Chiang Mai J. Sci.

2014; 41(2) 395

Chiang Mai J. Sci. 2014; 41(2) : 395-402
http://epg.science.cmu.ac.th/ejournal/
Contributed Paper
Analysis of Volatile Constituents of Fermented Tea
with Bacillus subtilis by SPME-GC-MS
Patcharee Pripdeevech
*
, Sakon Moonggoot, Siam Popluechai and Ekachai Chukeatirote
School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand.
*Author for correspondence; e-mail: patcharee.pri@mfu.ac.th
Received: 5 September 2012
Accepted: 12 December 2012
ABSTRACT
The volatile components of Green Oolong tea No. 12 fermented with culture
supernatants of five Bacillus subtilis strains were investigated. Initially, the culture supernatants
of five different strains of B. subtilis were prepared and subsequently used as crude enzymes to
ferment tea samples. After 2 h-fermentation, the volatile components were extracted using
solid phase microextraction (SPME) technique and determined by gas chromatography-mass
spectrometry (GC-MS). At least 54 components were identified in all samples. Linalool, hotrienol
and -terpinene were found to be the major components in dry Green Oolong tea while
B. subtilis-fermented teas provided 2-pentylfuran and limonene in higher amounts. The contents
of most major volatiles increased remarkably in the fermented tea samples. Superior quantity
of volatile components was related to the use of B. subtilis culture supernatants whereas
2-pentylfuran and limonene were responsible for the special odor of B. subtilis-fermented teas.
Keywords: Camellia sinensis, Bacillus subtilis, SPME, GC-MS
1. INTRODUCTION
Tea (Camellia sinensis) is a popular drink
worldwide and more than 3 million hectares
has been planted with tea [1]. Tea is applied
in pharmaceutical products [2-4]. Green tea
production does not involve fermentation
whereas Oolong and red tea are produced
through semi-fermentation. Black tea is
obtained though a complete fermentation
process. The odors and flavors of tea result
from important components such as terpenes,
caffeine, organic acids and polyphenols [5-11].
There have been many attempts to develop
new tea products especially those with distinct
aromas. One simple method is to include
edible essential oils into the tea product to
improve its aroma [12]. Other approaches
include modification of the tea production
process (i.e., withering, rolling, and
fermentation) which result in aroma changes
by promoting and/or inhibiting the enzymes
in the tea leaves [13,14]. Key odor compounds
detected from these experiments showed that
monosaccharide or disaccharide flavorless
glycoside precursors were present in fresh tea
leaves [15-21]. Free aroma constituents are
then released by hydrolysis of glycoside
precursors by -D-glycosidase enzymes
[13,14]. In addition, the addition of external
enzymes (i.e., pectinase and glucosidase) may
improve tea aromas [12,22].
396 Chiang Mai J. Sci. 2014; 41(2)
Thua nao is a conventional fermented
soybean generally used as a flavor enhancer in
dishes mainly in the northern part of Thailand.
Cooked soybean is fermented with Bacillus
subtilis and related bacilli [23]. It has been
reported that Bacillus species are capable of
synthesis a wide range of enzymes that can
be used in industry [24]. A dramatic increase
of several volatile components was found in
soybean fermentation when using this bacterial
strain as a starter culture [25-28]. Owens and
co-workers [26] reported large amounts of
3-hydroxy-2-butanone, 2, 5-dimethylpyrazine
and trimethylpyrazine during fermentation of
soy-daddawa. Ouoba et al. [29] also noted that
the highest contents of pyrazines in African
soumbala, fermented by pure-starter B. subtilis,
were detected significantly. It is therefore
evident that enzymatic action from B. subtilis
can increase the amounts of volatiles in
different soybeans products. However, there
is no report describing the application of
B. subtilis on tea. In order to develop and
improve aroma quality in tea product, the aim
of the present study is to investigate volatile
odor components of B. subtilis-fermented teas
obtained from Chiang Rai province which is
one of best place for planting tea in Thailand
[30].
2. MATERIALS AND METHODS
2.1 Tea Samples
Green Oolong tea No. 12 (Camellia sinensis
var. sinensis) samples obtained from Boonrod
farm, Chiang Rai, Thailand was used in this
study. The sample was stored below 5C
prior to fermentation with culture supernatants
of various Bacillus strains. Mixtures of C
8
to
C
19
n-alkanes were purchased from Merck
(Darmstadt, Germany).
2.2 Bacterial Strains, Culture Conditions
and Crude Extract Preparation
Five strains of Bacillus subtilis were used
in this present study including B. subtilis TN51
isolated from thua nao, a Thai fermented
soybean [3], B. subtilis ASA and B. subtilis
BEST195 isolated from Japanese natto [3,31],
B. subtilis S1-13 isolated from terasi, an
Indonesia shrimp paste [32], and B. subtilis
TISTR008 obtained from Thailand Institute
of Scientific and Technological Research
(TISTR). Each bacterial strain was routinely
cultured on nutrient agar (NA) and, for stock
culture, the 20% glycerol bacterial culture was
prepared and stored at -20C. For inoculum
preparation, a single colony of each bacterial
strain was subcultured to a test tube containing
3 ml of nutrient broth (NB) and incubated
at 37C for 24 h. One milliliter of the cell
suspension was then transferred to a flask
containing 250 ml of NB and then incubated
by shaking (170 rpm) at 37C. After
approximately 24 h of incubation (the A
600
values were ~ 1.0), the bacterial cells were
harvested from the culture media by
centrifugation (8,500 rpm at 4C for 10 min).
The supernatant was then collected to a sterile
media bottle and was used as crude enzymes
for tea fermentation. Alternatively, the crude
culture supernatants were kept at 4C until
required.
2.3 Fermentation of Tea
Tea sample was ground into very small
particles (almost a powder) using an electric
grinder. For each fermentation process,
one hundred grams of powdered tea was
inoculated with 100 ml of the various B. subtilis
supernatant. For mixture of B. subtilis TN51
and ASA, 100 ml of each strain was added
into 100 g of various tea samples. All samples
were fermented with different B. subtilis strains
for 2 h prior to extraction by SPME.
The experiment was carried out in triplicate.
2.4 Analysis of Volatile Constituents
- Solid-phase microextraction (SPME)
Chiang Mai J. Sci. 2014; 41(2) 397
The SPME apparatus with a SPME fiber
assembly holding 1.0 cm fused-silica fibers
was purchased from Supelco, Bellefonte, PA,
USA. A 50/30 m divinylbenzene-carboxen-
polydimethylsiloxane (DVB-CAR-PDMS)
fiber was selected to extract the volatile
components from tea leaf fermented with
various Bacillus strains. The fiber was
mounted in the manual SPME holder and
preconditioned for 2 h in a GC injection port
set at 250C. For each extraction, the sample
bottle was equilibrated at room temperature
around 25C for 2 h. By insertion through
the septum of the sample bottle, the fiber was
then exposed to the sample headspace for
30 min prior to desorption of the volatiles
into the splitless injection port of the GC-MS
instrument for 5 min.
- Gas Chromatography-Mass Spectrometry
(GC-MS)
The volatile constituents of tea leaves
fermented with various Bacillus strains obtained
from the SPME extracts with DVB-CAR-
PDMS fiber were analyzed using a Hewlett
Packard model HP6890 gas chromatograph
(Agilent Technologies, Palo Alto, CA, USA).
It was equipped with an HP-5MS (5% phenyl-
polymethylsiloxane) capillary column (30 m
0.25 mm i.d., film thickness 0.25 m;
Agilent Technologies, USA) interfaced to an
HP model 5973 mass-selective detector. The
oven temperature was initially held at 40C
and then increased by 2C/min to 220C.
The injector and detector temperatures were
250 and 280C, respectively. Purified helium
was used as the carrier gas at a flow rate of
1 ml/min. EI mass spectra were collected at
70 eV ionization voltages over the range of
m/z 29-300. The electron multiplier voltage
was 1150 V. The ion source and quadrupole
temperatures were set at 230C and 150C,
respectively. Identification of volatile
components was performed by comparison
of their Kov t retention indices, relative
to C
8
-C
19
n-alkanes, and comparison of the
mass spectra of individual components with
the reference mass spectra in the Wiley 275
and NIST05 databases and 2007 [33] with
corresponding data of volatile flavor
components in tea.
3. RESULTS AND DISCUSSION
The fingerprints of volatile components
of dry Green Oolong tea No. 12 from
Boonrod farm fermented with bacterium
supernatants of various B. subtilis strains are
present in Figure 1. Percentages of peak area
of volatile compounds of Green Oolong tea
No. 12 fermented with various B. subtilis are
summarized in Table 1. There Similar
characteristics of all B. subtilis-fermented teas
were illustrated. Fifty-four volatiles were
identified among the Green Oolong tea
No. 12 samples. Increased amounts of most
volatile components occurred among different
B. subtilis-fermented teas as compared to the
dry tea sample. Linalool, hotrienol, -terpinene,
2-pentylfuran, -3-carene and endo-fenchol
were found to be the major components in
dry Green Oolong tea No. 12. Small amounts
of terpinolene, 1,8-cineole, cis-linalool oxide
(furanoid), limonene and trans-isolimonene
were also detected. Tea fermented with culture
supernatants of B. subtilis TN51 contained
limonene, 2-pentylfuran, -3-carene, E--
ocimene, hotrienol and linalool as the key
constituents, while monoterpene components
such as terpinolene, -terpinene, trans-
isolimonene, -terpinene, and allo-ocimene
were minor components. The dominant
components of B. subtilis ASA-fermented tea
were 2-pentylfuran, limonene, linalool,
hotrienol and -3-carene. They were
accompanied by the small amounts of E--
ocimene, terpinolene, trans-isolimonene, 1,
8-cineole and caffeine. Green Oolong tea
No. 12 fermented with B. subtilis BEST195
398 Chiang Mai J. Sci. 2014; 41(2)
and S1-13 culture supernatants produced
similar volatile profiles with the dominant
components of 2-pentylfuran, limonene,
linalool, hotrienol, -3-carene, E--ocimene,
terpinolene and trans-isolimonene. Other
components such as -terpinene, terpinolene,
-terpinene, endo-fenchol and allo-ocimene
were detected in lower amounts. 2-Pentylfuran
was found to be the principle constituent
in TISTR008-fermented tea followed by
-3-carene, hotrienol, limonene, E--ocimene,
caffeine, 1,8-cineole and terpinolene,
respectively. Dajanta et al. [3] previously noted
that Bacillus subtilis supernatants can cause a
change in quality and quantity of volatile
components in Green Oolong tea No. 12. As
the results, caffeine, bitter xanthine alkaloid,
impacted the higher value which was found
in fermented tea with B. subtilis ASA,
TISTR008 and S1-13 culture supernatants
compared to original sample.
It was found that greater intensity of
most volatile components was detected in
B. subtilis TN51-fermented tea while
tea fermented by B. subtilis ASA culture
supernatants provided highest amount of
caffeine. Volatile compounds of tea fermented
with various supernatants of B. subtilis in this
study were different from some previous
studies [12,14], which reported that major
compounds of geraniol, benzyl alcohol,
phenylethanol and Z-3-hexenol appeared
significantly in Oolong teas fermented with
enzyme. B. subtilis. Culture fermentation of tea
could induce amounts of various volatile
components due to enzyme production of
each strain. In overall, volatile components
significantly increased from their previous
concentrations in the original non-fermented
sample. Besides, the enzyme could show
different substrate specificity to different
aroma precursors. Increased amounts of
2-pentylfuran shown in all B. subtilis-fermented
teas may be related to enzymatic production
of soybean that Sugawara et al. [27] reported
that 2-pentylfuran was a key bean-like
odor compound of the soybean. Several
investigations also reported that 2-pentylfuran
was detected in soybeans [25,26]. It was
found that B. subtilis generated 2-pentylfuran
in different materials such as soybean and
tea. However, some major compounds found
in pure Bacillus-fermented thua nao and
in naturally fermented soybean such as 2,
5-dimethylpyrazine,2-methylbutanoic
acid, 2,3,5-trimethylpyrazine and 2-
methylpropanoic acid [3] were disappeared
in B. subtilis-fermented teas. It seems that
2-pentylfuran play important role in the
characteristic odor of B. subtilis-fermented
teas especially in BEST195-fermented tea.
Increased intensities of most components
might be also affected by enzymatic activities
produced by B. subtilis, such as protease,
amylase and galactosidase [34,35], that
Figure 1. GC-MS chromatograms of volatile
odor compounds of Green Oolong tea
No. 12 from Boonrod farm fermented
with various B. subtilis culture supernatants. 1;
Dry tea, 2; TN51, 3; ASA, 4; BEST195, 5;
S1-13 and 6; TISTR008.
Chiang Mai J. Sci. 2014; 41(2) 399
improved volatile components during
fermentation. Furthermore, various isolated
B. subtilis could produce several extracellular
enzymes with the same function, such as
nattokinase, protease, amylase, phytase, lipases
and glutamyl hydrolase [3,23]. Enzymatic
degradation products might be generated
further complex odorous compounds
through other reactions.
Table 1. Volatile compounds of Green Oolong tea No. 12 fermented with various B. subtilis.
Component
Linear
retention
index
% Relative peak area (meanSD)
Dry tea TN51 ASA BEST195 S1-13 TISTR008
trans-Isolimonene
2-Pentylfuran
-3-Carene
-Terpinene
p-Cymene
Limonene
1,8-Cineole
Z--Ocimene
E--Ocimene
-Terpinene
cis-Linalool oxide
(furanoid)
Terpinolene
Linalool
Hotrienol
endo-Fenchol
allo-Ocimene
Lavandulol
Methyl salicylate
Safranal
2,6,6-Trimethyl
cyclohexene
carboxaldehyde
Linalool formate
E-Ocimene
Isobornyl formate
Car-3-en-2-one
Linalool acetate
Geranial
Dihydro-linalool acetate
2-Ethyl menthone
p-Cymen-7-ol
-Elemene
-Cubebene
984
988
1011
1017
1024
1029
1031
1037
1050
1059
1072
1088
1096
1108
1116
1128
1181
1191
1196
1212
1216
1238
1239
1248
1257
1265
1275
1282
1290
1325
1338
1.100.32
2.780.05
2.550.11
0.530.20
0.400.12
1.160.06
1.670.13
0.250.24
0.870.31
2.930.11
1.330.22
1.700.08
4.270.07
3.630.17
1.960.23
0.210.14
0.340.14
0.210.32
0.640.15
0.510.11
0.230.17
0.250.13
0.210.20
0.520.17
0.120.16
0.140.07
0.120.14
0.350.22
0.080.14
0.480.31
0.110.06
3.220.08
8.770.14
2.000.07
3.960.14
0.200.09
20.430.27
0.240.08
2.740.09
8.380.11
3.080.09
0.400.17
5.700.15
7.150.09
7.280.05
2.260.17
2.800.12
1.020.11
1.590.17
1.250.08
1.290.07
0.140.09
0.560.11
0.380.13
0.160.07
0.340.07
0.250.11
0.150.08
0.130.05
0.230.07
0.660.10
0.100.09
1.960.12
8.840.20
5.110.11
1.310.08
0.880.09
7.140.10
1.730.25
0.830.11
3.410.13
1.330.17
1.160.09
3.320.08
6.450.41
5.280.14
1.070.21
1.250.09
0.900.11
1.090.22
0.610.12
0.790.11
0.060.07
0.270.09
0.310.21
0.040.02
0.300.08
0.170.09
0.110.08
0.060.03
0.230.07
0.610.04
0.120.05
3.300.08
14.780.07
7.450.11
2.430.05
1.650.07
11.680.10
2.960.08
2.070.04
5.920.14
2.610.04
2.260.08
5.910.17
9.940.11
9.430.08
2.370.05
2.230.06
1.140.04
1.730.08
1.250.04
1.410.09
0.130.05
0.480.08
0.530.07
0.120.04
0.420.08
0.280.07
0.190.07
0.110.05
0.260.04
1.210.09
0.140.07
1.970.07
9.280.04
5.340.05
1.720.04
0.930.08
8.230.07
1.480.05
0.840.07
3.700.06
3.100.08
0.970.05
2.870.02
5.780.09
4.300.10
1.150.11
1.120.09
0.620.08
0.750.05
0.660.04
0.460.02
0.160.04
0.120.08
0.010.01
0.510.07
0.090.04
0.160.04
0.130.02
0.410.04
0.140.01
0.480.07
0.150.06
1.030.07
5.880.03
4.930.04
0.710.05
0.660.08
3.270.13
1.190.07
0.370.10
1.350.07
0.850.05
0.040.02
1.170.04
0.950.04
3.460.03
0.830.09
0.530.04
0.560.02
0.130.04
0.390.03
0.370.04
0.140.06
0.010.02
0.110.02
0.030.02
0.100.03
0.070.04
0.040.02
0.040.02
0.120.05
0.220.14
0.040.02
400 Chiang Mai J. Sci. 2014; 41(2)
4. CONCLUSIONS
Increased contents of total volatiles were
detected in all B. subtilis culture supernatants
compared to the original dry tea. Among these,
the major volatiles were 2-pentylfuran,
limonene, linalool and -3-carene. All Green
Oolong tea No. 12 has similar volatile
profiles whist their amounts were different
according to the different origin, genotype
breeding and ratio of supernatants of
B. subtilis. The significant increase of volatiles
in fermented teas was affected by enzymatic
activities such as protease, amylase and
galactosidase and several extracellular
enzymes such as nattokinase, protease,
amylase, phytase, lipases and glutamyl
hydrolase improving volatile components
during fermentation. In addition, Bacillus strains
may be added to improve key aroma of tea
in non fermentation tea processing.
ACKNOWLEDGEMENTS
The authors would like to thank
Dr. Mitsuhiro Itaya of the Institute for
Advanced Biosciences, Keio University, Japan
for providing the B. subtilis (natto) strain
BEST195 and Institute of Scientific and
Technological Research (TISTR), Thailand for
providing B. subtilis TISTR008.
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Table 1 (continued)
Component
Linear
retention
index
% Relative peak area (meanSD)
Dry tea TN51 ASA BEST195 S1-13 TISTR008
Calacorene
-Ionene
-Longipinene
-Copaene
3Z-Hexenyl hexanoate
-Panasinsene
Z-Jasmone
-Gurjunene
2-epi--Funebrene
-Cedrene
Neryl acetone
-Elemene
Z-Jasmonyl acetate
9-epi-E-Caryophyllene
-Muurolene
Germacrene D
E--Ionone
-Muurolene
Germacrene A
Cubebol
trans-Calamenene
1342
1348
1352
1370
1380
1382
1392
1409
1412
1420
1436
1438
1455
1466
1479
1485
1489
1500
1509
1515
1521
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0.030.09
0.110.22
0.030.15
0.060.07
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0.070.09
0.050.05
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0.020.00
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0.430.05
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0.150.06
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1.530.17
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0.210.05
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0.720.16
0.220.11
0.250.09
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0.070.02
0.240.04
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0.080.04
0.190.07
1.420.09
0.150.08
0.130.06
0.140.05
0.120.08
0.080.08
0.090.04
0.230.05
0.120.04
0.470.09
0.270.11
0.310.13
0.170.09
0.390.08
0.090.08
0.100.07
0.080.07
0.430.09
0.240.07
0.150.02
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0.20 0.07
0.12 0.08
0.17 0.11
0.41 0.09
0.21 0.07
0.88 0.05
0.37 0.08
0.29 0.09
0.18 0.07
0.71 0.04
0.150.02
0.120.04
0.030.04
0.140.02
0.110.02
0.150.04
0.60 0.03
0.60 0.05
0.07 0.03
0.07 0.02
0.07 0.03
0.05 0.02
0.05 0.04
0.02 0.03
0.03 0.02
0.29 0.04
0.05 0.02
0.07 0.02
0.14 0.03
0.03 0.04
0.18 0.05
0.030.02
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0.010.02
0.080.02
0.070.02
0.020.03
0.10 0.04
0.52 0.05
0.04 0.03
0.06 0.02
0.06 0.03
0.02 0.02
0.05 0.02
0.01 0.02
0.05 0.03
0.02 0.02
0.28 0.08
0.06 0.02
0.13 0.03
0.02 0.03
0.16 0.04
Chiang Mai J. Sci. 2014; 41(2) 401
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