Process Biochemistry 39 (2004) 20432046

Extraction and fractionation of glucosyltransferase

inhibitors from cacao bean husk
Kyoung Heon Kim a , Ki Won Lee b , Dong Young Kim c ,
Hyung Hwan Park c , Ik Boo Kwon c , Hyong Joo Lee b,
a Division of Food Science, Korea University, Seoul 136-701, Korea
b Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
c Lotte R&D Center, Seoul 150-104, Korea

Received 24 February 2003; accepted 12 October 2003

Abstract

The re-utilization of cacao bean husk, a waste generated from the chocolate industry, would bring benefits both environmentaly and
economicaly. This study relates to a process for effectively separating and fractionating a cacao bean husk fraction having high inhibitory
activity against glucosyltransferase (GTF) for the prevention of tooth decay (anticaries activity). Since the GTF inhibitory activity is known to
be rendered by polyphenols, the separation process was also able to aim at high recovery of polyphenols which benefits human health. In this
study, cacao bean husk extract, obtained under optimal conditions, extraction with 50% (v/v) aq. (aqueous) acetone solution at 60 C for 4 h
followed by 50% (v/v) aq. ethanol using a styrene-based resin, showed significantly higher inhibitory activity (2 and 12 folds, respectively)
against GTF and a similar polyphenol content, compared to two other commercial anti-GTF polyphenol products.
2003 Elsevier Ltd. All rights reserved.

Keywords: Cacao bean husk; Theobroma cacao; Glucosyltransferase; Anticaries; Polyphenols

1. Introduction [2], and immunoregulatory activities [3], are possessed by

Cacao (Theobroma cacao) is an industrially important
crop since cacao bean is the active ingredient of chocolate
that is one of the most popular foods in the world. During
the manufacturing process of chocolate, the nib portion of
peeled-off cacao bean is used as the raw material for choco-
late or cocoa, while its husk (approximately 15 wt.% of to-
tal weight of cacao bean) is generated as a waste. Judging
from the current worldwide annual production of cacao bean
which accounts for about 2.5 106 tons, it is estimated that
4 105 tons of cacao bean husk are discharged from the
industry each year.
Cacao beans are known to be rich in polyphenols [1].
In particular, not only cacao nib but also cacao bean husk
contains a large amount of polyphenols. Recently, it is be-
ing revealed that various biological and health-beneficial ef-
fects, such as anticaries (i.e. anti-tooth decay), antioxidant
Corresponding author. Tel.: +82-2-880-4853;
fax: +82-2-873-5095.
E-mail address: leehyjo@snu.ac.kr (H.J. Lee).
polyphenols contained in cacao. The anticaries activity of
polyphenols is accomplished by inhibition of glucosyltrans-
ferase (GTF) which is the key enzyme in initiation, devel-
opment, and growth of dental plague glucans on the tooth
surface [46]. Extract of cacao obtained either from cacao
bean mass [7] or cacao husk [8] was found to show anti-
caries activity.
Meanwhile, due to the fact that conventional polyphe-
nol extracts produced from green tea and oolong tea are
expensive and their inhibitory activity against glucosyl-
transferase (GTF) is relatively low due to the high content
of monomers containing catechin and epicacatechin, the
re-utilization for the cacao bean husk is under active devel-
opment in the industry [9,10]. Previously, we have reported
that cacao extraction from bean husk obtained during the
manufacturing process of chocolate was economically fea-
sible [9].
The object of this study was to develop a process for effec-
tively separating and fractionating cacao bean husk extract
having potent activity against GTF, for dental application,
along with a high polyphenol content. More specifically,

0032-9592/$ see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2003.10.006
2044 K.H. Kim et al. / Process Biochemistry 39 (2004) 20432046
the GTF, inhibitory activity and polyphenol content of
each extract obtained at various operating conditions were
compared with those of other commercial products.
2. Experimental
2.1. Preparation of GTF
GTF derived from Streptococcus mutans, known to be the
major microorganism for tooth decay, was prepared by the
method of Fukushima and Motoda [11] with a slight mod-
ification. S. mutans ATCC 6715 was cultivated at 37 C for
24 h. 2% (v/v) of the seed culture broth was then inoculated
into a 4.5 l brainheart infusion medium to perform the incu-
bation under the same conditions. After the incubation, the
culture broth was centrifuged at 6000 rpm at room temper-
ature for 20 min. Three litres of ethanol, previously cooled,
were added to the supernatant to precipitate proteins, and left
at 4 C overnight. The residue was centrifuged at 8000 rpm
for 30 min to obtain a precipitate which was re-suspended
in 10 ml of 0.05 M phosphate-buffered solution (pH 6.8) to
make a crude enzyme solution, then stored at 20 C. An
appropriate amount of the crude enzyme solution diluted
with 0.05 M phosphate-buffered solution (pH 6.5) was used
for enzyme reaction for the evaluation of inhibitory activi-
ties of extract samples.
2.2. Extraction of cacao bean husk
The cacao bean husk used in this study, were discharged
from the manufacturing process of chocolate or cocoa drinks
and obtained from Lotte Co. (Seoul, S. Korea). Thirty grams
of completely dried cacao bean husk with sizes of less than
2 mm was extracted with 300 ml of one of the following aq.
(aqueous) solvents: methanol (50 and 100%, v/v); ethanol
(50 and 100%, v/v); and acetone (40, 50, and 60%, v/v) un-
der reflux. The extraction temperature and time were cho-
sen in the ranges of 4080 C and 46 h, respectively. After
the extraction, the solution was filtered off to collect the ex-
traction solution only. The above procedures were repeated
twice. Cacao bean husk extract was finally obtained by con-
centrating the extraction solution using a vacuum evaporator
at either 40 or 50 C.
2.3. Fractionation of extract of cacao bean husk
Seventy grams (by dry wt.) of extract of cacao bean husk
obtained as the above was loaded onto a styrene-based ad-
sorption resin column (60 mm 450 mm, HP-20, Mitsubishi
Co., Japan), washed with 1400 ml of 20% (v/v) aq. ethanol
and eluted with 1400 ml of 50% (v/v) aq. ethanol. The eluted
fraction was concentrated at 40 or 50 C under reduced pres-
sure and used for the determination of inhibitory activity
against GTF and polyphenols content.
2.4. Determination of inhibitory activity of cacao bean
husk extract
The inhibitory activities of each extract against GTF were
performed as follows. Substrate solution was prepared by
dissolving 12.5 g of sugar with 0.25 g of sodium azide as
an antimicrobial agent in 1000 ml of a 62.5 mM phosphate
buffer solution (pH 6.5). 0.1 ml substrate solution, 0.025 ml
GTF solution, and 0.175 ml of extract sample were put into
a test tube to make a final volume of 1 ml of a reaction mix-
ture. A control reaction mixture was constituted in the same
manner as the sample reaction mixtures except for replacing
the extract sample with the same amount of distilled water.
Test tubes were inclined at a slope of about 30 and re-
actions were performed at 37 C for 16 h. After the reac-
tion was completed, the supernatant contained in each test
tube was decanted and glucan, the reaction product, was dis-
persed using an ultrasonicator for 5 s following the addition
of 3 ml of distilled water. To quantify the inhibitory activity
of the extract samples, the amount of glucan was determined
by measuring the absorbance of the diluted reaction mix-
ture at 550 nm using a UV-Vis spectrophotometer (UV-260,
Shimadzu Co., Japan).
2.5. Determination of polyphenol content in cacao bean
husk extract
The polyphenol contents of each sample were measured
by a slightly modified Folin-Ciocalteu method [12]. First,
750 l of distilled water was put into an Eppendorf tube,
followed by the addition of 100 l of sample solution dis-
solved at an appropriate concentration. A mixture of 50 l of
Folin Denis reagent and 100 l of saturated sodium carbon-
ate solution was then added to the reaction mixture, mixed
well, and reacted at room temperature for 1 h. After the re-
action was completed, the absorbance of the reaction mix-
ture was measured at 725 nm to determine the polyphenol
content using a standard curve prepared using epicatechin.
3. Results and discussion
3.1. Effects of extraction solvent
To find an effective solvent for the extraction of a frac-
tion showing high inhibitory activity against GTF and also
containing a high content of polyphenols, various solvents
were tested as shown in Table 1. In this experiment, 30 g of
dried cacao bean husk was extracted twice with 300 ml of
one of the solvents listed in Table 1 at 60 C for 4 h with stir-
ring. The extract solution was concentrated at 50 C using a
vacuum evaporator.
The recovery yields and the polyphenols contents of
extracts were approximately 23 times higher when the
solvents including methanol, ethanol, and acetone were
4060% (v/v) aqueous solutions, compared with the pure
 K.H. Kim et al. / Process Biochemistry 39 (2004) 20432046 2045

Table 1 Table 3
Effects of extraction solvents on extract yield, inhibitory activities against Effects of extraction time on extract yield, inhibitory activity against
GTF, and polyphenol content in extract of cacao bean husk glucosyltransferase (GTF), and polyphenol content in extract of cacao
bean husk
Aqueous solution Yield of extract IC50 against Polyphenol
(%, w/w)a GTF (g/ml)b (wt.%)c Extraction Yield of extract IC50 against Polyphenol
time (h) (%, w/w)a GTF (g/ml)b (wt.%)c
Methanol (100%, v/v) 8 100 8
Methanol (50%, v/v) 17 100 14 1 20 65 14
Ethanol (100%, v/v) 9 100 8 2 21 60 14
Ethanol (50%, v/v) 22 100 15 3 24 60 14
Acetone (60%, v/v) 25 90 18 4 26 57 14
Acetone (50%, v/v) 26 80 21 5 25 59 14
Acetone (40%, v/v) 19 95 19 6 25 60 14
7 24 67 13
a Dry weight of extract per dry weight of total cacao bean husk used
a Dry weight of extract per dry weight of total cacao bean husk used
in percentage.
b Concentration that produces a 50% inhibitory effect on the evaluated in percentage.
b Concentration that produces a 50% inhibitory effect on the evaluated
parameter.
c Percentage of polyphenols in total extract on dry weight basis. parameter.
c Percentage of polyphenols in total extract on dry weight basis.

solvents. The higher extraction yields with the aqueous

solutions may be ascribed to an increase of polarity of the
solvents by adding water having a high dielectric constant.
When using the aqueous solutions, in spite of the significant
increases in extract yield, the IC50 values of the extracts
decreased by 520%, compared with the use of pure sol-
vents. Therefore, using the aqueous solutions at 4060%
instead of using the pure solvents is advantageous for both
increasing the yields of extract and polyphenols and the
GTF inhibitory activity. 50% aq. acetone was selected as
the optimal extraction solvent for further experiments in
this study.
diffusivity of solute in liquid at a higher temperature. The
inhibitory activity of extract against GTF was significantly
higher at 4070 C compared to 30, 80, and 90 C. The low
inhibitory activity are considered to be due to the fact that
the GTF inhibitors are poorly extracted at the low temper-
ature and possibly denatured or destroyed at the high tem-
peratures. Therefore, 60 C, at which high values of GTF
inhibition and recovery yield of extract and polyphenols
were shown, was selected for further extraction experiments.
3.3. Effects of extraction time

3.2. Effects of extraction temperature
The extractions were carried out at various temperatures
under the conditions described in Section 3.1. The extract
recovery yield, the polyphenol content of extracts and the
inhibitory activity against GTF are shown in Table 2. The
recovery yield of extract and the polyphenols content gen-
erally increased with an increase in extraction temperature
and these results are probably due to a higher solubility and
Using the optimal extraction conditions selected in
Sections 3.1 and 3.2, extractions were conducted for various
extraction time lengths. The extract recovery yield appeared
to increase by increasing extraction time up to 4 h, but the
further extending of extraction time did not result in an
increase of extract recovery yield. The polyphenol content
and IC50 values of extracts were not much affected by vary-
ing extraction time. Therefore, the optimal extraction time
was considered to be 4 h (Table 3).

Table 2 3.4. Effects of fractionation of extract using a polymeric

Effects of extraction temperature on extract yield, inhibitory activity
against glucosyltransferase (GTF), and polyphenol content in extract of
adsorbent
cacao bean husk
Temperature Yield of extract IC50 against Polypheno1
Cacao bean husk was extracted under optimal conditions
( C) (%, w/w)a GTF (g/ml)b (wt.%)c (50% aq. acetone, 60 C, and 4 h) determined in the preced-
30 18 90 11 ing sections. Results are listed as Sample no. 3 in Table 4.
40 21 66 14 The inhibitory activity of Sample no. 3 was higher than that
50 20 59 15 of green tea extract and lower than that of oolong tea extract.
60 24 58 18 Both commercially available for the prevention of tooth de-
70 24 63 19
cay. The polyphenol content of Sample no. 3 was lower than
80 27 90 18
90 25 90 16 those of the commercial products.
a Dry weight of extract per dry weight of total cacao bean husk used
To increase the concentration of GTF inhibitors in the ex-
tract, cacao bean extract obtained under the conditions used
in percentage.
b Concentration that produces a 50% inhibitory effect on the evaluated for Sample no. 3 was fractionated using the styrene-based
parameter. resin column such as washing and elution with 20 and 50%
c Percentage of polyphenols in total extract on dry weight basis.
aq. ethanol, respectively, at a flow rate of 50 ml/min for both
2046 K.H. Kim et al. / Process Biochemistry 39 (2004) 20432046
Table 4
Comparison of inhibitory activities against glucosyltransferase (GTF) and polyphenol contents of various extract samples
Sample Source Extraction condition
no.
Solvent
1 Green tea extracta Not applicable
2 Oolong tea extracta Not applicable
3 Cacao bean husk Acetone (50%, v/v)
4 Cacao bean husk Acetone (50%, v/v)
5 Cacao bean husk Methanol (50%, v/v)
6 Cacao bean husk Ethanol (50%, v/v)
a Commercial products available in Japan.
steps. When the elution flow rate exceeded 100 ml/min, the
yield of final product decreased, probably due to insufficient
adsorption time (data not shown). The eluate was referred to
as Sample no. 4 after vacuum concentration. By comparing
Sample nos. 3 and 4, it was found that both the GTF in-
hibitory activity and phenol content increased approximately
threefold.
In the column fractionation for Sample no. 4, the GTF
inhibitors and polyphenol contained in more hydrophobic
fraction, which eluted with 50% aq. ethanol, appeared to
be bound to the styrene-based resin without being washed
out with 20% aq. ethanol. However, when the extraction
solvent (i.e. 50% aq. acetone) was replaced with 50% aq.
methanol and ethanol as in the case of Sample nos. 5 and
6, both the GTF inhibitory activity and the phenol content
were significantly lower than Sample no. 4 that was ex-
tracted with 50% aq. acetone before column fractionation.
Therefore, the GTP inhibitor- and polyphenol-containing
fraction is considered to be more soluble in less polar sol-
vents such 50% aq. acetone and ethanol solutions than 20%
aq. ethanol.
In Table 4, the GTP inhibitory activity of Sample no. 4
was much higher than the commercial products developed
for fighting tooth decay. The polyphenol content in Sample
no. 4 was similar to those of the two commercial products
from Japan. These results suggest that the GTP inhibitory
fraction extracted from cacao bean husk followed by col-
umn fractionation is feasible for commercialization due to its
high GTF inhibitory activity and polyphenol content. This
process is also considered to be economically feasible since
the raw materials are abundantly available from the choco-
late industry at a low cost.
4. Conclusion
This study demonstrates to an effective process for sepa-
rating and fractionating a polyphenol extract with high in-
hibitory activity against GTF from cacao bean husk which
is a waste from the chocolate industry. Optimal conditions
for the recovery of cacao bean husk with a high anti-GTF
Column fractionation IC50 against Polyphenol
using ethanol GTF (g/ml) (wt.%)
Time (h) Temperature ( C)
250 40
40 40
4 60 No 57 14
4 60 Yes 20 39
4 50 Yes 85 15
4 50 Yes 81 18
activity and a high polyphenol content was determined to be
extraction with 50% aq. acetone at 60 C for 4 h, followed
by fractionation with 50% aq. ethanol using styrene-based
resin. The cacao bean husk obtained in these conditions
showed 2 and 12 times higher inhibitory activity against
GTF and a similar polyphenol content compared to two other
commercially-available products.
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