Biotechnology Letters 23: 17191722, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.

1719
Isolation and characterization of a clay-dispersing polysaccharide
produced by the phytopathogenic fungus, Colletotrichum gloeosporioides
Sang Ho Park
1,
, Chang Won Choi
1
, Myoung Yong Shim
1
, Won Mok Park
2
, Soon Seop Hwang
1
& Jung-Mo Koo
1
1
BioMed RRC and Department of Biology and Medicinal Science, Pai Chai University, Taejon 302-735, Korea
2
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea

Author for correspondence (Fax: 82-42-520-5617; E-mail: shparkbs@dreamwiz.com)

Received 31 May 2001; Revisions requested 22 June 2001/31 July 2001; Revisions received 27 July 2001/20 August 2001; Accepted 20 August
2001
Key words: Colletotrichum gloeosporioides, dispersion, kaolin, polysaccharide
Abstract
The fungus, Colletotrichum gloeosporioides, which is pathogenic to peppers produced an extracellular polysac-
charide in liquid culture which possessed clay-dispersing activity. The polysaccharide could bind cationic dyes,
Ruthenium Red and Alcian Blue, indicating it to be polyanionic. The polysaccharide dispersed kaolin in water and
the dispersion was maintained for more than 7 days at 25

C. Kaolin dispersion by the polysaccharide was stable

from pH 3 to 10 but the addition of divalent metals at 1 mM inhibited half of the dispersion activity comparing
to the control. The polysaccharide could disperse bentonite, calcium carbonate and other ne particles but did not
possess emulsifying activity.
Introduction
Microorganisms produce various kinds of polysac-
charides outside the cells. Since the polysaccharides
from microbial sources have characteristic properties
different from synthetic polymers, some polysaccha-
rides such as alginate (Carlson & Mathews 1966)
and xanthan (Sloneker & Jeanes 1962) are added to
foods as thickening agents or stabilizers. Furthermore,
certain polysaccharides from microbial sources are
surface-active, and thus they have been attempted to
use as metal-chelators (Sutherland 1998), emulsiers
(Cirigliano & Carman 1984), and occulants (Bar-
Or & Shilo 1987, Choi et al. 1998) in industrial and
environmental elds.
In the course of our screening work to isolate
surface-active polysaccharides, we found that the phy-
topathogenic fungus, Colletotrichum gloeosporioides,
isolated from the pepper plant (Hwang 2000), pro-
duced the anionic extracellular polysaccharide in liq-
uid culture. We have now isolated and characterized
this polysaccharide being capable of dispersing clay
particles in water.
Materials and methods
Chemicals and fungi
Alcian Blue 8 GX, Ruthenium Red, kaolin (particle
size: 0.14 m), bentonite, and calcium carbonate
were purchased from Sigma. Potato/dextrose broth
was from Difco. All other chemicals were of the
highest purity available.
Colletotrichum gloeosporioides (isolates A1/8,
A1/5, and A1/11) were collected from the infected
green or red fruits of the pepper plants, isolated by
monoconidial culture and maintained on potato dex-
trose agar medium. Their cultural, pathological, and
genetic characteristics have been described (Hwang
2000).
1720
Isolation of the extracellular polysaccharide
C. gloeosporioides was grown in a 250 ml Erlenmeyer
ask containing 100 ml of 2% potato/dextrose broth
with shaking at 120 rpm at 25

C. After 10 days, the

medium was ltered through four layers of cheese-
cloth and the ltrate was centrifuged at 6000 g for
25 min. The supernatant was added into 2 volumes of
chilled 95% ethanol under stirring. The slimy precip-
itated material was harvested by centrifugation (6000
g for 10 min), dissolved in, dialyzed against distilled
water, and dried.
Determination of cationic dye binding activity
The Alcian Blue binding assay was performed with a
minor modication by the method of Bar-Or & Shilo
(1988). Alcian Blue was dissolved in 0.5 M acetic acid
to give 1 mg ml
1
. After adding 700 l of 0.5 M
acetic acid to a 200 l sample, the solution was mixed,
100 l Alcian Blue solution was added, and the re-
sulting solution mixed again. After standing for 1 h
at room temperature, the solution was centrifuged at
12 000 g for 5 min, and then the absorbancy of the
supernatant was read at 610 nm.
For determining of the Ruthenium Red binding ac-
tivity, 10 l Ruthenium Red solution (5.5 mg in 1 ml
distilled water) was added to various concentrations
of samples in distilled water and the nal volume ad-
justed to 1 ml with distilled water. After standing for
1 h at room temperature the solution was centrifuged
at 10 000 g for 5 min, and then the absorbancy of the
supernatant was read at 530 nm.
Determination of clay dispersion activity
A stock solution of kaolin clay was prepared in dis-
tilled water at 5 mg ml
1
. Two hundred fty l of
the stock solution was added into test tubes containing
2.5 ml distilled water. Various concentrations of sam-
ples in distilled water were added and nal volume
was adjusted to 4 ml with distilled water. The tubes
were stirred with a Vortex mixer and left at 25

C for
6 h. The upper 2 ml of the suspension was then care-
fully removed, and the turbidity of the upper 2 ml was
measured at 550 nm. A control experiment without the
polysaccharide was carried out in the same manner.
The kaolin control at zero time yielded a turbidity of
1.
Fig. 1. Growth and production of the polysaccharide by
C. gloeosporioides A1/5. The cultivation was done with 100 ml
potato/dextrose broth under the conditions described in Materials
and methods. Fungal growth (); the extracellular polysaccharide
().
Analytical methods
The protein concentration was determined by Brad-
ford (1976) method using a Bio-Rad dye reagent.
Neutral sugar determination was done by the method
of Dubois (1956) using glucose as a standard. For
estimation of fatty acid, the polysaccharides were hy-
drolyzed with 0.5 M KOH for 90 min at 50

C, the
extracted with petroleum ether. The solvent extraction
was evaporated, and the residue was weighed (Bar-Or
& Shilo 1987).
Results and discussion
Three isolates of C. gloeosporioides produced an ex-
tracellular polysaccharide that could disperse spores
and kaolin clays in distilled water (Table 1). Isolate
A1/5 was chosen for further experiments because of
its highest dispersing activity. For the assay of spore
dispersion by the polysaccharide, spores (average size,
13.2 5 m) were harvested at 5000 g for 5 min from
the 14-day-old culture, washed twice by centrifuga-
tion and resuspended in distilled water. At zero time
the turbidity of spore suspensions was adjusted to 1 at
550 nm. When the spores were incubated for 6 h with
0, 25, and 50 g polysaccharide ml
1
, the turbidities
were 0.11, 0.63, and 0.93, respectively.
When the fungus was grown on potato/dextrose
broth at 25

C for 3 weeks, the production of the

1721
Table 1. Production of extracellular polysaccharides by
C. gloeosporioides.
Fungal isolate
a
Dry wt of the Kaolin Spore
polysaccharide dispersion dispersion
(mg l
1
)
A1/8 68 + +
A1/5 72 + +
A1/11 70 + +
a
Each fungus was grown in 100 ml potato/dextrose broth for 2
weeks and the polysaccharide was obtained by ethanol precipita-
tion. The dispersion activity of kaolin and spore suspension was
determined in the presence of the polysaccharide; + indicates
positive dispersion.
polysaccharide increased steadily during 2 weeks of
incubation and remained constant (Figure 1). The
culture medium became very viscous during fungal
growth indicating the excretion of extracellular mate-
rials.
The clay-dispersing polysaccharide was isolated
by ethanol precipitation and found mainly to consist
of sugars with a trace amount of proteins, but not fatty
acids. Many surface-active polysaccharides have neg-
ative charges (Bar-Or & Shilo 1987, Choi et al. 1998,
Cirigliano & Carman 1984). Alcian Blue is a cationic
dye with a high afnity for polyanions along with
sulfates. This reagent has been used for an indirect as-
say for calculation for comparing anionic densities of
different polysaccharides (Bar-Or & Shilo 1987). Fur-
thermore, Ruthenium Red has been used to stain and
quantify acidic polysaccharides in algal cells (Hoff-
man & Bauknecht 1999). Therefore, the dye binding
activity of the polysaccharide was assayed and the re-
sults are shown in Figure 2. When increasing amounts
of the polysaccharide were added to a constant amount
of dye, both dyes bound to the polysaccharide. How-
ever, the polysaccharide had much higher afnity for
Ruthenium Red than Alcian Blue.
Aqueous suspensions of clays occulate and settle
rapidly, leaving a clear upper phase. In the presence of
a dispersant, the occulation is prevented so that the
individual clay particles settle relatively slow (Choi
et al. 1998, Rosenberg et al. 1988). Figure 3 shows
the clay dispersing activity of the polysaccharide from
C. gloeosporioides. When kaolin was suspended in
water, this clay was effectively dispersed with increas-
ing amounts of the polysaccharide. Furthermore, in
the presence of the polysaccharide at 200 g ml
1
or
above, the dispersion of kaolin lasted more than 7 days
at room temperature.
Fig. 2. Binding of Ruthenium Red () and Alcian Blue () by the
polysaccharide.
Fig. 3. Dispersion of kaolin suspension by the polysaccharide.
1722
Table 2. The functional spectrum of the polysaccharide pro-
duced by C. gloeosporioides A1/5.
Chemicals Dispersing activity
a
Kaolin ++
Bentonite ++
Calcium carbonate ++
Celite +
Activated charcoal +
Olive oil
Hexadecane
Hexadecane/2-methylnaphthalene
b

a
Dispersion maintained more than 24 h (++); less than 6 h
(+); no activity ().
b
Emulsifying activity was determined by the method of
Cirigliano & Carman (1984).
To further characterize the physical properties of
this polysaccharide, the effects of pH and divalent
metals on dispersing activity were determined us-
ing kaolin clay suspension. The dispersing activity
remained relatively unchanged over pH 3 to 10, sug-
gesting that the polysaccharide has a broad pH range.
The effect of divalent metal ions was also investigated
at various concentrations. All metal ions at 1 mM in-
hibited half of the dispersion activity comparing to the
control.
The spectrum and activity of the polysaccharide
are summarized in Table 2. The polysaccharide from
C. gloeosporioides could disperse other particle mate-
rials. Bentonite and calcium carbonate were dispersed
as much as kaolin for more than a week. Activated
charcoal and Celite werd also dispersed but olive oil
and hexadecane were not emulsied. Therefore, the
polysaccharide fromC. gloeosporioides seems to have
only a dispersing activity, being specic to clays or
other ne particles.
In this study, we demonstrated that the phytopatho-
genic fungus, C. gloeosporioides produced a clay-
dispersing polysaccharide. There are many types of
occulating or emulsifying polysaccharides from mi-
crobial sources but there are relatively few records on
a dispersing agent from microorganisms. Until now,
only Acinetobacter calcaoaceticus A2 is known to
produce such a dispersing polysaccharide (Rosenberg
et al. 1988). This polysaccharide possesses limestone-
dispersing activity and its dispersion is optimum be-
tween pH 9 and 12. Phosphate (2 mM) and Mg
2+
(8 mM) caused a 50% inhibition of activity.
Dispersion of clays has been the subject of con-
siderable applications in many elds, such as drilling
uids, pesticide and pharmaceutical formulations, ce-
ments, and ceramic additives. Therefore, the polysac-
charide from C. gloeosporioides may be exploited for
potential uses for dispersing agents of biological ori-
gin. Elucidation of the composition and structure of
the polysaccharide is necessary for further study and
mechanisms of action of clay dispersion might be
important for improving their activity.
Acknowledgement
This work was supported by Korea Research Founda-
tion Grant (KRF-1999-015-DP0400).
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