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ORIGINAL ARTICLE
Tetsuya Takahashi · Yuji Aso · Wakako Kasai
Tetsuo Kondo
Synergetic deodorant effect and antibacterial activity of composite paper
containing waste tea leaves
Abstract Composite paper containing waste tea leaves was
prepared to investigate the effective utilization of waste tea
leaves as deodorant material. Paper containing waste green
tea leaves did not have increased deodorizing ability com-
pared with controls either against acidic odors such as
hydrogen sulfi de and acetic acid gases or against neutral
odors such as formaldehyde and acetaldehyde gases. In con-
trast, the paper had excellent deodorizing ability against
basic odors such as ammonia and trimethylamine gases. It
was observed during additional tests conducted on paper
samples containing 60 wt% waste leaves of oolong tea,
black tea, pu-erh tea, or hojicha, that all the samples reduced
the ammonia concentration to below 1 ppm, which is the
threshold concentration for olfactory recognition, within
30 min. Further, paper containing waste green tea leaves
was found to decrease the odor residual rate to 5.1% in
30 min even for a waste tea leaf content of 10 wt%. The
excellent deodorizing ability of the paper could be attrib-
uted to the chemical reactions between odorous substances
and the catechins found in tea leaves. After the deodoriza-
tion of ammonia, paper containing waste green tea leaves
was found to have increased antibacterial activity against
Staphylococcus aureus.
Key words Waste tea leaves · Composite paper · Deodorant
effect · Ammonia gas
T. Takahashi (*)
Faculty of Education, Shimane University, 1060 Nishikawatsu-cho,
Matsue, Shimane 690-8504, Japan
Tel. +81-852-32-6350; Fax +81-852-32-6350
e-mail: ttetsuya@edu.shimane-u.ac.jp
Y. Aso
Department of Biobased Materials Science, Kyoto Institute of
Technology, Kyoto 606-8585, Japan
W. Kasai · T. Kondo
Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Introduction
The present authors have examined composite paper con-
taining waste tea leaves with the objective of effectively
utilizing industrial drink wastes.1–5 Composite paper con-
taining waste leaves of green tea, oolong tea, black tea,
hojicha, and pu-erh tea were found to possess antibacterial
activities.1,3 The deodorizing action of waste tea leaves is
well known, and waste tea leaves are used in several appli-
cations in everyday life. For example, waste tea leaves used
to be strewn over fl oors before sweeping or to remove the
smells of raw fi sh and meat after cooking. The deodorizing
action of tea leaves has attracted much attention for its
application in environmental hygiene.6 Thus, the number of
applications of composite paper containing waste tea leaves,
which possess a deodorizing ability in addition to antibacte-
rial activity, is expected to rise. In this study, the authors
examined the deodorant effect of composite paper contain-
ing waste green tea leaves against a variety of odorous
substances. The authors also examined the synergetic effect
on antibacterial activity of composite paper containing
waste tea leaves after the deodorization of odorous sub-
stances, as previously reported.
Materials and methods
Preparation of waste tea leaves
The study involved the following fi ve kinds of tea leaves:
green tea (sencha), oolong tea, black tea, pu-erh tea, and
hojicha. These tea leaves are fermented to different degrees
by different methods. Green tea leaves were obtained from
Ito En, oolong tea leaves were from Nihon-cha Hanbai,
black tea leaves were from Mitsui Norin, hojicha leaves
were from Nihon-cha Hanbai, and pu-erh tea leaves were
from Tenjin Seicha.
To prepare waste tea leaves, a prescribed amount of tea
leaves was added to a specifi c volume of hot distilled
water in an enamel kettle and boiled for 30 min. To 4.0 l of
Received: September 7, 2010 / Accepted: December 21, 2010 / Published online: April 13, 2011
J Wood Sci (2011) 57:308–316 © The Japan Wood Research Society 2011
DOI 10.1007/s10086-010-1171-9
309
distilled water, 400 g of green tea (sencha) leaves was added
and the mixture was heated to 70°C for extraction of tea.
In the case of oolong and black teas, the heating tempera-
ture was set to 90°C for the extraction. For hojicha, 300 g of
tea leaves was added to 3.0 l of distilled water, and the
mixture was heated to 90°C for the extraction. In the case
of pu-erh tea, 250 g of tea leaves in 2.5 l of distilled water
was heated to 90°C for the extraction. A fi ne stainless steel
mesh ball was used to fi lter the liquid.
Preparation of paper containing waste tea leaves
Paper containing waste tea leaves was prepared by the
method reported previously.1 Briefl y, waste tea leaves were
ground under wet conditions with a mass colloider (stone
mill type crusher) with a clearance of 40 μm, before blend-
ing with desired amounts of pulp and latex binder. The leaf
content was adjusted to 60 wt% in the formulation, whereas
the latex binder content was 0.3 wt% of the total amount
of the waste tea leaves and the pulp in all the formulations.
The pulp used was prepared by refi ning Canadian bleached
conifer kraft pulp to a Canadian freeness of 550 ml with a
refi ner. A latex binder (Aica Aibon RAX117 from Aica
Kogyo) made of styrene-butadiene rubber (SBR) was used
to improve the adhesion between pulp and waste tea leaves.
The binder is mainly used for paper processing, which com-
monly involves strengthening and coating of paper. After
distilled water was added to the above mixtures, they were
stirred with a mixer for 10 s to obtain slurries of homoge-
neous dispersion. An angular-sheeting machine (PU-401)
from Tester Sangyo was used to adjust the slurries to a basis
weight of 100 g/m2 and to prepare paper sheets of size 25 ×
25 cm. The sheets were pressed at a pressure of 410 kPa at
room temperature and dried at approximately 120°C with
a rotating drier.
Preparation of odorous gases
A stainless-steel teaspoon was used to transfer four spoon-
fuls of hydrogen sulfi de to a rubber-stoppered test tube. The
test tube was heated on a gas burner to produce hydrogen
sulfi de gas. The hydrogen sulfi de gas was captured in a tube,
and its concentration was adjusted to 20 ± 2 ppm with pure
air. A quantity of 400 ml each of acetic acid, ammonia, tri-
methylamine, acetaldehyde, and formaldehyde was placed
in a separate 500-ml glass bottle at room temperature. A
2.0-ml syringe was used to take an appropriate amount of
saturating vapor from the bottle via suction.
Each of the highly concentrated odorous gases men-
tioned above was injected via a syringe into a separate
Tedlar bag containing 20 l of pure air. Additional pure
air was added to the Tedlar bags to adjust the concentra-
tions of the gases to the following values: acetic acid,
25 ± 0.5 ppm; ammonia, 60 ± 2 ppm; trimethylamine,
60 ± 2 ppm; acetaldehyde, 20 ± 2 ppm; and formaldehyde,
5.0 ± 0.1 ppm.
Evaluation of deodorizing ability
A 1 ± 0.0001 g sample of paper or natural fi ber cloth (cotton,
silk, or wool) was placed in a 5-l Tedlar bag, to which 3.0 l
of an odorous gas, at the concentrations specifi ed in the
previous section, was transferred at a fl ow rate of 500 ml/
min with a measuring pump. The gas concentration in the
bag was measured using a Gastec gas detector tube at
10 min, 30 min, 1 h, 3 h, 6 h, and 24 h from the time of the
gas injection into the Tedlar bag. To evaluate the deodor-
izing effect of a composite paper sample on formaldehyde,
5.0 l of the gas, adjusted to 5 ppm concentration, was
injected to a Tedlar bag containing a 1.7 ± 0.0001 g paper
sample. The initial concentration of the gas was converted
to 100% and a plot of the odor residual rate was mapped
against time. The odor residual rate of the sample was cal-
culated by the equation given below.
Odor residual rate
The measured gas concentration
Initi
%
()
=
aal concentration ×100
Equilibrium regain
Samples of paper and natural fi ber cloth weighing approxi-
mately 5.0 g were dried in a dryer at 70°C for 3 h; the sample
weight was measured in grams, accurate to four decimal
places, before moisture absorption took place. Dried samples
were placed in desiccators set at different levels of relative
humidity and left in the same condition for 3 days. Samples
were then taken out of the desiccators for weight measure-
ment. The equilibrium regain of the samples was calculated
by the equation given below. Different mixing ratios of dis-
tilled water and glycerin were used to adjust the humidity
with the saturating vapor in the desiccators.
Equilibrium regain BA
A
%
()
=−×100
where A is the sample weight before moisture absorption
and B is the sample weight after moisture absorption
pH of aqueous extracts of paper
A piece of paper was fi nely cut into a piece approximately
5 × 5 mm2, corresponding to an absolute dry weight of
1 g. The sample was shaken for 60 min with 25 ml of
distilled water in a 100-ml weighing bottle. It was left stand-
ing for 1 hour at 23°C. The supernatant was used for the
measurement of pH at 23°C with a glass electrode pH
meter.
Tests of antibacterial properties
Staphylococcus aureus (NBRC 12732) used for the antibac-
terial evaluation was obtained from the Incorporated
Administrative Agency, National Institute of Technology
310
and Evaluation. The bacterium is a gram-negative coccus
and is known as a source of purulent diseases as well as a
pathogen of food poisoning. Japanese Industrial Standards
were used as the reference for testing antibacterial proper-
ties. A sample of 0.20 g of composite paper containing waste
tea leaves was transferred into a vial. Vials containing
samples were autoclaved in a BS-245 autoclave from Tomy
Kogyo at 121°C for 15 min. Peptone (1.0 wt%) and yeast
extract (0.5 wt%) from Becton Dickinson and sodium chlo-
ride (0.5 wt%) were used to prepare peptone water of a
prescribed concentration. The peptone water was used to
prepare a Staphylococcus aureus (NBRC 12732) suspension
at a concentration of 1.0 ± 0.3 × 105 colony forming units /
milliliter (CFU/ml). Each autoclaved sample was inoculated
with 0.10 ml of the suspension, tightly sealed, and incubated
at 37° ± 1°C for 18 h.
To each of the incubated vials, 10 ml of a rinsing physi-
ological saline, adjusted to a prescribed concentration with
sodium chloride (0.85 wt%) and Tween 80 (0.20 wt%) from
Sigma Chemicals, was added and the vials were shaken to
disperse the bacterial cells. A physiological saline prepared
with sodium chloride (0.85 wt%) was added to the stock
dispersion of each sample to dilute them to the desired
concentrations of up to 107-fold. A mannitol salt medium
(Ganule) from Nissui, adjusted to 11.1 wt%, was inoculated
with each of the diluted bacterial suspensions. The adopted
inoculation method7 involved dropping 5 μl of the diluted
suspension at fi ve locations in each of the four sections of
the medium, as described previously.1 The Petri dishes were
placed inverted in an incubator at 37° ± 1°C for 44 h. Grown
colonies were counted and multiplied by the dilution ratios
to calculate the numbers of viable cells.
log C = the common logarithm of the viable cell number
Bacteriostatic activity = log N2 − log N3
Bactericidal activity = log N1 − log N3
where N1 is the initial cell number, N2 is the viable cell
number dropped on 100-wt%-pulp paper after 18 h of incu-
bation, and N3 is the viable cell number dropped on paper
samples after 18 h of incubation.
Thin-layer chromatography for examining changes in
epigallocatechin gallate
Epigallocatechin gallate [(−)-EGCg] was dissolved in
diethyl ether and spotted on a thin-layer chromatography
(TLC) aluminum sheet of silica gel 60F254 (Merck, Japan)
for thin-layer chromatography. After confi rming that the
spots were completely dry, the thin-layer chromatography
sheets were slowly placed in a glass container with a solvent
for separation. The solvent used was a mixture of chloro-
form, methanol, and distilled water (59 : 32 : 9).8 The thin-
layer chromatography sheets were dried and then
photographed under an incandescent UV light source
(254 nm). (−)-EGCg (99.8% purity) from Nagara Science
was used.
Mass spectrometry
The molecular mass of (−)-EGCg adsorbed ammonium gas
was determined by using an Agilent LCMSD mass spec-
trometer equipped with an electrospray ionization source
(Agilent Technologies, Type 6320, United States). Before
injection, the (−)-EGCg sample was dissolved in 50% ace-
tonitrite/0.05% trifl uoroacetic acid.
Results and discussion
Deodorant effect of composite paper containing waste
tea leaves
Effect on acidic odors
A variety of smells are recognized in our daily lives, some
of which people fi nd displeasing. The olfactory sense in
human beings is responsible for assessing the magnitude
and nature of different smells. However, various analytical
techniques have been devised for the assessment of smells
to simplify and improve the reproducibility of measure-
ments.9 In this study, a gas detector tube was employed to
measure the concentrations of odorous substances. The
odors were categorized into acidic, basic, and neutral odor
groups according to their chemical properties. First, hydro-
gen sulfi de gas, which has an offensive acidic odor, was used
to test the deodorizing ability of the composite paper con-
taining waste green tea leaves. To obtain a reference, similar
tests were performed with cloths made of natural fi bers,
such as cotton, silk, and wool. The measurements in the case
of the cloths were taken using a Tedlar bag without paper
samples.
Figure 1a indicated that silk and wool had a relatively
good deodorant ability for hydrogen sulfi de gas. The odor
residual rate for silk decreased to 75.6% after 24 h. There
was no clear difference in the deodorizing ability of cotton
and that of paper made from 100 wt% pulp paper. The
composite paper containing 60 wt% waste green tea leaves
had an odor residual rate of 81.9% even at 24 h after gas
injection. This rate was almost the same as that of paper
made from 100 wt% pulp paper. The 85.6% odor residual
rate in the case of the empty bag was not substantially dif-
ferent from that of the composite paper, indicating that the
waste green tea leaves did not have a deodorizing effect on
hydrogen sulfi de gas.
The deodorizing ability of the comparative samples
against acetic acid (a component of a variety of smells) is
shown in Fig. 1b. Compared to their deodorizing ability
against hydrogen sulfi de gas, each sample exhibited an
improved deodorizing ability against acetic acid gas. The
residual odor decreased to 8%–10% in the fi rst hour and
fi nally reached close to 0% after 6 h. There was no signifi -
cant difference between the behavior of paper made from
100 wt% pulp paper and that of the composite paper con-
taining 60 wt% waste green tea leaves, indicating that the
incorporated waste green tea leaves did not have a major
deodorizing effect on acetic acid gas. Acetic acid gas tends
311
to be adsorbed by the bag relatively easily since the odor
residual rate in the case of an empty bag decreased to 32%
in 24 h.
Effect on neutral odors
Formaldehyde is a neutral odor associated with the “sick
building syndrome.” It is released into the air by construc-
tion materials such as adhesives, paints, and preservatives.
It can cause the sick house syndrome even at low concentra-
tion values. Therefore, it would be benefi cial if composite
paper containing waste green tea leaves could adsorb form-
aldehyde. Figure 2a shows the odor residual rate against
time for each sample. The results clearly show that all tested
fi brous samples had good deodorant effects on formalde-
hyde and decreased its odor residual rate to as low as 2.0%
in 3 h after gas injection. Natural fi bers and 100 wt% pulp
paper were similar to the composite paper containing waste
green tea leaves in deodorant ability, i.e., composite paper
containing waste green tea leaves did not have improved
deodorant ability. It may be safe to state that waste tea
leaves do not deodorize formaldehyde.
In the case of acetaldehyde, as shown in Fig. 2b, the
natural fi bers and the composite paper containing waste
green tea leaves exhibited different deodorant effects
against formaldehyde. Judging from the above results, com-
posite paper containing waste green tea leaves did not have
a substantially enhanced deodorant effect on neutral odors.
Effects for basic odors
Ammonia gas, as a basic odor, is one of the 22 offensive
odorifi c substances specifi ed by the Offensive Odor Control
Law.10 Ammonia gas is a component of the objectionable
smells that emanate from feces, rotten meat, and tobacco.
Ammonia gas is also the main component of smells emanat-
ing from the body, such as urine, sweat, and body odor.
As shown in Fig. 3a, ammonia gas was adsorbed to the
empty bag relatively easily, reaching residual rates of 75.6%
in 10 min and 53.3% in 24 h. Among natural fi bers, animal
fi bers such as wool and silk were found to have higher
deodorant abilities that decreased the residual rates to
8%–10% in 24 h; in contrast, the residual rate in the case of
cotton, which is a natural plant fi ber, decreased to 36.1% in
24 h. Among natural fi bers, the residual rate in the case of
Fig. 1a,b. Time dependence of odor residual rate for acidic odors. a
Hydrogen sulfi de gas (initial concentration: 20 ± 2 ppm), b acetic acid
gas (initial concentration: 25 ± 0.5 ppm)
Fig. 2a,b. Time dependence of odor residual rate for neutral odors. a
Formaldehyde gas (initial concentration: 5.0 ± 0.1 ppm), b acetalde-
hyde gas (initial concentration: 20 ± 2 ppm)
312
wool was 7.5% (4.42 ppm); wool had the highest deodor-
izing ability according to the results of the 24-h test.
According to the Weber–Fechner law, the perceived odor
intensity is proportional to the logarithm of the concentra-
tion of the odorous substance.11 When wool, which has a
good deodorizing ability against ammonia gas, decreased
the odor residual rate from 100% to 7.5%, the perceived
odor intensity would decrease to approximately half its
value in accordance with the Law. This implies that even
wool does not deodorize ammonia gas suffi ciently.
In contrast, composite paper containing 60 wt% waste
green tea leaves decreased the odor residual rate to 2.9%
in 6 min and to 0% in 1 h; for paper made from 100 wt%
pulp, the odor residual rate remained at 19.1% after 24 h.
This result suggests that the waste tea leaves in the compos-
ite paper can absorb ammonia gas.
The composite paper showed a similar superior deodor-
izing ability against trimethylamine, as shown in Fig. 3b. For
natural fi bers, the odor residual rate remained greater than
50%. The above results clearly show that composite paper
containing waste green tea leaves potentially has a very high
deodorizing ability against basic odors.
Deodorant effects of composite paper containing different
waste tea leaves on ammonia gas
As previously described, composite paper containing waste
green tea leaves had a good deodorizing ability against
ammonia gas. Paper composites with several kinds of waste
green tea leaves at ratios varying from 10 to 60 wt%, showed
excellent deodorant abilities on ammonia gas as shown in
Figs. 4a and b.
Ammonia gas at a concentration of 1 ppm (the threshold
concentration) is known to be detectable by the human
olfactory system.9 According to the evaluation method stip-
ulated for deodorant fi bers by the Japan Textile Evaluation
Technology Council, a fi ber material that can reduce the
concentration of ammonia gas originally at 40 ppm or more
to the detection threshold concentration level, or lower,
within 24 h, is considered to be a deodorant fi ber.6 There-
fore, all the composite paper examined in this study can be
considered deodorant fi ber materials with a good deodor-
izing ability against ammonia gas.
Fig. 3a,b. Time dependence of odor residual rate for basic odors. a
Ammonia gas (initial concentration: 60 ± 2 ppm), b trimethylamine gas
(initial concentration: 60 ± 2 ppm)
Fig. 4a,b. The deodorant properties of papers containing waste tea
leaves against ammonia gas (weight of paper sample: 1 ± 0.0001 g,
container: 5 l bag, size of container: 3 l, initial concentration of ammonia:
60 ± 2 ppm). a For different values of waste green tea leaf contents,
b for different types of waste tea leaves at a contents of 60 wt%
313
Next, the dependence of the deodorizing ability of tea
leaves on their fermentation level was examined, as shown
in Fig. 4b. All types of tea leaves in the composite paper,
including green tea, oolong tea, black tea, pu-erh tea, and
hojicha, reduced the initial concentration of ammonia gas
[60 ± 2 ppm (100%)], to 2 ppm (3%) or less within 10 min.
Thirty minutes after gas injection, very little ammonia gas
remained in the bags because of the deodorizing effect of
the leaves. The teas did not differ in deodorant ability,
showing good deodorant action within a short time. The fact
that the waste tea leaves took a short time to act on the
ammonia gas indicates that they have a good deodorizing
ability against this substance. To study the deodorizing
ability of each kind of tea in composite paper containing
waste tea leaves in the future, it is planned to adopt mea-
surement methods of higher precision than the gas detector
tube method.
Feasible reasons for the superior deodorizing ability of
composite paper containing waste tea leaves
As described above, composite paper containing any kind
of waste tea leaves has a good deodorizing ability against
basic odors. The following three factors can be considered
as feasible reasons for the deodorizing ability of composite
paper containing waste tea leaves: (1) the effect of the
porous surface structure of waste tea leaves may have
induced the physical adsorption of odors,12,13 (2) the high
equilibrium regain of waste tea leaves may have contributed
to the adsorption of odorous substances dissolved in mois-
ture in the air, and (3) the chemical reactions of waste tea
leaf components with basic odorous substances incorporate
the odorous substances into waste tea leaves.
Considering that composite paper has a deodorizing
ability against basic odors and no clearly enhanced deodor-
ant effect on acidic or neutral odors, reasons (2) and (3)
could, in theory, be the cause of the deodorizing ability of
composite paper containing waste tea leaves. Therefore, to
examine the probability of reason (2) being the cause of the
deodorizing ability, equilibrium regain against the relative
humidity of the atmosphere was calculated for composite
paper containing waste green tea leaves. Composite paper
containing 60 wt% waste tea leaves and natural fi bers, such
as cotton, silk, and wool, were used as test samples. Paper
made from 100 wt% pulp was also used as a sample for
comparison. As shown in Fig. 5, composite paper containing
60 wt% waste green tea leaves has a higher equilibrium
regain than paper made from 100 wt% pulp and other
natural fi bers, indicating that the waste tea leaves cause an
increase in the equilibrium regain value. Presumably, this is
because the composite paper is superior, in a hygroscopic
sense, than the other natural fi bers, which can be considered
nearly equal as far as their hygroscopic properties are con-
cerned. Therefore, the cause of the superior deodorizing
ability of composite paper containing waste green tea leaves,
as compared to natural fi bers, is understood to be due to
some other factors.
As described above, the chemical reaction between
odorous substances and waste green tea leaves in composite
paper was considered the most feasible factor for the excel-
lent deodorizing ability of the composite paper. The odorous
substances adsorbed by the composite paper were extracted
using distilled water. The measured pH value of the extracts
helps determine whether an acid/base neutralization reac-
tion took place between the composite paper containing
waste green tea leaves and odorous substances. If an acid/
base neutralization reaction took place between the odorous
substances and composite paper containing waste green tea
leaves, the extract from the composite paper that adsorbed
basic ammonia gas would have a nearly neutral pH value.
For comparison, a similar measurement was taken for
100-wt%-pulp paper.
Table 1 gives the pH values of distilled-water extracts of
paper after the adsorption of acetic acid and ammonia
gases. The pH values of the extract from the composite
paper containing waste green tea leaves was not neutral,
indicating that no acid/base neutralization reaction took
place between the odorous substances and the composite
paper. Thus, it was proven that deodorization by composite
paper containing waste green tea leaves does not takes
place as a result of a simple acid/base neutralization reac-
tion. However, from the pH measurement of paper extract
alone, it can be qualitatively concluded that no neutraliza-
tion reaction takes place but the data are not suffi cient for
quantitative discussion. The authors plan to use other means,
such as titration methods, in future studies.
Fig. 5. Moisture absorption behavior of paper samples. a, Natural fi ber
fabrics; b, paper containing 60wt% wasted green tea leaves
Table 1. pH values of the distilled-water extract of paper that has
adsorbed different gases
Sample Adsorption gas
Acetic acid
gas Ammonia
gas Air
Paper containing 60 wt% of
waste green tea leaves 4.17 10.08 6.52
100-wt%-pulp paper 4.04 10.43 6.80
314
The change in hue of the composite paper was then
examined after ammonia gas was deodorized. Composite
paper containing 60 wt% waste green tea leaves was left in
a desiccator fi lled with saturated aqueous solutions for 24 h
to fully adsorb the ammonia, trimethylamine, and acetic
acid odors. The photographs in Fig. 6 show that the compos-
ite paper turned dark brown in hue after adsorbing the basic
odors of ammonia and trimethylamine gases. In contrast,
the hue of the paper did not change signifi cantly after treat-
ments with acetic acid gas as an acidic odor. The results
indicate that the constituents of the waste tea leaves reacted
with ammonia and trimethylamine gases.
Catechins are one of the main constituents of tea leaves
and are known to convert into theafl avins through the process
of fermentation in the presence of polyphenol oxidase. Chlo-
rophyll is another important constituent in the leaves and is
relatively stable in alkaline environments. In contrast, poly-
phenols, including catechins, are unstable and are easily
decomposed during the color development in alkaline envi-
ronments. It is also known that a part of the polyphenols
undergoes oxidation polymerization.14 In particular, cate-
chins are supposed to be polymerized in alkaline environ-
ments without an oxidase. The compounds in waste green tea
leaves are likely to be involved in the deodorization of basic
odors. The following process has been proposed as the mech-
anism responsible for the deodorizing ability of catechins
toward ammonia gas: phenolic hydroxyl groups of catechins
form hydrogen bonds with ammonia molecules from the air
to produce an adduct (O−NH4+) salt.15 The catechins are thus
considered to be polymerized by oxidation.
Therefore, it was necessary to further study the chemical
changes in catechins due to the adsorption of ammonia.
Epigallocatechin gallate [(−)-EGCg] is known to be the
most abundant constituent in green tea. It was spotted on
thin-layer chromatography plates left in desiccators fi lled
with ammonia gas for 24 h until the complete adsorption of
the ammonia odor.
Figure 7 shows photographs of the thin-layer chromatog-
raphy plates with ammonia odor adsorbed before separa-
tion (on the left) and after separation (on the right). The
samples were observed under incandescent light and 245-nm
ultraviolet light for easier observation. The (−)-EGCg was
clearly detected at the starting point before separation (on
the left, Fig. 7). The (−)-EGCg turned dark brown after
ammonia adsorption; therefore, it was detectable even
under an incandescent light (circled by a broken line). A
similar result was obtained when trimethylamine odor was
adsorbed. In contrast, the (−)-EGCg remained too white on
the TLC plate after the adsorption of acetic acid odor to be
detected by visual inspection.
The solvent water was then used for separation in thin-
layer chromatography in a subsequent experiment (on the
right in Fig. 7). After development, the upward migration of
(−)-EGCg alone was confi rmed from the thin-layer chroma-
tography plates. After the adsorption of acetic acid, an
upward migration of (−)-EGCg was found in the plates after
separation, indicating that the acetic acid gas had under-
gone no signifi cant chemical reaction with the (−)-EGCg.
When either ammonia gas or trimethylamine gas was
adsorbed, the (−)-EGCg still remained at the starting point
and an upward migration of the compound was observed.
In other words, when basic odors were adsorbed, the (−)-
EGCg was chemically modifi ed. This was because the com-
pound did not migrate on the TLC plate even when a
mixture of chloroform, methanol, and distilled water was
used as a solvent for separation.
To identify the compound, mass spectrometry was
employed. Unfortunately, the spectrum of the modifi ed (−)-
EGCg showed a large number of peaks in the molecular
weight range 500–1500; owing to their large number, these
peaks could not be identifi ed. These results indicate that the
catechins adsorbed basic odors to form many complex
structures. (−)-EGCg treatment of ammonia is estimated to
form a very large number of substances that have complex
structures. The authors have searched in vain for examples
of related studies and would like to study the relationship
between the structures and antimicrobial activity of the
substances in the future.
Fig. 6. Paper containing 60 wt%
of waste green tea leaves after
odor adsorption (i.e., after 24 h
in a saturated gas desiccator)
315
As described above, the incorporation of waste tea leaves
into paper dramatically increased the level of deodorant
effect of the paper. The phenomenon seems to involve
chemical reactions; however, it is also necessary to examine
the effects of the paper surface area on the deodorant effect.
The authors plan to study the effects of the paper surface
area in the future.
Antibacterial activity of composite paper containing waste
tea leaves after deodorization of ammonia gas
Composite paper containing waste green tea leaves was left
in a desiccator fi lled with ammonia gas for saturation, and
then for 24 h after the saturation for the complete adsorp-
tion of ammonia gas. Staphylococcus aureus was used to
evaluate the antibacterial activity of the composite paper
Table 2. Antibacterial properties (toward Staphylococcus aureus) of paper containing 60 wt% of waste green tea leaves after adsorbing ammonia
gas
Sample Adsorption of
ammonia gas Incubation
time (h) Antibacterial properties
Viable bacteria
(CFU/ml) Log C Bacteriostatic
activity Bactericidal
activity
0 1.00 × 1055–
a–
Paper containing tea leaves Before 18 5.00 × 1055.70 2.79 −0.70
100-wt%-pulp paper 3.08 × 1088.49 – –
Paper containing tea leaves After 18 <4.40 × 102<2.64 >5.85 >2.36
100-wt%-pulp paper 6.48 × 1088.81 −0.32 −3.81
a Non-numerical value
C, viable cell number
Fig. 7. Changes in
(−)-epigallocatechin gallate on
silica gel thin-layer
chromatography (TLC) plates
coated with a fl uorescent reagent
after ammonia adsorption (i.e.,
after 24 h in a saturated gas
desiccator). The extraction
solvent was diethyl ether; the
separation solvent was
chloroform, methanol, and water
(59 : 32 : 9); and the separation
time was 210 min. The (−)-EGCg
turned dark brown after
ammonia adsorption and was
detectable even under an
incandescent light (circled by a
broken line)
containing waste green tea leaves. As a reference, paper
made from 100 wt% pulp was allowed to achieve complete
adsorption of ammonia gas; it was also subjected to evalu-
ation of its antibacterial activity. Table 2 lists the evaluation
results of the antibacterial activities.
The number of viable bacteria in the culture at 18 h
increased from 1.00 × 105 to 5.00 × 105 CFU/ml for a bacte-
rial suspension that was dropped on the composite paper
containing waste green tea leaves before ammonia gas
adsorption. In contrast, it increased to 3.08 × 108 CFU/ml
for the bacterial suspension dropped on the paper made
from 100 wt% pulp before ammonia gas adsorption. The
composite paper containing waste green tea leaves appeared
to suppress bacterial multiplication. This antibacterial effect
was considered to be due to the catechins contained in the
waste green tea leaves.
316
When the above experiments were repeated with paper
samples that had been used to deodorize ammonia gas, no
viable bacteria were found in the composite paper contain-
ing waste green tea leaves after culture for 18 h. Further-
more, the antibacterial activity had increased to a higher
level than that of the reference. This could be due to the
polymerization of catechins in the ammonia environment
because these polymerized compounds are believed to
increase the hydrophobicity of cell walls and enhance the
interactions between the polymer and the cell membrane of
the bacterium. Here, since the number of viable bacteria
(6.48 × 108 CFU/ml) in the paper made from 100 wt% pulp
that had adsorbed ammonia gas was almost the same as that
in the paper made from 100-wt%-pulp paper without
adsorbed ammonia gas, it was evident that the ammonia gas
itself had little antibacterial effect on Staphylococcus aureus.
From the above experimental results, the catechins con-
tained in waste green tea leaves were inferred to have
polymerized in the alkaline environment with ammonia gas.
During the chemical reaction, some OH groups in the cat-
echins supposedly got chemically transferred into O−NH4+
as an intermediate for holding ammonia gas in the molecu-
lar structure; this was indirectly confi rmed by mass
spectrometry. As described previously, the details of the
molecular structures of the compounds formed in the reac-
tions between catechins and ammonia remain unknown and
will be a subject for future research. The characteristics
elucidated in these experiments are very useful and indicate
that composite paper containing waste tea leaves can be
used effectively in different applications.
Conclusions
Composite paper containing waste tea leaves was prepared
and its deodorizing ability was examined in an effort to
promote the effective utilization of used tea leaves. The
paper showed excellent deodorizing ability against ammonia
gas and trimethylamine gas, both of which are basic odors.
As described above, composite paper containing waste tea
leaves was found to possess not only good deodorizing
ability but also exhibited improved antibacterial activity
after it had adsorbed gas during deodorization. Thanks to
these characteristics, composite paper containing waste tea
leaves has several potential applications.
Acknowledgments We would like to thank Mr. Hiroshi Yokota and
Mr. Tetsunori Kunitake of Ehime Paper Mfg. Co., Ltd., for their kind
cooperation in providing samples and in measurement of physical
properties.
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