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The effect of green tea extract on the removal of sulfur-containing oral malodor volatiles in

vitro and its potential application in chewing gum

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2010 J. Breath Res. 4 036005

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IOP PUBLISHING JOURNAL OF BREATH RESEARCH
J. Breath Res. 4 (2010) 036005 (8pp) doi:10.1088/1752-7155/4/3/036005

The effect of green tea extract on the

removal of sulfur-containing oral malodor
volatiles in vitro and its potential
application in chewing gum
Q C Zeng, A Z Wu and J Pika
Firmenich Inc., PO Box 5880, Princeton, NJ 08543, USA
E-mail: jana.pika@firmenich.com

Received 8 March 2010

Accepted for publication 6 August 2010
Published 27 August 2010
Online at stacks.iop.org/JBR/4/036005

Abstract
Increasing pH solution from 7.5 to 8.0 was found to significantly improve the effectiveness of
green tea extract for methanethiol removal in vitro. Green tea extract was also found to remove
hydrogen sulfide and its effectiveness was greatly improved under alkaline conditions. It was
found that with green tea extract, maximum H2 S removal was achieved when the pH was
between 8.1 and 8.4 at 37 ◦ C for 5 min. Further increases in pH resulted in decrease of the
extract effectiveness. Vegetable acetone powders which contain polyphenol oxidases or
peroxidases were found to further enhance the effectiveness for the removal of thiols when
used in combination with green tea extracts at body temperature under alkaline conditions.
Adding 5% baking soda to green tea extract-containing chewing gum was found to buffer
saliva pHs to 8.0 during 10 min of chewing. However, severe discoloration was observed and
undesirable bitterness was perceived, most likely due to the polymerization of unencapsulated
green tea polyphenols. Therefore, encapsulation of green tea extract is recommended for
applications at elevated pHs.

Abbreviations Tea polyphenols have also been found to have inhibitory effects
on the growth of halitosis-causing bacteria [7].
AP acetone powder It was suggested that the deodorizing mechanism of tea
EGCG (–)-epigallocatechin gallate catechin (–)-epigallocatechin gallate (EGCG) was due to the
GTE green tea extract addition of a thiol group to ortho-quinone, generated from
PG propylene glycol the oxidation of EGCG by atmospheric oxygen [2]. The
PPO polyphenol oxidase formation of quinones was shown to be critical for the reaction
POD peroxidase between phenolic compounds with CH3 SH and the presence of
VSC volatile sulfur compound. oxygen was required. The oxidation reaction proceeded more
rapidly in a slightly alkaline (pH 7.5) than in a neutral solution.
Introduction Acidic conditions suppressed the oxidation of polyphenols to
the corresponding quinones, but the oxidation can still occur
Volatile sulfur compounds (VSCs), particularly hydrogen in the presence of polyphenol oxidases (PPOs) or peroxidases
sulfide and methanethiol (CH3 SH), are considered to be the (PODs) [2, 5, 6].
predominant gases causing human oral malodor or halitosis Applications of tea polyphenols in different oral care
[1]. It has been reported in the literature that green tea products for bad breath control have been reported [8–11].
catechins and other natural products exhibited deodorizing However, the effectiveness of green tea extract (GTE) on
effects against methanethiol and allium-specific VSCs [2–6]. thiol removal in oral care products relies on whether the

1752-7155/10/036005+08$30.00 1 © 2010 IOP Publishing Ltd Printed in the UK

J. Breath Res. 4 (2010) 036005
conditions in the oral cavity during consumption favor the
formation of the quinones, and this was not discussed in
these patent applications. The pH of individual human saliva
varies from 5.5 to 7.7 [12], and a study conducted by Stösser
showed that during the normal chewing of sugar-free gum
containing Firmenich Flexarome R
, the saliva pH was between
6.3 to 7.3, while the saliva pH could drop below 5 after the
mouth was rinsed with 10% sucrose solution [13]. For those
individuals with acidic saliva, or upon the use of products
containing sugars, the effect of GTE alone on thiol-containing
compounds in the mouth would probably be minimal, if any.
Previous studies have shown that the deodorizing reaction
proceeded faster at pH 7.5 than under neutral conditions and
the reaction was completely suppressed at pHs lower than
5 [2]. Therefore, in order to promote a reaction between
CH3 SH and polyphenols, it is necessary to adjust saliva pH to
values that are above neutral. However whether pH 7.5 is the
optimum condition for this effect has not been reported. On the
other hand, adding PPOs or PODs to the GTE containing oral
care products under the optimum pH conditions may further
improve their effectiveness. Hydrogen sulfide is also a thiol,
but whether it could be removed by green tea polyphenols in
a similar way has not been extensively studied [11]. We have
observed significant increases in the thiol removal capacity of
GTE at different alkaline pHs especially when PPO or POD
containing acetone powders (APs) were used [14]. In this
work, we studied the extent of the reaction between GTEs
and thiols including CH3 SH and H2 S at pHs greater than 7.5,
without and with the use of enzymes.
Our plan in this study was to develop an in vitro analytical
method for the measurement of methanethiol and hydrogen
sulfide concentrations in solutions containing GTEs with and
without APs. Additionally we checked the in vitro samples
for the oxidation of methanethiol to yield the odorant product,
dimethyl disulfide.
We used our analytical method to determine the optimum
solution pH for reaction of GTE with methanethiol and
hydrogen sulfide at physiological body temperature. At the
optimum pH we determined the optimum GTE concentrations
for reaction with the thiols of interest in the presence
and absence of PPO and POD containing vegetable APs.
We further studied the effect of preoxidizing the GTE to
determine whether this might affect the extent of reaction with
thiols.
Finally we studied whether adding a base, sodium
bicarbonate, to chewing gum, in vivo, would increase saliva
pH to a value at which reaction of GTE with thiols would be
expected to proceed in an efficient manner, based on the results
of our in vitro experiments.
Materials and methods
In vitro experiments
All of the sample vials were preheated in a water bath at 37 ◦ C
before aliquots of the stock CH3 SH or H2 S standard solutions
were introduced. For CH3 SH studies, the control vials were
flushed with nitrogen to remove oxygen before capping so that
2
Q C Zeng et al
the oxidation of CH3 SH to CH3 SSCH3 was minimized. The
vials containing GTEs were either flushed or not flushed with
nitrogen as noted in the text since oxygen had been reported
to be necessary for the reaction between tea polyphenols and
CH3 SH [2]. For the H2 S studies, none of the vials were
flushed with nitrogen. A small portion of the methanethiol
stock solution (30 μL) or a known volume (0.05 to 2 mL)
of hydrogen sulfide standard was introduced using a syringe.
For hydrogen sulfide, as the injected volume was relatively
large, an appropriate amount of air was removed from the
vials using a gastight syringe before the same volume of
5.00% H2 S was introduced into the vials. Equilibration times
of 5 and 10 min were chosen for CH3 SH and H2 S controls,
respectively, as the latter material was found to take longer
to reach equilibrium between the headspace and the solution
in the vial. Since the pKa1 for hydrogen sulfide is around
6.9, as the pH of our samples increased, more H2 S would
be expected to dissolve in the buffer and its concentration
in the headspace would decrease accordingly. In order to
determine if the removal of H2 S from the headspace was due
to the reaction with GTE rather than due to dissolution in the
buffer, acidification of the solution to a pH lower than 6 was
performed to ensure the partitioning of dissolved H2 S into
the headspace before sampling and injecting into the GC-MS.
Consequently, a small volume of the 0.5 N HCl solution (1.2–
2.0 mL) was injected into the vial to adjust the pH to around
6 before 1 mL of headspace was extracted and injected into
GC-MS.
Chemicals
99.5% methanethiol and 0.5 N hydrochloric acid solutions
were purchased from the Sigma-Aldrich Company (USA).
Na2 HPO4 · 7H2 O and NaH2 PO4 · H2 O were purchased from
the Sigma-Aldrich Company (USA). 5.00% Hydrogen sulfide
was purchased from Matheson Tri-Gas Inc. (NJ, USA).
GTEs Spec TP-80 (Wuxi Green Power Bio-Product Co., Wuxi,
China) used in this study had total catechin contents of 52.2%.
Glass vials
Glass vials (20 mL, metal seal LO, 20 mm with Tan
PTFE/White Sil) were purchased from MicroLiter Analytical
Supplies Inc. (GA, USA).
Preparation of methanethiol stock standards
The Sigma-Aldrich methanethiol came in a gas cylinder.
A portion of the gas was condensed into a glass ampoule
using dry ice in an acetone bath. A small portion of liquid
methanethiol (about 19.5 mg) was transferred using a cooled
syringe into a cooled (in dry ice) and pre-weighed capillary
glass tube with one end flame sealed. Then the open end
was also flame sealed. The amount of methanethiol in the
capillary tube was calculated from the weight of the tube. A
100 mL volumetric flask was filled with approximately 80 mL
of degassed 50% aqueous propylene glycol (PG) solution
(w/w). The solution was placed in a freezer until the
temperature dropped to slightly above 4 ◦ C. The capillary
J. Breath Res. 4 (2010) 036005
tube containing methanethiol was dropped into the flask, and
crushed to release methanethiol into the solution using a glass
rod. The glass rod was rinsed with some fresh 50% aqueous
PG solution and the flask was capped. When the solution
reached room temperature, the flask was filled to volume with
the degassed 50% aqueous PG solution. The solution was
mixed well, transferred to small GC vials in 1 mL portions
and stored in the freezer for later use. A small portion of
methanethiol stock solution (30 μL) was introduced to sample
vials using a syringe by piercing through the septum, and
the final concentration of methanethiol in the sample was
about 325 ppb. The vials of the stock standard solution were
discarded after sampling five to six times using a syringe.
Preparation of hydrogen sulfide stock standard
Hydrogen sulfide in a cylinder (5.00%) was loaded into an
empty PVF Tedlar R
bag with a nickel-plated brass on/off
valve (0.3 L, Chemware R
). A small length of TygonR
tubing
with a septum was attached to the valve and a known volume of
hydrogen sulfide was drawn from the septum using a gastight
syringe and introduced into sample vials by piercing through
the septum.
Preparation of acetone powder
AP was prepared using the procedure described by Negishi
et al [5]. Raw white mushrooms or eggplant (100 g each),
purchased from a local supermarket, were homogenized with
400 mL of cold acetone (−10 ◦ C) in a Waring blender. The
homogenates were filtered using a coffee filter paper, and
the vegetable residues that remained on the filter paper were
further washed by homogenizing with the cold aqueous 80%
acetone solution to remove aqueous acetone soluble substrates.
After filtration, the residual acetone in the vegetable residues
was removed using rotary evaporation. The vegetable residue
was lyophilized, ground using a coffee grinder and stored in a
−20 ◦ C freezer.
Preparation of phosphate buffer solutions
Solutions of 0.1 M phosphate buffer at different pHs were
prepared by weighing calculated amounts of Na2 HPO4 · 7H2 O
and NaH2 PO4 · 7H2 O into 100 mL volumetric flasks and filling
to the mark with deionized water. The 0.1 M NaOH solution
was used to adjust the pH when necessary. Phosphate solutions
or phosphate buffer must be prepared fresh and stored in a
refrigerator if short-term storage is necessary.
Control samples
The degassed phosphate buffer solution (10 g) was weighed
into a 20 mL glass vial (MicroLiter Analytical Supplier, Inc.,
metal seal LO, 20 mm with Tan PTFE/White Sil). The
headspace was flushed with nitrogen before sealing with a
crimp cap possessing a silicon septum with PTFE liner. A
small portion of methanethiol stock solution (30 μL) or a
known volume (0.05 to 2 mL) of hydrogen sulfide standard
was introduced using a syringe.
3
Q C Zeng et al
Samples with green tea extract
A small amount of GTE was weighed into a 20 mL glass vial
and a degassed phosphate buffer solution was added to 10 g.
The vial was sealed with a crimp top. A small portion of the
methanethiol stock solution (30 μL) or a known volume of
hydrogen sulfide standard was introduced using a syringe.
Samples with green tea extract and acetone powder
A small amount of GTE was weighed into a 20 mL glass vial
which contained 5 to 20 mg of AP and the degassed phosphate
buffer solution was added to 10 g. The vial was sealed with a
crimp top. A small portion of the methanethiol stock solution
(30 μL) or a known volume of hydrogen sulfide standard was
introduced using a syringe.
Pre-oxidized green tea extract
Air was slowly bubbled through the tea extract in the buffer
solution (1 mg mL−1 , pH 7.4) for 3 h and then the solutions
were stored in capped bottles on the counter for up to 6 days.
Headspace GC-MS-SIM analysis
The thiol removal capacity of each sample was assessed
by a headspace-GC-MS for CH3 SH, CH3 SSCH3 and H2 S.
An Agilent GC-5973 was used in Selected Ion Mode (m/z
45, 47 and 48 for CH3 SH, m/z 79 and 94 for CH3 SSCH3,
and m/z 33 and 34 for H2 S). An SPB-1 column (30 m ×
0.25 mm × 0.25 μm) was used for CH3 SH and CH3 SSCH3 .
The oven temperature was held at 32 ◦ C for 2.28 min, and
then increased to 100 ◦ C at 25 ◦ C min−1 . The runtime was
5 min. The inlet split ratio was set at 20:1 and a solvent delay
of 1.55 min was used to avoid introducing excess air into
the mass spectrometer. For H2 S, a SOLGEL-WAX column
(30 m × 0.25 mm × 0.25 μm) was used as it was found that
the degree of separation of the air peak from H2 S was better
on a polar column. The air peak was used in this study as a
reference to check the repeatability of the auto sampler. For
studying the effect of GTE, an amount of H2 S within the linear
range was used. The oven temperature was held at 33 ◦ C for
4.75 min, and then increased to 150 ◦ C at 30 ◦ C min−1 . The
runtime was 8.65 min. The inlet split ratio was set at 50:1
and a solvent delay of 1.45 min was used to avoid introducing
excess air into the mass spectrometer.
A 2.5 mL headspace syringe was used for the Leap
CombiPAL system. The syringe holder module was
maintained at a temperature of 45 ◦ C. The fill speed of the
syringe was set at 100 μL s−1 with one fill stroke. The injection
speed was set at 1000 μL s−1 and the injection volume was
1 mL. The sample solutions were stirred using an agitator speed
of 500 rpm with incubation times of 5 min at set temperatures.
The procedure that included sampling H2 S from the Tedlar R
bag using a gastight syringe, then injecting into glass vials with
crimp caps, and finally injecting the headspace into GC-MS
using a Leap CombiPAL system gave very good repeatability
as the relative standard deviation was less than 3% from six
replicate sample vials. The relative standard deviation for
J. Breath Res. 4 (2010) 036005 Q C Zeng et al

CH3 SH analysis from six replicate sample vials was less than
(a)
5%. The thiol removal capacity of the samples were shown by 120
the % remaining thiols, which was calculated as S/C × 100,

% CH3SH Remaining
where S represented the peak area of the remaining thiol in the
treated samples, and C represented the peak area of the thiol Control
80
in the control. GTE

40
Typical preparation of chewing gum containing baking soda
and green tea extract
0
Commercial chewing gum was purchased from a local CVS
0 2 4 6 8 10
store. Sodium bicarbonate was purchased from Westro time (min)
(b)
Chemical Inc. The chewing gums used in this study are 220
representative of those available on the market and were Control

prepared in accordance with industry guidelines and under
GMP conditions. The formula of the gum was as follows:
Mallorca R
gum (70.5 to 80.5%), sorbitol (5.6%), Syloid
R
244
(10.2%), magnesium stearate (2.0%), GTE (1.7%) and baking
soda (0, 2, 5 and 10%). Mallorca R
gum was from Cafosa,

R
Spain, and Syloid 244 was from WR Grace Company. All
materials were of food grade quality and satisfied ingredient
specifications. The sorbitol and SyloidR
244 were premixed.
The gum was sheared using a sigma-blade mixer until a
temperature of 50–55 ◦ C was reached. The premixed powder
was added and mixed with the gum base raw material until a
homogeneous system was obtained in the form of a stringy
paste, after approximately 5 min. The agglomerated mix
was removed from the sigma-blade mixer and left to cool to
room temperature. The coarse particles were then milled in a
hammer mill and the resulting powder was sieved through
a 1 mm sieve. The powder was then dry blended with
the remaining ingredients. The blend thus obtained was
compressed by means of a manual press (Specac R
machine)
under a compression force of between 1 and 4 tons. Tablets of
20 mm diameter were obtained.
Panelists
Ten panelists were recruited from the R&D group of Firmenich
Inc., Princeton, USA, following internal selection practices.
Panelists were not paid for their participation and were fully
informed of the nature of the experiment and the materials
being tested. The tasting protocol employed was approved by
Firmenich R&D management and complied with both internal
and external industry guidelines for the sensory analysis of
foods.
% Dimethyl disulfide
GTE
180
140
100
0 2 4 6 8 10
time (min)
Figure 1. (a) Effect of GTE on methanethiol (CH3 SH) removal in
pH 7.5 buffer at 37 ◦ C. (b) Relative amount of dimethyl disulfide in
vials containing CH3 SH with buffer only and buffer with GTE.
Control: pH 7.5 phosphate buffer; GTE: 1 mg mL−1 GTE in pH 7.5
phosphate buffer.
Statistical analysis
All experiments were conducted with three or two replicates
for each treatment. The resulting values were averaged and
standard error bars are shown in the figures. The data were
subjected to two-way analysis of variance without interaction,
with the dependent variable the percentage remaining of
CH3 SH or H2 S and the independent variable factors the green
tea and/or AP concentrations and the number of replications.
Tukey’s HSD was used to assess the significance of the
mean separation of the different treatments. An alpha level
of 0.05 was used to determine significance. The analyses
were conducted using the JMP software (version 5.1.1, SAS
Institute Inc., Cary, NC). Saliva pHs were analyzed using one-
tailed one sample Student’s t test with an alpha level of 0.01.
Results

Methanethiol removal capacity of green tea extracts

Measurement of saliva pHs
Ten panelists were asked to chew either a commercially or in-
house produced chewing gum for periods of 10 min. Panelists
were instructed not to swallow their saliva during the testing
period and saliva was collected in small plastic cups at 0, 1, 3,
5, 7 and 10 min intervals. The pHs of the collected saliva were
measured using a pH meter (Thermo Electron Corporation,
Orion 3 STAR) with a microelectrode (Orion 9802 BN).
During the course of our in vitro experiments to measure the
effect of GTE on CH3 SH concentrations, the residual CH3 SH
in the control samples, which did not contain the extract,
decreased slightly over the test period of 10 min (figure 1(a)).
When GTE was present, the residual CH3 SH declined with
time and, significantly, this decline was not accompanied by
an increase in the concentration of CH3 SSCH3 , an oxidation
product of CH3 SH (figure 1(b)). The concentrations of

4
J. Breath Res. 4 (2010) 036005
2.E+06
Signal Intensity
1.E+06
5.E+05
0.E+00
pH 5.78 pH 5.72
(Control)
Figure 2. The effect of pH on the signal intensities of methanethiol and dimethyl disulfide in the presence of GTE for 5 min at 37 ◦ C. The
vertical bars indicate the standard errors.
120
% CH3SH Remaining
90
60
30
0
Control
Figure 3. The effect of GTE concentration on the removal of methanethiol at pH 8.0 for 5 min at 37 ◦ C. The vertical bars indicate the
standard errors.
CH3 SSCH3 were observed to increase in the control samples,
however.
Figure 2 shows the effect of GTE on methanethiol removal
at different pHs after incubation of the samples at 37 ◦ C for
5 min. There was no effect under acidic conditions (pH =
5.72). The remaining methanethiol decreased significantly
with an increase in pH (p < 0.05), and greater than 90% of
methanethiol removal was achieved within 5 min when the
solution pHs were 8.0 or higher. Figure 2 also showed that
in the presence of GTE and after incubation at 37 ◦ C for
5 min, the level of dimethyl disulfide remained about the same
over the range of pHs tested, while the level of methanethiol
decreased.
To determine the amount of GTE needed for maximum
methanethiol removal at pH 8.0, samples containing different
amounts of GTE were evaluated and the results were shown
in figure 3. Further increasing the concentration of GTE
concentration to greater than 0.5 mg mL−1 did not provide
additional benefits as no significant differences in the %
remaining methanethiol concentrations were observed (p <
0.05). Similar results were obtained at pH 7.5 in terms of the
optimum GTE concentration (data not shown).
Figure 4 showed the effect of AP at different
concentrations in pH 7.4 solutions containing GTE. In
solutions without AP (bars labeled AP(0)), greater than 50%
Q C Zeng et al
Methanethiol
Dimethyl disulfide
pH 7.31 pH 7.50 pH 7.70 pH 8.02 pH 8.18
0.1 0.5 1.0 1.5
Green tea extract (mg/mL)
methanethiol remained even when GTE was present. With
an increase in the amount of AP from 5 to 10 and 15 mg,
the remaining methanethiol concentrations decreased to 20%,
11% and 5%, respectively. This trend leveled off and a
further increase in the amount of AP to 20 mg did not show
additional effect (p < 0.05). At each AP concentration, there
was no significant difference in the effectiveness of GTE at
concentrations of 0.1, 0.5 and 1.0 mg mL−1 (p < 0.05).
Figure 5 showed the effect of pre-oxidized GTE on the
removal of methanethiol. The data showed that pre-oxidized
GTE alone had almost no effect in reducing methanethiol
concentrations; however, in the presence of a small amount
of mushroom AP, it was significantly more effective than
the same concentration of freshly prepared GTE without AP
(p < 0.05). The pre-oxidized solutions after storage for 1 or 4
days significantly reduced the concentrations of methanethiol
in vitro when AP was used, and were still very effective with
the use of AP after 6 days.
Hydrogen sulfide removal capacity
Figure 6 showed the effect of GTE on hydrogen sulfide
concentrations when solutions at different pHs were incubated
at 37 ◦ C for 5 min. When only GTE was present, no effect
5
J. Breath Res. 4 (2010) 036005 Q C Zeng et al

120
A
100
B

%CH3SH Remaining
80
C C C
C
60
D D
40
E E,F E,F
20 E,F,G
G G G G G G
0 0

0
0.25

0.25

0.25

0.25
0.1

1.0

0.1
0.5
1.0

0.1

0.5
1.2

0.1

0.1

0.6
1.0
AP (0) AP (5) AP (10) AP (15) AP (20)
Green Tea Extract (mg/mL)
Figure 4. The effect of GTE and AP on methanethiol reduction at pH 7.4. The value in parenthesis after AP is the mg of AP in 10 mL
solution. Levels not connected by same letter are significantly different at 95% confidence level.

120
% CH3 SH Remaining

90

60

30

0
Control GTE GTE GTE GTE AP GTE GTE GTE GTE
(1d) (4d) (6d) (fresh) (fresh) (1d) (4d) (6d)
+ AP + AP + AP + AP

Figure 5. The effect of pre-oxidized GTE on methanethiol removal at 37 ◦ C for 5 min without or with the presence of AP in pH 7.4 buffer.
d is the number of days stored after pre-oxidation. The vertical bars indicate the standard errors.

120

A A GTE
100
A GTE + AP
% H2S Remaining

80 B
C C
60 C
C
D,E D
40 E,F E,F
G
D H,I
20 H,I H,I H,I,J H,I,J G,H,I J I,J
J,K
0
5.80 6.37 7.20 7.54 7.83 8.01 8.10 8.22 8.30 8.41 8.70 10.0 11.0
pH
Figure 6. The effect of pH on H2 S removal in the presence of GTE and AP (20 mg). Levels not connected by same letter are significantly
different at 95% confidence level.

was observed under acidic conditions. Residual H2 S decreased undetectable at pH 8.1 to 8.4. Again, further increases in
significantly from 100% to below 20% when pHs increased pH beyond 8.4 resulted in an increase in residual H2 S but the
from 5.8 to 8.1, and remained at below 20% from pH 8.1 to remaining H2 S was below 20%, which was lower than the
8.4 (p < 0.05). However, further increases in pH beyond 8.4 samples without AP. In the presence of GTE, H2 S removal of
resulted in an increase in residual H2 S. When AP (20 mg) about 85% was achieved when the pH was between 8.1 and
was added to the GTE solutions, residual H2 S decreased from 8.3. With further addition of AP, 100% removal of H2 S was
70% to 0% with pH increase from 5.8 to 8.1, and remained achieved between pH 8.0 and 8.4.

6
J. Breath Res. 4 (2010) 036005 Q C Zeng et al

Table 1. Saliva pHs measured while chewing a commercially confirmed that under acidic conditions, green tea polyphenols
available sugar-free mint-flavored chewing sum. did not react with methanethiol [2].
Saliva pH (8 panelists) The slight decline in the signal intensity of CH3 SH in the
Min 1 2 3 4 5 6 7 8
control solutions may be due to the oxidation by the residual
oxygen in the vials as the level of CH3 SSCH3 was found to
0 7.1 6.5 7.4 6.5 6.5 6.9 6.9 7.0 increase slightly (figure 1(b)). This also suggested that purging
1 7.7 8.5 8.5 8.3 8 8.7 8.2 7.9 the headspace with nitrogen did not completely remove the
3 7.9 8.5 8.5 8.5 8.5 8.5 8.4 8.3 oxygen in the vials. When GTE was present in solutions at
5 7.9 8.1 8.3 8.1 7.4 8.3 8.1 7.9
10 7.7 8.1 8.1 7.9 7.7 8.0 7.9 7.4
pH 7.5, whether or not samples were flushed with nitrogen,
the level of CH3 SH decreased significantly, but this decrease
was not accompanied by a correspondingly large increase in
Table 2. Saliva pHs measured while chewing gum which contained the concentration of CH3 SSCH3 (figure 1(b)). Therefore, it
5% baking soda for 10 min.
is assumed that the decrease of CH3 SH was due to reaction
Saliva pH at different chewing times with tea polyphenols, but not to reaction with oxygen to form
(min) CH3 SSCH3 . On the other hand, in the presence of GTE,
Panelist 0 0 to 1 1 to 3 3 to 5 5 to 10 even though these samples were not flushed with nitrogen,
much lower levels of CH3 SSCH3 were detected than in the
1 6.95 8.11 8.17 8.47 8.30 control samples which contained only buffer. These results
2 6.93 8.03 8.09 8.21 7.87
3 7.05 8.11 8.16 8.37 8.28 suggested that GTE acted as an antioxidant; specifically,
4 6.94 8.11 8.14 8.36 8.35 the reaction of CH3 SH with the tea polyphenols was faster
5 7.16 7.97 8.03 7.97 7.88 than with oxygen. This was further confirmed where the
6 7.13 8.22 8.15 8.37 8.40 level of dimethyl disulfide remained about the same over
7 7.45 7.91 8.09 8.31 8.12 the range of pHs tested when GTE was present, while the
8 6.89 7.97 8.12 8.22 8.19
9 6.25 7.73 8.10 8.46 8.47
level of residual methanethiol decreased (figure 2). This
10 7.74 7.98 8.07 8.06 8.16 again suggested that the disappearance of methanethiol was
not due to oxidation to form dimethyl disulfide, but was the
Average 7.05 8.01 8.11 8.28 8.20
result of a reaction with components of the GTE. We noticed
that without GTE, methanethiol was reduced about 25% in
Amount of baking soda needed in chewing gum applications pH 8.18 buffer but was converted to another odorant, dimethyl
disulfide. Therefore, a true deodorization at high pH without
A commercial chewing gum containing baking soda was found GTE did not occur. It seemed that higher pHs promoted the
to buffer the saliva of eight panelists to pHs between 7.4 and oxidation of methanethiol when there were no polyphenols
8.5 within 10 min of chewing (table 1). present.
The saliva pH of ten panelists before chewing house-made The effect of thiol removal increased with the increase
chewing gums containing baking soda and GTE ranged from of GTE concentration, but it leveled off at concentrations
6.72 to 7.74 (table 2). When there was no baking soda in the greater than 0.5 mg mL−1 (figure 3). Further reductions
chewing gum, none of the panelists’ saliva pH reached 8.0. in thiol concentrations could be achieved with the use of
When there was 2% baking soda in the chewing gum, only AP. Pre-oxidized GTE alone was found to have no effect in
four out of the ten panelists’ average saliva pH during 10 min reducing methanethiol; however, in the presence of a small
reached 8.0. When 5% baking soda was added to the chewing amount of mushroom AP, it was more effective than the same
gum, the average saliva pH from ten panelists was 8.16 during concentration of freshly prepared tea extract with mushroom
10 min of chewing, which was significantly greater than 8.0 AP. The pre-oxidized solution was still very effective after
(p < 0.01). 6 days with AP, which suggested that the pre-oxidized products
might be stable at pH 7.4. These results suggested that
the pre-oxidation of GTE by air generated compounds that
Discussion were not as reactive as polyphenols, but better substrates
for enzymes to produce more reactive chemicals to capture
Effectiveness of green tea extract against thiols methanethiol. Whether the oxidation of polyphenols stopped
upon formation of quinones or whether these underwent
The disappearance of methanethiol observed in the course of
further polymerization was not clear. Studying these oxidation
our in vitro experiments may have been due to the reaction products may lead to the discovery of more reactive chemicals
with GTE, or due to the formation of another odorant, to capture methanethiol. It is likely that tea extracts from
dimethyl disulfide, resulting from the oxidation reaction of fermented teas such as oolong and black teas may be more
methanethiol. Therefore, simply measuring methanethiol effective than or as effective as GTE in the presence of AP.
without monitoring dimethyl disulfide was inadequate
[4–6]. When samples were not buffered, the pH of the GTE
Hydrogen sulfide removal capacity
solution was about 6.5 and no significant decrease in the
concentrations of methanethiol and concomitant deodorizing Similar to our observations for solutions which contained
effect were observed even when GTE was present. This methanethiol, H2 S removal by GTE increased with pH

7
J. Breath Res. 4 (2010) 036005
increase and complete removal could be achieved when AP
was added (figure 6). In our in vitro experiments, increases
in pH beyond 8.4 resulted in a loss of effectiveness and the
mechanism was not clear.
Baking soda in chewing gum applications
The pH of individual human saliva varies from 5.5 to 7.7 [12].
Our study showed that a saliva pH of 8.0 would be desirable for
GTE to react with thiols and adding baking soda to chewing
gum is a practical way to achieve this goal. The saliva pH
was greater than 8.0 within 10 min of chewing 5% baking
soda containing chewing gum. Typically gum chewing lasts
for less than 5 min; therefore, periods of chewing longer than
10 min were not evaluated. As 5% baking soda was basic
enough to buffer the saliva pH to the target 8.0, gum samples
containing 10% baking soda were not evaluated.
We have noticed that the GTE containing chewing gums
turned brown during storage. This was probably due to
polymerization of green tea polyphenols at alkaline pHs. The
higher the load of baking soda, the darker the gums became.
The dark chewing gums also gave an undesirable bitter taste.
To avoid the discoloration, it is important to keep the green
tea polyphenols from contacting the baking soda. Therefore,
encapsulation of GTEs would be necessary for this type of
application [14].
Conclusions
In in vitro experiments, increasing the pH of a buffer solution
from 7.5 to 8.0 resulted in significant improvements in the
removal of methanethiol by GTE. GTE was also found to
remove hydrogen sulfide with the optimum effectiveness
observed at pH 8.1 in our assay. The addition of mushroom
AP, which contained PPOs or PODs, was found to further
enhance the effectiveness of GTEs for the removal of thiols
at body temperature under alkaline conditions. In a proof-
of-concept-type experiment we demonstrated that adding 5%
baking soda to GTE containing chewing gum resulted in
buffering of the saliva pH to 8.0 during 10 min of chewing.
Our experiments demonstrated that the use of GTEs in chewing
gum which also contained baking soda resulted in unappetizing
darkening of the gum. Consequently, we recommend the use
of encapsulated GTEs for baking soda-containing gum. In
order to develop a product such as a chewing gum which
contains baking soda and GTEs for removal of thiols from the
saliva, in vivo experiments should be conducted to determine
Q C Zeng et al
if the reaction proceeds in the mouth in the same manner as
we observed in our in vitro experiments to give a perceptible
and beneficial effect.
Acknowledgments
We thank Ken Dougherty for valuable discussion. We
appreciate the contributions of Marc Meyers, who prepared
the chewing gum samples, and Bill Adams, who provided
instrument support for the analyses.
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