro-wave ex-
traction, and agitation extraction, among others, could be used to ef-
ficiently recover target metabolites, essential oils, and fragrances (Da
Porto et al., 2014; Das & Eun, 2018; Ravi, Breil, Vian, Chemat, &
Venskutonis, 2018).
In a previous study, we performed a comparative analysis of ultra-
sonication extraction, agitation extraction, and conventional extraction
methods in a time- and temperature-dependent manner to determine
optimum extraction conditions for releasing green tea metabolites (Das
& Eun, 2018). We found that ultra-sonication and agitation extraction
techniques were equally efficient for maximal extraction of green tea

⁎
Corresponding author at: Department of Food Science and Technology, Chonnam National University, 77 Yongbong-ro Buk-gu, Gwangju 61186, South Korea.
E-mail address: jbeun@jnu.ac.kr (J.-B. Eun).

https://doi.org/10.1016/j.foodchem.2019.05.194
Received 23 January 2019; Received in revised form 26 May 2019; Accepted 28 May 2019
Available online 29 May 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
P.R. Das, et al.
metabolites, with particular reference to catechins. Besides phenolic
metabolites, volatile compounds and non-phenolic metabolites are also
considered as quality markers of green tea extracts in addition to having
health functionality. Nevertheless, to the best of our knowledge, ultra-
sonication and agitation extraction technologies have not yet been
rigorously compared with conventional extraction methods in regard to
the extraction efficiencies of green tea volatile compounds and non-
phenolic metabolites. Accordingly, the objective of the current study
was to investigate the volatile profiles and non-phenolic metabolites in
aqueous green tea extracts obtained via ultra-sonication extraction
(UE), agitation extraction (AE), hot water extraction (HWE), and con-
ventional extraction (CE) methods, under optimum extraction condi-
tions (Das & Eun, 2018). Multivariate analysis was conducted to explore
the positive correlation between the extraction yields of all metabolites
and functional volatile groups obtained via these extraction techniques.
The findings were expected to provide useful insights for extraction
procedures in the food, cosmetic, and pharmaceutical/nutraceutical
sectors.
2. Materials and methods
2.1. Material and reagents
Dried green tea leaves (Camellia sinensis O. Kuntze) were purchased
from a local Korean farm (Boseong Green Tea, South Jeolla Province,
South Korea). n-alkanes (C10–C40) were purchased from Restek
Corporation (Bellefonte, PA, USA). All other standard compounds were
purchased from Sigma Aldrich (St. Louis, MO, USA).
2.2. Green tea extracts preparation
An extraction temperature of 80 °C and an extraction time of 20 min
were selected for all extractions based on the highest green tea meta-
bolite yields reported in our previous study (Das & Eun, 2018). Briefly,
100 mL of double distilled water (80 °C) was added to 1 g of powdered
green tea leaves and placed in an ultrasonic bath (RK 510H model;
Bandelin, Berlin, Germany) at 35 kHz for UE. For AE, samples were
placed in a shaking water bath (JSSB-50T, 3.2 KW, 14.5 A 1P, JS Re-
search Inc., Gongju, South Korea) at 100 rpm, whereas they were placed
in a water bath (model JSWB-11, 1.0 kW, 5 A 1P, JSR Inc., Gongju-City,
Korea) for HWE. All UE, AE, and HWE samples were maintained at a
constant temperature of 80 °C for 20 min. For CE, 100 mL of double
distilled water (80 °C) was added to 1 g of powdered green tea leaves
and kept at room temperature for 20 min. After 20 min of extraction,
and all obtained green tea extracts were filtered through Whatman No.
1 filter paper.
2.3. Identification and quantification of free amino acids
A model S430 automatic amino acid analyzer (Sykam, Eresing,
Germany) coupled with a LCA K07/Li spherical cation exchanger
column based on polystyrol (4.6 × 150 mm; 7 μm particle size, 10%
cross- linked), maintained between 37 °C and 74 °C was used. The flow
rate was 0.45 mL/min for buffer (pH range was maintained between
2.90 and 7.95) and 0.25 mL/min for reagent. Chromatographic signals
were determined at wavelengths of 440 nm and 570 nm. Identification
and quantification of amino acids was performed based on standards.
The results were expressed as mg/100 g.
2.4. Identification and quantification of organic acids
A high-performance liquid chromatography (HPLC) apparatus
(Prominence; Shimadzu Co., Kyoto, Japan) equipped with an electro-
conductivity detector, a SCR-102H two shim-pack column
(300 × 8.0 mm), and a SCR-102H shim-pack guard column
(300 × 8.0 mm) was used at an oven temperature of 40 °C. Sample
70
Food Chemistry 296 (2019) 69–77
aliquots of 20 µL were injected. The mobile phase was 4 mM p-tolue-
nesulfonic acid at a flow rate of 0.8 mL/min. The reaction reagent was a
16 mM Bis-Tris aqueous solution containing 4 mM p-toluenesulfonic
acid and 100 μM EDTA. Identification and quantification of organic
acids were conducted based on the standards. The results were ex-
pressed as mg/100 g.
2.5. Identification and quantification of sugars and determination of total
soluble solids (°Brix)
HPLC was performed using a 200 series apparatus (PerkinElmer,
Waltham, MA, USA) equipped with a refractive index detector and a
610H column (300 mm; Shodex, Tokyo, Japan). The mobile phase was
100% water at a flow rate of 0.6 mL/min. Identification and quantifi-
cation of sugars were performed based on the standards. The results
were expressed as mg/100 g. Total soluble solid (°Brix) contents were
measured using a model HI96801 refractometer (Hanna Instruments,
Woonsocket, RI, USA).
2.6. Identification and quantification of volatile compounds
Volatile compounds were determined via solid-phase microextrac-
tion coupled to a gas chromatography-mass spectrometer (SPME-
GC–MS), according to the method previously described by Kim, Lee,
and Kim (2016). A 4.9 mL sample of phenol-d6 (100000 µg/100 g,
50 µL) was added as the internal standard. The analysis was performed
using a model 7890A gas chromatograph (Agilent, Santa Clara, CA,
USA) in splitless mode using a 60 m × 0.25 µm RTX-5 ms column with a
helium flow rate of 1.25 mL/min. The transfer line to the TruTOF MS
(Leco, St. Joseph, MN, USA) was held at 240 °C and the data was col-
lected for 20–250 m/z at an acquisition rate of 10 spectra/sec. Com-
pounds were identified based on matches with NIST14, a MS library
database, combined with reported retention indices (RI) and authentic
standards. n-alkanes (C10–C40) were run under the same conditions as
the samples and the RI of compounds were calculated based on the
standard run. The tentative quantification of volatile compounds in
green tea extracts was based on the internal standard (phenol-d6)
concentration and the results were expressed as µg/100 g.
2.7. Quantification of phenolic metabolites
In this study, the phenolic metabolite contents in green tea extracts
were only determined to find out their correlations with volatile pro-
files. Total phenolic, flavonoid, and individual catechin contents of
green tea extracts were determined based on the method as described in
our previous study (Das & Eun, 2018).
2.8. Statistical analysis and multivariate analysis
All experiments were performed in triplicate and values are re-
presented as the mean ± standard deviation (SD). SAS 9.1.3 software
(North Carolina, Cary, USA) was used for statistical analysis. Duncan's
multiple range tests were used to compare the means of the three re-
plicates. Statistical significance was set at P < 0.05. Multivariate
analysis using partial least squares-discriminate analysis (PLS-DA) score
plot, dendrogram analysis, and Pearson’s correlation by heat map
analysis were performed using online versions of MetaboAnalyst
(http://www.metaboanalyst.ca/). Principal component analysis (PCA)
was performed using a FactoMineR package (factor analysis and data
analysis in R) via the method previously described by Le, Josse, and
Husson (2008).
P.R. Das, et al. Food Chemistry 296 (2019) 69–77

Table 1
Amino acid composition of green tea extracts.
No. Amino acids CE HWE UE AE

(mg/100 g)

1 Phosphoethanolamine ND ND 0.10 ± 0.00a 0.04 ± 0.00b

2 Urea 0.06 ± 0.00c 0.07 ± 0.00c 0.28 ± 0.01a 0.29 ± 0.01b
3 Aspartic acid 0.30 ± 0.00c 0.52 ± 0.00b 1.20 ± 0.09a 1.23 ± 0.12a
4 Threonine 0.06 ± 0.00d 0.09 ± 0.00c 0.17 ± 0.01b 0.24 ± 0.02a
5 Serine 0.21 ± 0.00c 0.32 ± 0.00d 0.67 ± 0.01b 0.79 ± 0.01a
6 Asparagine ND 0.05 ± 0.01c 0.06 ± 0.00b 0.20 ± 0.00a
7 Glutamic acid 0.43 ± 0.01d 0.74 ± 0.01c 1.55 ± 0.01b 1.62 ± 0.01a
8 Glycine 0.03 ± 0.00d 0.05 ± 0.01c 0.09 ± 0.00a 0.08 ± 0.01b
9 Alanine 0.13 ± 0.00d 0.25 ± 0.01c 0.69 ± 0.01a 0.59 ± 0.02b
10 Citrulline ND 0.02 ± 0.00b 0.07 ± 0.00a ND
11 Valine ND 0.02 ± 0.00b ND 0.05 ± 0.01a
12 Cystine ND ND 0.05 ± 0.00a ND
13 Methionine ND ND 0.01 ± 0.00a ND
14 L-Theanine 11.05 ± 0.00c 17.66 ± 0.01b 41.58 ± 0.06a 41.61 ± 0.01a
15 Leucine 0.03 ± 0.00c 0.03 ± 0.00c 0.09 ± 0.00b 0.15 ± 0.04a
16 Tyrosine 0.02 ± 0.00c 0.02 ± 0.00c 0.09 ± 0.01a 0.08 ± 0.01b
17 Phenylalanine 0.03 ± 0.01b 0.09 ± 0.00b 0.36 ± 0.05a 0.37 ± 0.02a
18 β-Alanine ND 0.01 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a
19 β-Aminoisobutyric acid ND ND 0.03 ± 0.00a 0.01 ± 0.00b
20 γ-Amino-n-butyric acid 0.20 ± 0.00c 0.32 ± 0.00b 0.75 ± 0.01a 0.74 ± 0.01a
21 Histidine 0.02 ± 0.00c 0.03 ± 0.00c 0.08 ± 0.01a 0.07 ± 0.01b
22 1-Methylhistidine 0.01 ± 0.00a ND 0.01 ± 0.00a ND
23 3-Methylhistidine 0.01 ± 0.00a ND 0.01 ± 0.00a ND
24 Carnosine ND 0.02 ± 0.00b 0.03 ± 0.00a 0.03 ± 0.00a
25 Tryptophan ND 0.03 ± 0.00b 0.05 ± 0.01a 0.04 ± 0.01b
26 Ornithine 0.02 ± 0.01c 0.03 ± 0.00b 0.52 ± 0.00a 0.34 ± 0.01c
27 Lysine ND 0.03 ± 0.00c 1.47 ± 0.30a 1.22 ± 0.16b
28 Arginine ND 1.35 ± 0.01c 4.65 ± 0.01a 4.49 ± 0.07b
29 Isoleucine 0.01 ± 0.00c 0.02 ± 0.00bc 0.03 ± 0.00b 0.06 ± 0.02a
Total 12.67 ± 2.05c 27.11 ± 3.27b 54.57 ± 7.69a 54.35 ± 8.00a

(a-d) Mean ± standard deviation (SD) followed by a common letter within a row are significantly different (P < 0.05). CE: conventional extraction, HWE: hot water
extraction, UE: ultra-sonication extraction, AE: agitation extraction, ND: not detected.

3. Results and discussion
3.1. Identification and quantification of free amino acids
Besides phenolic metabolites, non-phenolic metabolites in green tea
have significant health functionalities (Nimse & Pal, 2015; Saeed et al.,
2017; Son, Satsu, & Shimizu, 2005). Amino acids in tea not only con-
tribute the quality attributes of tea infusion but also exert biological
effects on brain function, inflammation, and oxidation (Saeed et al.,
2017; Son et al., 2005). A total of 29 amino acids were detected in green
tea extracts (Table 1). The amino acids detected in our study were
consistent with those found in other studies (Horanni & Engelhardt,
2013; Kausar et al., 2013). Of these 29 amino acids, 28 were de-
termined via UE, 24 via AE, 23 via HWE, and 17 via CE. Total (sum)
yields were significantly higher (P < 0.05) in green tea extracts ob-
tained via UE (54. 57 mg/100 g), AE (54. 35 mg/100 g), HWE
(27.11 mg/100 g), and CE (12.67 mg/100 g). L-Theanine was identified
as the major amino acid with significantly higher (P < 0.05) con-
centrations of 41.58 and 41.61 (mg/100 g) in UE and AE, respectively,
followed by 17.66 (mg/100 g) in HWE and 11.05 (mg/100 g) in CE.
Subsequently, arginine, glutamic acid, lysine, and aspartic acid had
higher contents than other amino acids in all green tea extracts. The
threonine content could increase the tastiness intensity of green tea
infusion by contributing a complex sweet and bitter taste mixed umami
(Narukawa et al., 2008). By contrast, aspartic acid, and glutamic acid
act synergistically with threonine to contribute to the umami taste.
Sweet taste was contributed mainly by alanine, glycine, threonine, and
serine. In contrast, valine, methionine, leucine, phenylalanine, histi-
dine, tryptophan, arginine, and isoleucine contributed to bitterness or
astringency (Kirimura, Simizu, Kimizuka, Ninomiya, & Katsuya, 1969).
Results of the present study confirmed the efficacy of UE and AE in
increasing amino acid yields. This finding is consistent with other
reports that described a significantly higher release of amino acids by
UE from grape (Carrera, Ruiz-Rodríguez, Palma, & Barroso, 2015) and
moromi (Goh, Lai, Abas, & Tan, 2017). We have previously found that
UE and AE had significantly higher total free amino acids yields
(P < 0.05) compared with CE, in a temperature- and time-dependent
manner (Das & Eun, 2018).
3.2. Identification and quantification of free organic acids, sugars, and total
soluble sugar (Brix)
Extracted minor organic acid contents of green tea displayed trends
similar to those of the extraction efficiency of amino acids. The organic
acids identified were citric acid, malic acid, succinic acid, lactic acid,
and formic acid (Fig. 1a). These organic acids have been reported to
exert antioxidant effects (Nimse & Pal, 2015) as well as other biological
effects on cardiovascular diseases and cellular metabolism (Gundogdu
et al., 2014). In this study, all organic acids were extracted via UE and
AE. Lactic acid was not extracted via HWE. Interestingly, only citric
acid and malic acid were extracted via CE. Significantly higher
(P < 0.05) contents (sum) of organic acid were found in green tea
extracts obtained from UE (5.96 mg/100 g) and AE (6.19 mg/100 g),
compared with HWE (3.81 mg/100 g) and CE (1.68 mg/100 g). Among
the organic acids, citric acid was the major organic acid in all green tea
extracts followed by malic acid, succinic acid, formic acid, and lactic
acid. Citric acid content was significantly higher (P < 0.05) in UE
(2.59 mg/100 g) and AE (2.61 mg/100 g), followed by HWE (1.78 mg/
100 g) and CE (0.75 mg/100 g). The presence of citric acid largely re-
sponsible for the sweet taste of green tea infusion (Horie et al., 1998).
Sugar content is responsible for the production of acid during proces-
sing (Ginz, Balzer, Bradbury, & Maier, 2000). Significantly higher
(P < 0.05) glucose, fructose, and sucrose contents were associated
with UE and AE in our study (Fig. 1b). These results substantiated

71
P.R. Das, et al.
Fig. 1. Organic acid contents (a), sugar contents (b), and total soluble solids (c) of green tea extracts. CE: conventional extraction; HWE: hot water extraction; UE:
ultra-sonication extraction; AE: agitation extraction. Data are presented as mean ± standard deviation (SD); n = 3.
previous reports that increased levels of formic acid, acetic acid, gly-
colic acid, and lactic acid were detected in coffee during the roasting
process due to the addition of sucrose, glucose, or fructose with green
beans (Ginz et al., 2000). Data related to the extraction efficiencies of
green tea organic acids is limited. Thus, present data has limited
comparative value.
The main sugar constituents were glucose, sucrose, and fructose
(Fig. 1b). These 3 sugar constituents were significantly increased
(P < 0.05) by UE and AE, compared to that by HWE and CE. The
predominant sugar was sucrose, followed by glucose and fructose.
Fructose was present in low quantities and was not extracted by CE.
Significantly higher (P < 0.05) yields (sum) of sugars were observed in
UE (24.39 mg/100 g), and AE (23.91 mg/100 g) followed by HWE
(11.67 (mg/100 g), and CE (6.60 mg/100 g). Soluble solid contents
(Brix) followed a trend similar to that of individual sugar contents
(Fig. 1c) and were significantly higher (P < 0.05) in UE and AE than
both conventional extraction methods. In general, total soluble solid
contents comprised soluble sugar contents. This finding is consistent
with the prior result showing that increased yields of soluble solid and
sugar contents were obtained from blueberry juice subjected to ultra-
sonic treatment (Zou & Hou, 2017).
72
Food Chemistry 296 (2019) 69–77
3.3. Identification and quantification of volatile compounds
The changes of volatiles in tea mainly occurred during processing
(Ho, Zheng, & Li, 2015). In ancient folk medicine, tea aroma was used
to treat anxiety and also known to have other biological functions in-
cluding antioxidant effects (Li, Kawasaki, Tomita, & Kawai, 2017). In
the current study, a total of 267 volatile compounds were identified and
quantified. Of these, 212 were detected in UE, 201 in AE, 103 in HWE,
and 65 in CE. Values of volatile compounds in green tea extracts higher
than or equal to 10 µg/100 g (≥10 µg/100 g) are presented in Table 2.
Volatile compounds with values lower than 10 µg/100 g (< 10 µg/
100 g) are presented in Supplementary Table S1. Green tea extracts
obtained via UE and AE tended to contain significantly higher
(P < 0.05) volatile compounds than HWE and CE. Total (sum) yields of
volatile contents were significantly higher (P < 0.05) in UE (4494 µg/
100 g), and AE (4482 µg/100 g), than HWE (2538 µg/100 g), and CE
(1443 µg/100 g). The identified volatile compounds belonged to 8
functional groups, including aldehydes, esters, alcohols, hydrocarbons,
ketones, acids, nitrogen-containing compounds, and other mis-
cellaneous compounds. Among the functional volatile groups, alcohols
were the most prominent in all green tea extracts, followed by acids,
P.R. Das, et al. Food Chemistry 296 (2019) 69–77

Table 2
Volatile compounds of green tea extracts with values equal to, or greater than, 10 µg/100g (≤10 µg/100 g).
No. Volatile compounds RI MF MW CE HWE UE AE

Aldehydes (µg/100g)
1 Nonanal 1097 C9H18O 142 – 4.01 ± 0.01c 10.01 ± 1.21b 14.72 ± 1.10a
Esters
1 Methyl salicylate 1232 C8H8O3 152 – 48.54 ± 3.33c 72.52 ± 4.34a 67.01 ± 09.34b

Alcohols
1 2-Ethyl-1-hexanol 1019 C8H18O 130 41.13 ± 0.94d 65.01 ± 1.03c 75.02 ± 1.60b 93.33 ± 06.13a
2 Linalool 1093 C10H18O 154 1.00 ± 0.01d 4.51 ± 0.01c 14.52 ± 1.01b 17.02 ± 0.09a
3 Caryophyllenyl alcohol 1552 C15H26O 222 69.22 ± 1.03c 162 ± 6.70b 315 ± 6.81a 327 ± 6.42a
4 Junenol 1575 C15H26O 222 1.11 ± 0.00c 2.02 ± 0.00b 96.01 ± 4.24a 95.05 ± 1.01a
5 Di-epi-1,10-cubenol 1577 C15H26O 222 24.04 ± 1.01d 51.52 ± 5.15c 118.4 ± 1.01b 145 ± 12.12a
6 α-Cadinol 1588 C15H26O 222 7.27 ± 1.12c 46.10 ± 1.01b 56.01 ± 1.01a 57.01 ± 7.17a
7 (R)-2-((4aS,8aR)-4a-Methyl-8-methylene-1,4,4a,5,6,7,8,8a- 1561 C15H24O 220 – 19.5 ± 1.34c 48.63 ± 1.82b 51.52 ± 6.116a
octahydronaphthalen-2-yl)propan-1-ol
8 (1aR,3aS,7S,7aS,7bR)-1,1,3a,7-Tetramethyldecahydro-1H-cyclopropa 1541 C15H26O 222 – – 15.03 ± 11.10a 5.03 ± 0.01b
[a]naphthalen-7-ol
9 6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalen- 1560 C15H24O 220 – – 16.52 ± 1.13b 18.04 ± 0.01a
2-ol
10 4a,5-Dimethyl-3-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7- 1610 C15H24O 220 – – 18.00 ± 0.09a 2.34 ± 0.00b
octahydronaphthalen-1-ol
11 Dimethylsilanediol 743 C2H8O2Si 92 139 ± 1.11a 59.63 ± 6.24d 113 ± 3.09b 110 ± 5.02c
12 2-((3R,3aR,3bS,4R,7R,7aS)-3,7-Dimethyloctahydro-1H-cyclopenta 1563 C15H26O 222 – – 17.52 ± 1.04a 18.72 ± 1.02a
[1,3]cyclopropa[1,2]benzen-4-yl)propan-2-ol
13 2,4-Di-tert-butylphenol 1512 C14H22O 206 245 ± 1.03d 269 ± 13.11c 294 ± 1.03b 299 ± 1.03a
14 2-Methylbutanol, diisopropylsilyl ether 1610 C11H25OSi 201 - – 41.01 ± 1.02a 22.72 ± 8.11b

Hydrocarbons
1 Butylated Hydroxytoluene 1514 C15H24O 220 12.32 ± 0.04d 23.4 ± 0.0c 25.31 ± 0.05b 27.02 ± 1.02a
2 α-Muurolene 1509 C15H24 204 - – 17.04 ± 0.04b 16.54 ± 0.09a
3 α-Calacorene 1535 C15H20 200 4.31 ± 0.01d 9.02 ± 0.0c 18.00 ± 5.13a 14.02 ± 1.01b
4 Benzene, 1-(1-methylethenyl)-4-(1-methylethyl)- 1548 C12H16 160 10.23 ± 0.03c 25.7 ± 1.41b 30.73 ± 1.08a 31.53 ± 3.08a
5 Naphthalene, 1,6-dimethyl-4-(1-methylethyl)- 1597 C15H18 198 2.04 ± 0.00d 7.25 ± 0.0c 24.02 ± 1.04b 32.12 ± 1.04a
6 2,4-Diphenyl-4-methyl-1-pentene 1642 C18H20 236 10.02 ± 0.05d 38.21 ± 3.03c 96.83 ± 1.11a 94.01 ± 1.03b
7 trans-Calamenene 1523 C15H22 202 – – 88.21 ± 2.09b 19.01 ± 1.11a
8 1,2,3,4-Tetrahydro-3-isopropyl-5-methyl-1-oxonaphthalene 2,4- 1610 C20H22N4O4 382 – 16.30 ± 0.09c 17.02 ± 1.05b 18.11 ± 0.08a
dinitrophenylhydrazone
9 Hexamethylcyclotrisiloxane 807 C6H18O3Si3 222 32.02 ± 0.06d 37.1 ± 1.13c 41.24 ± 2.05b 47.51 ± 1.414a
10 Silane, trimethyl-3-penten-2-yl-, trans 1171 C8H18Si 142 24.03 ± 1.02a – 19.01 ± 1.03b 18.21 ± 0.06b
11 1,1'-Biphenyl, 2,2',5,5'-tetramethyl- 1597 C16H18 210 2.11 ± 0.01d 11.10 ± 0.09b 8.01 ± 6.13c 16.02 ± 0.59a

Ketones
1 5,9-Undecadien-2-one, 6,10-dimethyl- 1469 C13H22O 194 – 7.91 ± 0.01b 19.10 ± 2.02a 3.03 ± 0.20c
2 2,5-cyclohexadien-1-one, 2,6-bis(1,1-dimethylethyl)-4-hydroxy-4- 1486 C15H24O2 236 3.42 ± 1.04c 1.10 ± 0.00d 18.11 ± 1.01a 4.51 ± 0.08b
methyl-
3 2,5-Dimethyl-4-nitro-3-hexanone 1556 C8H15NO3 173 371 ± 22.03d 406 ± 0.06c 735 ± 3.73b 773 ± 8.02a

Acids
1 Butyric acid, thio-, S-hexyl ester 1311 C10H20OS 188 – 309 ± 9.12c 413 ± 8.09b 517 ± 1.71a
2 Pentanoic acid, 2,2,4-trimethyl-3-hydroxy-, isobutyl ester 1364 C12H24O3 216 179 ± 3.02d 318 ± 6.15c 412 ± 7.09a 406 ± 1.94b
3 Propanoic acid, 2-methyl-, 3-hydroxy-2,2,4-trimethylpentyl ester 1385 C12H24O3 216 78.01 ± 2.11d 298 ± 9.14c 431 ± 6.08b 457 ± 1.01a
4 Benzoic acid, 2-ethylhexyl ester 1605 C15H22O2 234 – 5.01 ± 0.02b 56.54 ± 1.01a 57.11 ± 1.31a
5 1,2-Benzenedicarboxylic acid, bis(1-methylethyl) ester 1663 C14H18O4 250 3.14 ± 0.01d 21.80 ± 0.06b 37.04 ± 22.63a 14.52 ± 0.90c

Nitrogen-containing compounds
1 Caffeine 1667 C8H10N4O2 194 0.93 ± 0.00a 34.20 ± 0.08b 48.00 ± 0.09a 47.01 ± 1.07a
2 2-Pentanamine, 4-methyl- 666 C6H15N 101 62.84 ± 1.02a - 34.54 ± 1.06b 31.01 ± 1.10c

Miscellaneous
1 Argon 667 Ar 39 11.91 ± 0.0d 24.91 ± 0.07c 29.01 ± 1.91a 26.33 ± 3.23b
2 2H-1-Benzopyran, 3,4,4a,5,6,8a-hexahydro-2,5,5,8a-tetramethyl-, 1545 C13H22O 194 – 7.11 ± 5.02c 14.11 ± 0.09b 15.72 ± 1.02a
(2α,4aα,8aα)-

Total yield 1335 ± 53.15c 2332 ± 105b 3954 ± 95.21a 4028 ± 113a

(a–d) Means ± standard deviation (SD) followed by the common letter within a row are significantly different (P < 0.05). CE: conventional extraction, HWE: hot
water extraction, UE: ultra-sonication extraction, AE: agitation extraction, –: not detected. RI: retention indices, MF: molecular formula, MW: molecular weight.

ketones, hydrocarbons, esters, nitrogen-containing compounds, and
aldehydes (Fig. 2).
In general, the presence of alcohols in green tea infusion contributes
floral, lemon, fresh, green, sweet, fruity, buttery, and wet earthy flavors
(Kim et al., 2016). A total of 66 identified and quantified volatile
compounds belonged to alcohol groups. Among them, UE yielded 59
volatiles, AE yielded 50, and HWE yielded 20, while CE yielded only 16
compounds. The method of extraction significantly influenced the re-
lease of alcohol compounds. Green tea extracts obtained via UE
(1381 µg/100 g) and AE (1352 µg/100 g) had significantly higher
(P < 0.05) total (sum) alcohol contents than that of HWE (719 µg/
100 g), and CE (549 µg/100 g). The alcohols which were abundant were
caryophyllenyl alcohol, 2,4-Di-tert-butylphenol, di-epi-1,10-cubenol, 2-
Ethyl-1-hexanol, junenol, and dimethylsilanediol. Common volatile al-
cohols, linalool, terpineol, benzyl alcohols, and hexanols, were sig-
nificantly higher (P < 0.05) in green tea extracts obtained via UE and
AE, whereas terpineol and benzyl alcohols were not detected in HWE
and CE. Linalool is an odorant that imparts a floral lemon flavor.

73
P.R. Das, et al.
Fig. 2. Functional groups of volatile compounds identified in green tea extracts.
CE: conventional extraction; HWE: hot water extraction; UE: ultra-sonication
extraction; AE: agitation extraction; N-containing compounds: nitrogen-con-
taining compounds. Data are presented as mean ± standard deviation (SD);
n = 3.
Hexanol, benzyl alcohol, and terpineol impart green, mild sweet, and
floral scents, respectively (Yang, Yin, Yuan, Dong, & Deng, 2018).
A total of 24 acids were identified and quantified, among which 17
volatiles were detected in UE, 16 in AE, 12 in HWE, and 7 in CE.
Significantly increased (P < 0.05) concentrations of total (sum) acids
were observed in UE (1380 µg/100 g) and AE (1472 µg/100 g), fol-
lowed by HWE (975 µg/100 g) and CE (265 µg/100 g). Butyric acid,
pentanoic acid, and propanoic acid were consistently the most abun-
dant.
In regard to ketones, a total of 51 volatiles were identified and
quantified. Of these, 31 were detected in UE, 38 in AE, 10 in HWE, and
8 in CE. Total (sum) yields were significantly (P < 0.05) higher in UE
(855 µg/100 g), and AE (857 µg/100 g), followed by HWE (437 µg/
100 g), and CE (388 µg/100 g). The highest ketone concentrations
among all ketones, were 2,5-Dimethyl-4-nitro-3-hexanone, 5,9-
Undecadien-2-one, 6,10-dimethyl-, and 2,5-cyclohexadien-1-one, 2,6-
bis(1,1-dimethylethyl)-4-hydroxy-4-methyl-. The presence of ketone
especially 1-penten-3-one in green tea extracts is considered to be
mainly responsible for its green, metallic, mustard-like, and pungent
odor (Kim et al., 2016). The presence of 1-hepten-3-one in green tea
imparts floral and woody odors and was only detected in UE and AE.
Among the 86 identified and quantified hydrocarbons, significantly
higher (P < 0.05) number were identified in UE (67), followed by 64
in AE, 37 in HWE, and 25 in CE. Total (sum) yields were significantly
higher (P < 0.05) in UE (590 µg/100 g), and AE (521 µg/100 g), than
that of HWE (274 µg/100 g), and CE (138 µg/100 g). Abundant hydro-
carbons were butylated hydroxytoluene, muurolene, naphthalene, 2,4-
diphenyl-4-methyl-1-pentene, trans-calamenene, and hexamethylcy-
clotrisiloxane.
A total of 8 volatile esters were identified and quantified. Of these, 6
volatiles were detected in UE and 7 volatiles in AE, while 1 volatile was
detected in HWE and 2 volatiles in CE. Total (sum) ester concentrations
were also significantly higher (P < 0.05) in UE (104 µg/100 g), and AE
(99.21 µg/100 g), than that in HWE (48.54 µg/100 g) and CE (15.02 µg/
100 g). Methyl salicylate and ethyl acetate were identified as the major
constituents. Methyl salicylate was not detected in CE, while ethyl
acetate was not detected in HWE. Methyl salicylate imparts a holly oil
herbal fragrance (Ahmad et al., 2018).
A total of 13 nitrogen volatiles was identified and quantified. Of
these, UE and AE released 8 volatiles, HWE released 3 volatiles, and CE
released 5 volatiles. Caffeine and 2-Pentanamine, 4-methyl- were found
in higher concentrations. Generally, caffeine in green tea infusion is
considered as contributing to taste, while being minimally related to
74
Food Chemistry 296 (2019) 69–77
aroma. The total concentration of nitrogen volatiles was also sig-
nificantly higher (P < 0.05) in UE (103 µg/100 g) and AE (98.02 µg/
100 g) than in HWE (44.21 µg/100 g) and CE (74.78 µg/100 g).
A total of 12 aldehyde volatiles were identified and quantified in
green tea extracts. Of these, 11 volatiles were detected in UE and AE,
while 2 volatiles (3,5-di-tert-butyl-4-hydroxybenzaldehyde and non-
anal) were detected in HWE, and 1 volatile (3,5-di-tert-butyl-4-hydro-
xybenz-1-aldehyde) in CE. The common aldehyde volatiles were hep-
tanal, (E)-2-hexanal, pentanal, and nonanal. Likewise, total
concentration of aldehyde volatiles was significantly higher (P < 0.05)
in UE (33.04 µg/100 g), and AE (32.24 µg/100 g), than HWE (6.10 µg/
100 g), and CE (1.01 µg/100 g). The 7 remaining identified compounds
were categorized as miscellaneous. One furan (2,5-dibutyl-furan),
argon, and iron were identified among these miscellaneous compounds.
3.4. Effects of different extraction techniques on amino acids, organic acids,
sugars, and volatile profiles
The presence of metabolites in green tea infusions mainly corre-
spond to their health-promoting effects. Therefore, an efficient extrac-
tion strategy which increases the yields of bioactive metabolites in
green tea infusion may be useful. Tea metabolites are greatly influenced
by manufacturing and processing methods (Das & Eun, 2018; Kraujalyte
et al., 2016). In the current study, UE and AE displayed potential to
increase the yields of amino acids, organic acids, sugars, and volatile
compounds in green tea. This may be due to the effects of mechanical
stress caused by sound waves and continuous shaking, which enabled
cell membrane disruption. This resulted in higher mass transfer ki-
netics, which allowed more solvent access to cellular matrices by in-
creasing the surface area. This may result in a greater release of me-
tabolites (Chlopicka, Dobrowolska-Iwanek, Wozniakiewicz, &
Zagrodzki, 2014; Das & Eun, 2018).
Generally, soluble metabolites are located in plant vacuoles,
whereas cell wall-bound metabolites are present in cell wall membranes
(Jia, Chawhuaymak, Riley, Zimmt, & Ogden, 2013). Sugar molecules
are located inside the cell matrix (Das & Eun, 2016), while acid groups,
such as phenolic acids, amino acids, and organic acids among others are
normally present as cell wall-bound compounds in plant cells (Das &
Eun, 2016; Jia et al., 2013). It is very difficult to extract such meta-
bolites using conventional methods. Application of any mechanical
stress may enhance cell wall destruction and increase solvent diffusion
through cells, solubilizing soluble solids and cell wall-bound metabo-
lites (Jerkovic, Mastelic, Marijanović, Klein, & Jelic, 2007; Zou & Hou,
2017). The presence of higher yields of green tea non-phenolic meta-
bolites in UE and AE extracts than those in HWE and CE in the current
study may be reasonably explained by the fact that application of me-
chanical stress may degrade cell wall membranes and increase the so-
lubility of metabolites in green tea leaves.
Similarly, the higher release of volatile compounds by UE and AE
may be due to degassing and agitation intensity, which enhances mass
transfer rates by cell wall degradation, enabling more interaction be-
tween solvents and cellular materials (Da Porto et al., 2014). Another
possibility is that an increase in the yields of amino acids, organic acids,
sugars, and phenolic compounds may enhance higher volatile com-
pounds. Current results are substantiated by the prior findings that UE
extracted more honey volatiles than hydrodistillation (Jerkovic et al.,
2007) and extracted increased yields of Cannabis stevia volatiles com-
pared with maceration (Da Porto et al., 2014). Comparative analyses of
volatile metabolite contents extracted from green tea by different ex-
traction techniques are rare. Thus, data presented by the current study
may provide useful information.
It may be hypothesized that the significantly higher yields extracted
via UE and AE, as compared to HWE, may be due to the lower mass
diffusion rate of the conventional thermal extraction method, wherein
heat transfer occurs via convection and conduction. Poor interaction
between solvents and solids results in lower yields. Therefore,
P.R. Das, et al.
Fig. 3. Heat map analysis for Pearson’s correlation analysis of green tea non-phenolic metabolites and volatile functional groups. CE: conventional extraction; HWE:
hot water extraction; UE: ultra-sonication extraction; AE: agitation extraction; AABA: alpha amino-n-butyric acid; N-compounds: nitrogen-containing compounds.
application of any mechanical stress as seen in UE and AE may over-
come these limitations. One study reported that constant thermal ex-
traction produced lower yields of metabolites from Withania somnifera
compared to ultrasound extraction (Dhanani, Shah, Gajbhiye, & Kumar,
2017). In contrast, higher metabolite yields were detected by HWE than
CE in our study. These results clearly demonstrated that constant
thermal extraction exerted stronger effects on metabolite yields com-
pared with the sole effect of initial temperature (Perva-Uzunalic et al.,
2006).
3.5. Correlation of amino acids, organic acids, and sugars with volatile
functional groups
Amino acids, organic acids, and sugars, which release volatiles
during the manufacturing and brewing process of tea infusions, are
considered aroma precursors (Dursun, Güler, & Şekerli, 2017; Ho et al.,
2015; Yang et al., 2018). Heat map analysis based Pearson’s correlation
analysis was performed for amino acids (values ≥0.02 mg/100 g), or-
ganic acids, sugars, and functional volatile groups (Fig. 3). Most volatile
groups (except nitrogen-containing compounds) were positively corre-
lated with amino acids, organic acids, and sugars. Amino acids, mainly
75
Food Chemistry 296 (2019) 69–77
gamma amino-n-butyric acid, alanine, aspartic acid, glutamic acid, L-
theanine, and arginine, were highly correlated with volatile groups,
including alcohols, ketones, hydrocarbons, esters, and aldehydes
(P < 0.001). The Millard reaction, which occurs between reducing
sugar and amino acids, generates flavor compounds during the process
of tea manufacturing by adding nucleophilic amine groups to carbonyl
groups, defined as Strecker aldehydes (Ho et al., 2015). Phenolic me-
tabolites in green tea act as strong scavengers of carbonyl compounds,
thus inhibiting glyoxal formation as well as retarding the Millard re-
action. In the current study, higher contents of volatile alcohols sup-
porting free amino acids in tea infusions greatly influenced the gen-
eration of volatile alcohols (Yang et al., 2018). In our previous study,
we determined total and individual phenolic metabolites of aqueous
green tea extracts (Das & Eun, 2018). Therefore, in this study, we fo-
cused on non-phenolic metabolites and volatiles. However, in order to
determine the correlation between green tea phenolics and volatile
functional groups, we determined the contents of total and major
phenolic metabolites including epigallocatechin gallate (EGCG), epi-
gallocatechin (EGC), epicatechin gallate (ECG), gallocatechin gallate
(GCG), and epicatechin (EC). The phenolic metabolite contents in UE
and AE techniques were significantly higher (P < 0.05) than that in
P.R. Das, et al. Food Chemistry 296 (2019) 69–77

Fig. 4. 2D (a) and 3D (b) score plot of partial least squares-discriminate analysis (PLS-DA), individual factor map, (c) plot of principal component analysis (PCA), and
(d) cluster dendrogram analysis of green tea metabolites obtained by different extraction techniques. CE: conventional extraction; HWE: hot water extraction; UE:
ultra-sonication extraction; AE: agitation extraction; N-compounds: nitrogen-containing compounds.

HWE and CE (Fig. S1). Correlation analysis indicated that EGCG, EGC, 2017; Ho et al., 2015; Kausar et al., 2013).
ECG, GCG, and EC were highly correlated (P < 0.001) with most
functional volatile groups except nitrogen-containing groups
3.6. Multivariate analysis of green tea metabolites extraction efficiency
(P < 0.01) (Fig. S2). This result demonstrates that the phenolic me-
tabolites in green tea are responsible for aroma (Ho et al., 2015). Su-
PLS-DA, PCA, and cluster dendrogram analysis were performed
gars, especially sucrose, were positively correlated with alcohols, ke-
using amino acids (values ≥0.02 mg/100 g), organic acids, sugars, and
tones, hydrocarbons, esters, and aldehydes (Fig. 3). The breakdown of
functional volatile groups (Fig. 4). PCA individual factor map (Fig. 4c)
the cellulosic polysaccharide and glucoside bonds due to any stress
revealed that extraction techniques were the primary differentiator
caused by mechanical shear during processing may enhance the release
based on the yields of metabolites. UE and AE techniques showed re-
of free sugars, which induces the formation of heterocyclic flavor
latively higher extraction efficiency in releasing green tea volatile and
compounds, especially aromatic alcohols in tea liquor (Ho et al., 2015;
non-phenolic metabolites as demonstrated by close clustering and dis-
Kausar et al., 2013). Organic acids were positively related to volatile
tance from conventional extraction methods (HWE and CE). The con-
alcohols, acids, hydrocarbons, esters, and aldehydes (Fig. 3). The higher
tribution of the first principal component was 92.6%, whereas, that of
extraction yields of organic acids were mainly positively related to in-
the second principal component was 5.1%. The separation of UE and AE
creased yields of acid volatiles (Dursun et al., 2017). In green tea in-
techniques from HWE and CE along with the principal component 1,
fusion, the formation of volatile aroma, especially six to ten carbon
explained 92.6% of the variation in data, indicating the considerable
aroma compounds, is related to the oxidation of unsaturated fatty acids
differences in metabolite yields produced by different extraction
(organic acid). Fatty acid oxidation may be initiated by auto-oxidation/
methods. Volatile functional groups were found to be highly correlated
photo-oxidation/thermal oxidation or lipoxygenase mediated oxidation
(> 0.95) with principal component 1, except nitrogen-containing
during processing (Ho et al., 2015). Collectively, this correlation pro-
compounds (0.72). Organic acids, such as malic acid (0.96) and citric
vided a clear demonstration that extraction yields of green tea non-
acid (0.97), and sugars such as sucrose (0.99) and glucose (0.97) were
phenolic metabolites including amino acids, organic acids, sugars, and
found to contribute positively to principal component 1. Likewise, two-
phenolic metabolites (catechins) were positively correlated with the
and three-dimensional score plots of PLS-DA revealed that the extrac-
volatile profiles, which is consistent with prior findings (Dursun et al.,
tion yields of green metabolites mainly separated based on extraction

76
P.R. Das, et al.
techniques (Fig. 4a and b). The compounds which have more than 1 in
the VIP scores plot were L-theanine, sucrose, and arginine. This may be
due to the higher content of L-theanine and arginine found among all
amino acids. Similarly, sucrose was highest among sugars in all green
tea extracts. The cluster dendrogram analysis also revealed that UE and
AE were closely clustered based on extraction efficiency (Fig. 4d),
whereas HWE and CE were clustered separately from UE and AE. Col-
lectively, the multivariate analysis indicated a very easily-visualized
distinction between extraction techniques due to significant variation in
metabolite yields. Therefore, the findings of the current study suggest
that the UE and AE techniques may be applied to quantify volatiles and
other bioactive metabolites in green tea as well as in other plant ma-
terials.
4. Conclusion
This study provided conclusive evidence that the type of extraction
technique used may positively influence yields of green tea metabolites.
Application of mechanical force by UE and AE may enhance extraction
yields of green tea volatile profiles and non-phenolic metabolites in-
cluding amino acids, organic acid, and sugars, compared with con-
ventional HWE and CE methods. The results also revealed that release
of volatile compounds in green tea extracts is positively correlated with
amino acids, organic acids, sugars, and phenolic metabolites. These
findings may be useful to food, cosmetic, and nutraceutical/pharma-
ceutical industries for developing methods for extraction of target green
tea bioactive metabolites/fragrances or for the preparation of metabo-
lite-rich green tea extract/beverages or extract powder.
Declaration of Competing Interest
None.
Acknowledgements
This work was supported by the “Cooperative Research Program for
Agriculture Science & Technology Development (Project No.
PJ01256503)” Rural Development Administration, Republic of Korea.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.05.194.
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